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Mutant projectGeneMuation typeBone &
Cartilage
BehaviourNeurologyEye &
Vision
NociceptionEnergy
Metabolism
Clinical
Chemistry
Immun-
ology
AllergySteroid
Metabolism
Cardio-
vascular
Lung
Function
Expression
Profiling
PathologyBodyweightReport
KTA041Scube3Induced mutation
Bone & Cartilage

Summary:

In the morphological investigation via visual inspection, we observed a smaller body size and tail
length. In the X-ray analysis, we found short femora and malformations of the thoracic and
lumbar vertebrae. Also the ratio of the femur length to the body length was decreased
in mutants compared to controls.  In the DXA analysis, we detected a significantly decreased BMD,
BMC, and bone content in mutants compared to controls. Concurrently body length, body weight and
fat mass were significantly decreased in mutants.



Discussion:

The KTA041 mutant mouse line was analyzed in the Dysmorphology, Bone and Cartilage module of
the German Mouse Clinic. In the morphological investigation via visual inspection, we observed a smaller
body size and tail length. In the X-ray analysis, we found short femora and malformations
of the thoracic and lumbar vertebrae. In the DXA analysis, we detected a significantly decreased
BMD, BMC, and bone content in mutants compared to controls. Concurrently, body length, body weight
and fat mass were significantly decreased in mutants.

Behaviour

Summary:

In the open field, the mutant mice showed decreased locomotor and exploratory activity and features
that may indicate an increase in anxiety-related behaviour. There was increased acoustic startle reactivity by
the mutant mice.



Discussion:

In the open field, our test of spontaneous reactions to a novel environment, the mutant
mice showed hypoactivity and decreased movement velocity and exploration. This hypoactivity ties in with the
hypoactivity observed by the Neurology screen in the Shirpa protocol. Furthermore, the Energy Metabolism screen
found decreased exploratory behaviour (rearing) in the home cage environment during the indirect calorimetry measurement.
Reasons for this hypoactivity are not fully clear at present. Previously, we demonstrated that the
adult KTA014 mutant mice were moving slower than controls in the modified holeboard suggestive of
limited mobility. This may be due to the skeletal abnormalites described in these mice by
the Dysmorphology screen. Thus, this impeded movement phenotype and inability to assume the rearing posture
could be secondary to these changes.

We also found that the mutant mice were
spending less time and moving less distance in the central more aversive zone of the
open field. This effect may be secondary to the decreased ambulatory behaviour of these mice.
Nevertheless, we previously observed that the male mutant mice were exhibiting an increased risk assessment
behaviour in the modified holeboard. Such a behavioural pattern suggested that these male mice were
more anxious. Thus, the decreased centre time and distance could also be an effect on
anxiety. This would tally with the increased corticosterone, the stress hormone, reported by the Steroid
Metabolism screen. Therefore, further anxiety-related tests should be performed on these mice to determine whether
this is a true effect on anxiety.

One potential test of anxiety-related behaviour that
is not confounded by the effects of decreased movement ability is the acoustic startle reactivity
test. With this test we observed that the mutant mice showed increased acoustic startle. An
enhanced acoustic startle may be due to improved neuromuscular recruitment, hearing ability or increased anxiety.
The results of the Neurology screen do not suggest that neuromuscular recruitment was markedly affected
in these mice. They did however show that there was a problem with hearing ability
at lower sound pressure levels by measuring acoustic brainstem responses. Whether this is then an
improved hearing ability at these higher sound pressure levels requires more research. Nevertheless, this could
also be increased anxiety-related behaviour, which would be consistent with the decreased centre time in
the open field and increased corticosterone (in the males). It may be worth challenging these
mice with an acute stress challenge to determine whether there are differences in stress reactivity.


 

 

Neurology

Summary:

We detected reduced locomotor activity (SHIRPA) and reduced hearing sensitivity (ABR), Grip strength was reduced
due to the body mass reduction and no differences were detected for lactate levels or
rotarod performance.



Discussion:

Scube 3 is a signal peptide involved in embryonic development in several tissues. A published
mouse model with mutation of this gene had not shown any obvious phenotype (Xavier et
al., 2013). In that publication there was also no phenotype detected in bones of the
middle and inner ear analyzed in skeletal preparations of E17.5 old embryos.

The ABR
difference especially in female mutants shows a general shift of the hearing curve towards higher
threshold irrespective of the frequency used. The shift is not very pronounced so a confirmation
of the findings is suggested. If this could be confirmed, a closer look on the
ossicles of the middle ear would help to test the hypothesis that a malformation of
those impaired hearing sensitivity in the KTA041 mice.

Eye & Vision

Summary:

We did not identify ocular irregularities in the primary Eye Screen of the KTA041-mice.



Discussion:

Our data clearly demonstrate that eye development is not affected by the KTA041 mutation. The
observed retinal degenerations in controls and mutants that go along with blindness represent typical cosequences
of the C3H background (Pde6brd1-phenotype; Dalke and Graw, 2005).
 

Nociception

Summary:

We did not detect any genotype effect on the pain reaction.



Discussion:

The mutation had no effect on the pain reactivity of the animals.

Energy Metabolism

Summary:

Mutant mice showed significantly reduced body mass and a shift in body composition. During the
indirect calorimetry trial no major effects on energy turnover could be detected when VO2 was
adjusted to body mass. However, rearing behavior was significantly decreased. Based on FT/IR analysis we
could not detect any effect of the mutation on feces composition.



Discussion:

The mutation caused complex effects on energy metabolism related parameters. Body mass was significantly reduced
and the relation between body fat content respectively lean mass was significantly different between control
and mutant mice. Not only reduced body mass but also a shift in body composition
indicates mid- or long-term effects of the mutation on energy partitioning. However, during the indirect
calorimetry trial we could not detect clear effects on energy balance regulation.

Interestingly, rearing
behavior was reduced in mutant mice similar as reported for the open field test. This
result requires confirmation as the accuracy of the detection method may be reduced due to
the reduced body size. However, contrary to the findings of the behavior screen locomotor behavior
under home cage conditions did not differ between wildtype and mutant mice.

These findings
in combination with the improved glucose clearance in the GTT suggest that the Scube3 mutation
may have effects on energy balance regulation, glucose metabolism and insulin function that should be
followed up.

 

Clinical Chemistry

Summary:

In the first screen of KTA041 mice we found hints towards impaired kidney function, as
well as changes in fat/energy metabolism and/or pancreas function.

Most of the findings detected
in the first screen were reproduced in the second cohort: Mutant animals have increased urea
and creatinine values, decreased plasma lipid and glucose levels, improved glucose tolerance, decreased plasma phosphate
levels and increased ALP and alpha-amylase activities in blood.

Mutant mice consume more food
and produce significantly more feces. Additionally, urine excretion is increased, associated with an increased daily
loss of electrolytes (Na, K, Cl), calcium, magnesium and urea. Therefore, polyuria might be due
to osmotic effects secondary to impaired reabsorption of electrolytes.



Discussion:

The most prominent feature of mutant KTA041 mice is the small body size suggesting predominantly
developmental defects caused by the scube3 mutation. Sube 3 is a secreted and cell surface
associated glycoprotein, which is expressed in multiple tissues during embryonal development and seems to be
involved in hedgehog and TGFbeta1 signalling (Yang et al. 2007; Haworth et al. 2007; Johnson
et al. 2012; Xavier et al. 2013). Since potassium, urea and creatinine levels were increased
in mutant animals in both, the first and the second cohort, we decided to analyse
renal function in this line. Our results indicate, that creatinine clearance was quite variable in
both, mutant and control mice, but still within the range that can be expected for
mice of the respective size. We conclude, that increased urea and creatinine concentrations were not
due to renal failure due to reduced glomerular perfusion or filtration. In fact, our data
suggest that related to body mass mutant mice excrete higher amounts of creatinine than control
animals. This is likely to be due to the higher plasma concentration consequently leading to
higher concentrations in primary urine. Since creatinine is a product of muscle energy metabolism, the
increased plasma values must not necessarily be due to creatinine retention, but can also be
caused by increased production. It has been observed that scube3 is expressed in human cultured
coronary smooth muscle cells and overexpression leads to cardiac hypertrophy in transgenic mice, while expression
in other adult tissues seems to be low (Wu et al. 2004; Yang et al.
2007). Although the Pathology screen did not observe structural defects of muscle tissue, the observed
reduced locomotor activity and grip strength (Behaviour Screen, Neurology Screen) might be indications of functional
defects, which might also be associated with an abnormal muscle energy metabolism. Our results furthermore
suggest that tubular reabsorption of most electrolytes and urea is less effective in mutants than
in controls, leading to an increased 24h electrolyte and urea excretion which likely secondarily increases
water excretion (osmotic diuresis). Our data collected on increased food uptake and feces production, together
with the finding that feces composition does not show substantial genotype related differences (Metabolism Screen),
suggest that nutrient uptake might also be lowered in mutants. It has to be clarified,
whether the differences found are secondary to developmental defects and/or smaller body size, resulting in
reduced gut length and/or tubule length in the kidneys, or if they represent specific functional
defects.

Immunology

Summary:

We found decreased levels of IgG3 and in female mutants increased levels of IgG1. In
mutants of both sexes we found furthermore a decreased frequency of  monocytes,  decreased frequencies of
L-selectin expressing cells within the T cell populations and an increased frequency of CD11b expressing
cells within the NK cell compartment. In male mutants we found furthermore an increased frequency
of NK cells and in female mutants a decreased frequency of B cells.



Discussion:

In mutants of both sexes we observed a strongly reduced proportion of L-selectin expressing T
cells. L-selectin is important for the regulation of leukocyte migration and homing to lymph nodes
(Wedepohl et al. 2012). It is constitutively expressed and rapidly shed from the leukocyte membrane
following activation (Yang et al. 2012). Such shedding occurs also during sample preparation due to
effects of the erythrocyte lysis; this phenomena is at least partly caused by the release
of NAD+ and ATP (Scheuplein et al. 2009). In blood samples which do not undergo
erythrocyte lysis, we observe high frequencies of L-selectin expressing cells within the T cell compartment (>95%, unpublished
data). We therefore consider differences in the frequencies of L-selectin expressing T cells in samples
which underwent erythrocyte lysis as the reflexion of differences in the grade of erythrocyte-lysis-induced L-selectin shedding.
There is a speculative link between the observed phenotype of an increased L-selectin shedding to the
underlying mutation in the scube3 gene: L-selectin shedding is mediated by metalloproteases most importantly via
ADAM17 (Dreymueller et al., 2012) but also via other metalloproteases (Walcheck et al., 2003).  Scube3
is a ligand of TGF-beta (Wu et al., 2011) and TGF-beta has been described to
be involved in the regulation of matrix metalloproteinases (Philips et al., 2004; Wiercinska et al., 2004).
Moreover, ADAM17 is involved into the regulation of TGF-ß signaling via shedding of the ectodomain
of its type I receptor (Liu et al., 2009) and the generation of a TGF-ß-inhibiting
Vasorin fragment (Malapeira et al., 2011). A direct connection of TGF-beta to L-selectin shedding, however,
has not yet been described. We furthermore found a slightly increased frequency of CD11b
expressing cells within the NK cell cluster. CD11b is expressed on NK cells upon maturation.
Again, scube3 might be related to NK cell maturation via TGF-beta. Marcoe et al., 2012,
found that TGF-beta inhibits NK cell maturation.

Beside, we found decreased levels of IgG3
in mutants. TGF-beta is known to induce the isotype switch to IgG2b (Park et al., 2005).  Deenick
et al., 1999, analyzed the influence of TGF-ß on the production of IgG3. She described
a TGF-beta induced decrease of the frequency of IgG3 expressing cells in LPS-stimulated B cell
cultures, which was explained as a switch to IgG2b (downstream to the IgG3 switching). 


Taken together, our findings hint towards TGF-beta-related mechanisms in the phenotype of scube3 mutants.


 

 

 

 

Allergy

Summary:

The primary screen did not uncover an influence of the mutation on total plasma IgE
levels.



Discussion:

The analysis of total IgE levels in plasma mice did not reveal genotype-specific differences. Levels
of total IgE were higher in females than in males of both genotypes, a well-known
finding for many inbred strains (Allessandrini et al., 2001; Corteling et al., 2004; Seymour et
al., 2002; Melgert et al., 2005).

Steroid Metabolism

Summary:

The analysis of steroid hormones in plasma samples of KTA041-mice revealed differences between mutant and
control animals. Significant differences in the concentration of corticosterone have been found in male mice.




Discussion:

Genotype-specific differences for corticosterone concentrations in blood plasma were found.

Therefore, the KTA041-mutation seems
to affect steroid metabolism.

Cardiovascular

Summary:

The cardiovascular functions were investigated  by awake echocardiography and electrocradiography. The major findings were: decreased
interventricular septum width; decreased left ventricular posterior wall thickness in male mutants; decreased distolic ventricular
dimension; decreased respiration rate in female mutants; decreased left ventricular mass (corrected) mainly in male
mutants and prolonged QRS interval duration in male mutants.

Echocardiography and Electrocardiography of mutant
mice revealed several mild alterations. However, from the functional data there was no clear hint
for a physiological relevant constraint of cardiovascular function due to the mutation.

 

 


Discussion:

The observed alterations are probably a result of high differences in bodyweight.

In addition,
only subtle alterations were found by echocardiography and electrocardiography. Thus,  relevance of the mutation on
cardiovascular function is not given.

Lung Function

Summary:

Pulmonary function testing did not reveal any genotype-related differences.



Discussion:

The mutation in KTA041 mice is suggested not to affect pulmonary function. Body weight reduction
was significant in the heterozygout mice.

Expression Profiling

Summary:

In this report, we describe the results of transcript profiling of the KTA041 mutant mice.
Kidney was chosen for transcriptome analysis due to evidence to impaired renal functions. Experiments were
performed for four male mutant and four male control mice. Data analysis using various statistical
methods detected significant differences in gene expression patterns in kidney. Genes differentially expressed are associated
with distinct biological process and with kidney functions.



Discussion:

The transcriptome profiling of the KTA041 mice revealed alterations in kidney between mutant and
control animals. The differences on molecular level give evidence for alteration of kidney functions, which
might be associated with the results of the Clinical Chemical screen. The dataset of regulated
genes include genes associated with kidney function: Adamts1, Mme, Tnn, Slc12a1, Cldn10, Ccr1, Slc39a8, Tcf21,
Man1a, Met, Trpc1, Foxc1, Fmo5, Umod, Hif1a, Acta2, Ehd3, Ghrhr, Mep1b, Has2, Hmox1, Kcnj15, Havcr1,
and Slc2a12.

With regard to the preparation of a manuscript including transcriptome profiling
data we offer additional functional analysis of the dataset using distinct software tools (lngenuity, Genomatix,
Panther Classifications system, String, GESA,...) and will support writing a manuscript.

Please, contact
us (Marion Horsch (horsch@helmholtz-muenchen.de) or Johannes Beckers (beckers@helmholtz-muenchen.de)) if you have questions concerning this analysis.


Pathology

Summary:

Reduced body weight and tibia length as well as skeletal abnormalities were confirmed.



Discussion:

As in the first screen of the KTA041 mutant mouse line the pathology screen found
obvious changes in the body length and body weight of the mutant mice. All measured
parameters were decreased in male and female mutants.

X-ray analysis was not performed during
this screen since the known skeletal phenotype was already confirmed in 2005.

The changes
found in the clinical chemistry screen indicating an impaired kidney and pancreas function could not
be correlated to a corresponding histological finding. As in 2005, all investigated animals showed no
pathological alterations.

Perhaps the animals studied here are too young and the changes have
not manifested in organ histology detectable with light microscopy.

Focal microgranulomas as detected in
a total of five male mice are a common accidental finding in the murine liver.
The cause is usually unknown but may be related to infections with bacteria, e.g. Helicobacter
hepaticus
. It cannot be associated to the genotype of the mice.

The same is
true for the inflammatory reaction found in the arterial wall of two KTA041 mutant animals
in 2005. Since the current screening revealed a control animal with the lesion this is
a secondary finding with no relevant biological significance concerning the mutation of the mice.

Bodyweight
Hmgn1/Hmgn2Hmgn1, Hmgn2Targeted mutation
Bone & Cartilage

Summary:

In the analysis for dysmorphology, bone and cartilage, we did not detect any alterations that
we consider as interesting phenotypes.



Discussion:

We did not find any abnormalities or deviations from expected values for the genetic background
of the mice. Body weight, body length as well as lean mass showed up in
the statistcal analysis, but we do not consider these subtle alterations as interesting phenotypes that
are worth to follow up.

Behaviour

Summary:

The mutant mice showed decreased rearing activity in the open field. They also showed decreased
acoustic startle and prepulse inhibition responding.



Discussion:

In the open field, our test of spontaneous reactions to a novel environment, there were
no clear genotype effects on forward locomotor activity. There was a clear decrease in rearing
activity and resting time. This indicates that these mice were less exploratory in these novel
surroundings. These mice did not show ostensible differences in anxiety responding (centre time, centre distance)
in this test. The decrease in rearing activity may correspond to the impaired rotarod ability
determined by the Neurology screen. Thus, these mutant mice may have impaired motor coordination and
balance. Hmgn1 mutant mice also showed this decreased rearing activity in the open field.


We also observed a decrease in acoustic startle reactivity. Impaired acoustic startle may be attribued
to reduced hearing ability and/or neuromuscular recruitment or decreased anxiety responding. There were no clear
changes in anxiety-related behaviour in the open field to suggest that the latter underlies the
effect on acoustic startle. Furthermore, there were no clear differences in acoustic brainstem responses in
the Neurology screen to indicate that hearing is markedly affected in these mice. The results
from the Neurology screen in terms of rotarod and grip may infer that these mice
have a problem in neuromuscular recruitment. This may also be supported by the results of
the Nociception screen showing that these mice are less sensitive to pain. Overall, there may
be a sensorimotor recruitment abnormality that is also apparent in the acoustic startle response. A
decreased acoustic startle response was evident in both the Hmgn1 and Hmgn2 single mutant mice.


In terms of prepulse inhibition, there was a small signficant decrease in the mutant
mice at the 67 dB intensity with a tendency globally. In theory, alterations in PPI
indicates a change in sensorimotor integration, hence alterations in brain function, which could be due
to transformations in several neurochemical systems (see Geyer et al., 2001 for review). PPI is
largely regulated by neuronal connections between the limbic cortex (including the entorhinal cortex, hippocampus and
amygdala), ventral striatum, ventral pallidum as well as the pontine tegmentum. Glutamatergic fibers from the
limbic cortex converge at the nucleus accumbens within the PPI regulatory circuitry. GABAergic projections, originating
in the nucleus accumbens, then project to the globus pallidus. Regulation of PPI is carried
out by GABAergic projections from the globus pallidus to the pedunculopontine nucleus. Increased PPI is
associated with a reduction of GABAergic projections from the globus pallidus while decreases produce the
reverse effect (Geyer and Dulawa, 2003; Swerdlow et al., 2001 for review). This effect on
PPI was small, however, a similar effect (significant decrease at 67 dB and a tendency
globally) was found in the Hmgn2 mice.

 

Neurology

Summary:

Mutants of both sexes show reduced locomotor activity and impaired rotarod performance. In addition, only
the lighter male mutants display reduced plasma lactate levels and slightly reduced grip strength.



Discussion:

Hmgn1 mutants had not shown neurological alterations, Hmgn2 mutants were lighter, had less muscle force
and showed less transfer arousal.

In the double knockout only the males were lighter,
had less grip strength and less plasma lactate. This sex-specific difference could be linked to
the changes detected in energy metabolism, indicating that especially male mice might have a problem
in energetic fueling of muscle function.

In addition to the previous findings, mutants of
both sexes showed hypoactivity, a trend towards less transfer arousal as in Hmgn2 as well
as impaired rotarod performance. These findings could be related to energy metabolism as well, but
rotarod performance is predominantly an indicator for impaired motor coordination and balance. Since Hmgn1 as
well as Hmgn2 are expressesd in neuronal tissue, a neurological consequence of the knockout could
be possible. At the moment there is no respective link found in the literature. However,
the clear effect of the double knockout on nociception also indicates a neurological involvement.

Eye & Vision

Summary:

We found significantly increased eye sizes in Hmgn1/Hmgn2 mutants. The corneal thickness was measured and
found to be significantly reduced in the mutants as it was also shown for the
Hmgn1 mutants in Yehudit Birger et al., 2006. Furthermore the number of the main blood
vessels in the fundi was increased but not significant.



Discussion:

We found corneal thinning and increased eye sizes in the Hmgn1/Hmgn2 mutants. The corneal thinning
comes as a confirmation of the already published data on the effects of the Hmgn1
mutation on the maturation of the cornea (Yehudit Birger et al., 2006). Our data further
prove a role of Hgmn1 in corneal development. Increased eye sizes have not yet been
associated with Hgmn1/Hmgn2 mutations. This might be a secondary effect of corneal pathologies.

The
posterior eye morphology in the mutants did not present much irregularities, except for the number
of the main blood vessels, that is increased, but not significant.

Nociception

Summary:

The mutant animals showed hypoaglesia.



Discussion:

This phenotype can be considered as an interesting new phenotype. There is no known pathway involved in the
found hypoalgesia. It would be interesting to perform further pain-related studies to find out more details
that are responsible for this phenotype.

Energy Metabolism

Summary:

Body mass was slightly but significantly decreased mainly in male mutants. Fat content in males
was significantly decreased even when adjusted for the body mass difference. During the indirect calorimetry
trial energy expenditure (daily energy expenditure and resting metabolic rate) were significantly increased in mutants
(hypermetabolism). Mutants were also hyperphagic and showed increased RER.



Discussion:

In Hmgn1/2 mutant mice similar differences could be detected between control and mutant mice as
in the mouse line Hmgn2. Mutant mice were slightly underweight, showed reduced fat content, were
hyperphagic and showed a slight shift towards carbohydrate oxidation rates. Interestingly, in Hmgn1/2 mice not
only energy uptake but also energy expenditure was increased in mutant mice. We could not
detect clear differences in physical activity indicating that the hypermetabolism was not just secondary to
increased activity levels. Notably, hyperphagia was linked to an increased RER similar as in Hmgn2
suggesting that substrate utilization was affected by the mutation. So far, a systematic evaluation of
metabolic fuel utilization based on gas exchange data is still outstanding. However, it could be
hypothesized that the mutation had an impact on substrate oxidation.

Clinical Chemistry

Summary:

IpGTT: We saw increased body mass loss in male mutants and increased basal glucose levels,
but rather decreased AUC values in female mutants.

Insulin: Insulin level in plasma after
overnight food deprivation was very low in mutant males.

Clinical chemistry: Fastig plasma glucose
and cholesterol (total, HDL and non-HDL) were decreased in male mutants. In ad libitum fed
mice we found decreased albumin and alpha amylase activities in plasma of mutant mice, as
well as increased triglycerides in both sexes and only in females elevated cholesterol and calcium
levels.

Hematology: We found microcytic blood cell counts - indicated by low MCV and
MCH, but increased RBC and MCHC values, which were not associated with changes in hemoglobin
levels indicating anemia - and an increased proportion of large platelets in mutants.

In
total our results indicate possible pleiotropic effects of the double knockout on energy, protein and
mineral metabolism as well as hematopoiesis.



Discussion:

As expected we saw more and stronger effects in Hmgn1/Hmgn2 double knockout mice than in
Hmgn1 or Hmgn2 single knockout lines. As in Hmgn2 knckout mice, the results of the
Energy metabolism screen indicated specific effects on the regulation of energy metabolism, and that were 
in this line with several, partly sex-specific effects on plasma lipid and glucose levels and
glucose clearance in the IpGTT. However, we also observed effectsthat were not detected in any
of the single knockout lines: Albumin levels were slightly decreased in mutant animals of both
sexes compared to controls, but calcium concentration was higher in female mutants than in wild-type
females. Since biologically inactive calcium is bound to proteins, mainly albumin, in plasma, this could
indicate significantly elevated levels of ionized calcium in plasma. However, ALP activity did not show
a clear increase and also the results of the Dysmorphology screen did not provide evidence
of genotype-related effects on bone metabolism. Additionally we observed a decreased activity of alpha-amylase in
plasma of mutant mice compared to controls, that, together with the decreased in albumin levels
might be due to effects on liver protein metabolism. That correlates with the high number
of genes found to be differentially regulated in the liver ( s.results Molecular phenotyping screen).
Furthermore we found alterations of blood cell morphology likely indicating effects on erythropoiesis and probably
osmotic effects on blood cells. The latter can be due to changes in cellular membranes
influencing its properties or to changes in plasma composition. In total our results indicate possible
pleiotropic effects of the double knockout on energy, protein and mineral metabolism as well as
hematopoiesis.

Immunology

Summary:

Our analysis of leukocyte subpopulations in blood revealed differences between mutants and controls in the
frequencies of several leukocyte subpopulations. Interestingly, most of our findings refer to male mutant mice,
namely higher frequencies of T cells, NK cells and NKT cells, which went together with
a higher CD4:CD8 T cell ratio and a decreased frequency of CD44 expressing cells within
the CD8 T cell compartment. Furthermore we found a lower frequency of CD11c expressing cells
within the NK cell compartment in male mutants. Within the B cell compartment we found
a lower frequency of IgD expressing cells in mutants of both sexes. This change could
be assigned to differences in the frequencies of tiny distinct B cell subpopulations. We did
not see differences in the levels of the immunoglobulins IgG1, IgG3, IgG2b, IgM or IgA.




Discussion:

Sex-dependent differences is a common finding in most mouse strains (Petkova et al. 2008). We
generally observe a lower T cell frequency in males compared to females in wildtype mice which
is also seen in controls of this line and which might reflect that the age-related
thymus involution occurs earlier in male mice compared to females (Gui et al., 2012). The
higher CD4:CD8 T cell ratio in male mutants fits to the hypothesis of a lower
thymic T cell production in male controls, as the CD4:CD8 ratio is higher upon thymic
versus peripheral proliferation (Thomas-Vaslin et al., 2008). Also the frequency of NK cells in the
periphery declinces upon aging in mice and is correlated to a loss of the maturation
marker complement receptor CD11b (Beli, E. et al., 2013).
Seeing in our data that the frequency of NK cells was lower in control males
compared to females and to male mutants, one might therefore consider, that the higher frequency
of NK cells and the higher frequency of T cells in male mutants compared to
male controls reflects a lower grade of ageing or stress-related effects in these mice. We
found, however, in mutants of both sexes a similar reduction in the frequency of IgD
expressing cells within the B cell compartment. This echoed in changes in the frequencies of
some tiny B cell populations. In summary, we found subtle sex-and leukocyte subset-specific changes in
Hmgn1/2 mutants; compared to the immunophenotyping results of the Hmgn2 mutant mouse we have more
pronounced changes in the Hmgn1/2 mutants, with one overlapping finding, the  lower frequency of CD11c
expressing cells within the NK cell cluster. 

 

Allergy

Summary:

The screening did not uncover an IgE phenotype or disturbances in the skin.



Discussion:

The analysis of basal total IgE levels in plasma revealed values within the range expected
for mice on C57BL/6 genetic background.

Steroid Metabolism

Summary:

The analysis of steroids was not performed for this mouse line.




Cardiovascular

Summary:

By electrocardiography, increased HR (reduced RR), reduced PQ and PR interval duration in males, and 
increased SR and R amplitides in females were found.

By echocardiography, reduced LV dimension,
LV mass and SV were found in females. For males increased HR was found.


All these mostly quite small differences are findings of unclear relevance. In conclusion, no major
effects on the cardiovascular system could be detected.



Discussion:

All these mostly quite small differences are findings of unclear relevance. In conclusion, no major
effects on the cardiovascular system could be detected.

Lung Function

Summary:

The Lung screen performs an Elastase Challenge.




Expression Profiling

Summary:

In this report we describe the results of transcript profiling of the Hmgn1/2 mutant mice.
Brain, liver, spleen and thymus were chosen for transcriptome analysis. Data analysis using various statistical
methods detected significant differences in gene expression patterns in all analyzed organs. Hierarchical cluster analysis
revealed tissue-specific expression patterns and distinct functional annotations of the differentially expressed genes e.g. neurogenesis
in brain, response to stimulus and cell differentiation in liver and defense response and catabolic
processes in thymus.



Discussion:

Transcriptome profiling of brain, liver, spleen and thymus revealed significantly differentially expressed genes in all
organs. 77 - 93 % of the regulated genes in the different organs displayed decreased
expression levels. In each organ Hmgn2 was the strongest down-regulated gene (fold change up to
-172x), while no significant regulation at all was found for Hmgn1 (magnitude of fold change
0 to -1.34). The overlap of regulated genes between the organs was quite low. Only
brain, thymus and liver shared a group of 7-8 genes, frequently displaying the same tendency
of regulation. Hierarchical cluster analysis of all differentially expressed genes indicated tissue-specific expression patterns. As
expected, the functional annotations of those genes regulated in the four organs were quite different.


In summary, the transcriptome profiling analysis of four organs of the double knock out
mutant line Hmgn1/Hmgn2 affects specific cellular function in a tissue-specific manner.

Pathology

Summary:

The Hmgn1/Hmgn2 DKO mice showed no histopathological phenotype in the primary screen.

Male mutant
mice showed a statistically significant decrease in body weight.



Discussion:

The Hmgn1/Hmgn2 DKO mice showed no histopathological phenotype in the primary screen.

Male mutant
mice showed a statistically significant decrease in body weight. Decreased organ weights are consistent with
reduced body weight in these animals.

No morphological correlation was seen for reduced heart
weight in mutant mice.

Mutant and control mice revealed subtle background and spontaneous lesions.


Bodyweight
Rosa26 AoxAoxTransgenic
Bone & Cartilage

Summary:

In the analysis for dysmorphology, bone and cartilage, we did not detect any alterations.



Discussion:

In the Rosa26 mutant line we did not find any abnormalities or deviations from expected
values for the genetic background of the mice. There is no hint for changes in
bone development or bone metabolism.

Behaviour

Summary:

There were subtle changes in the open field that included a small decrease in movement
speed. In addition, prepulse inhibition was slightly decreased at certain prepulse intensities.



Discussion:

In the open field, our test of spontaneous reactions to a novel environment, we did
not detect any clear genotype-related differences. There was a tendency for the the mutant mice
to move and rear less in this arena and locomotor speed was reduced. No major
differences in the anxiety-related measures of centre time and distance were revealed. These subtle decreases
in locomotor activity and speed appear consistent with the decreased locomotor activity observed in the
Shirpa protocol by the Neurology screen. Furthermore, these mice were also rearing less during the
indirect calorimetry test of the Energy Metabolism screen.

While no differences were detected in
terms of acoustic startle reactivity, there were significant decreases in prepulse inhibition. This effect was
not pronounced and was only significant at two of the four prepulse intensities measured. Thus,
to determine whether this is a real effect it would need to be replicated in
additional mice before considering further.

Neurology

Summary:

At the modified SHIRPA, mutants showed a trend towards reduced locomotor activity. Grip strength, rotarod
performance as well as plasma lactate  were without genotype-related differences. There was a very subtle
shift in hearing theresholds in male mutants.



Discussion:

There was a trend towards reduced locomotor activity (SHIRPA), as also detected in the OF.
This could be an adaptation to changes in cellular energy metabolism.

There was a
shift in ABR thresholds in male mutants hinting towards reduced sound pressure thresholds needed for
the response towards higher frequencies. This was far from beeing statistically significant.

Usually higher
frequencies got lost first during the age-related sensorineural hearing loss observed in several background strains
like C57BL/6J (Johnson et al., 2006). This can have multiple origins including genetic factors and
oxidative stress leading to apoptosis (Yamasoba et al., 2013; Op de Beeck et al., 2011).


So even if the differences are small, in the context of introducing Aox in
the mutants, this should be kept in mind for further studies. If the pattern observed
for the Aox mice would be confirmed in additional experiments, the result could indicate less
pronounced or later onset of age-related hearing loss.

Eye & Vision

Summary:

No major eye phenotype was identified in the Aox expressing mutants.

A mild decrease
in males mutants eye size was found.



Discussion:

Our data demonstrated that the eye axial length is decreased in the Aox expressing mutants,
while eye morphology and visual properties were not affected by the Rosa26 Aox mutation.

Nociception

Summary:

The mutation did not influence the pain sensitivity of the animals.



Discussion:

We could not detect any genotype-related effect on the first pain sensitivity. We do not
plan any further pain related study. 

Energy Metabolism

Summary:

No clear effects of the mutation could be detected on body mass, body composition, rectal
body temperature, energy turnover, locomotor activity and metabolic fuel utilization.



Discussion:

We could not detect major effects of the mutation on whole body energy balance regulation,
body mass, and body composition. Only rearing behavior but not distance travelled was reduced in
mutants confirming findings from the behavior and neurology screens. However, the relevance of this difference
remains unclear so far.

Clinical Chemistry

Summary:

Results obtained from glucose tolerance tests, plasma clinical chemistry and hematological analyses did not provide
clear evidence of significant effects of the genotype on any of the parameters investigated.


The only parameter showing a common trend in both sexes was plasma fructosamine, leading to
a statistically borderline significant genotype effect. Additionally we saw trends towards higher AUC 30-120 values
in the IpGTT and lower fasting cholesterol levels compared to controls in the male group.




Discussion:

Results obtained by screening clinical chemical and hematological values in mouse blood as well as
glucose tolerance in Rosa26 AOX transgenic mice did not reveal major effects of the mutation
on any of the parameters screened. Very mild genotype-related differences were seen for some parameters
of the glucose tolerance test, namely a trend towards larger areas under the glucose curves
for the second part of the test (T30 - T120), and a trend towards higher
fructosamine levels in both sexes. Both findings together could indicate a very mild effect on
the regulation of glucose metabolism. However, fructosamine levels also depend on plasma protein levels. In
females we also found a mild but not significant elevation of plasma albumin concentrations. Therefore
the difference seen in fructosamine levels could also be secondary to mild effects on plasma
protein levels. Overall, differences detected between genotypes were very small and reproducibility should be tested
in an independent cohort of mice.

Immunology

Summary:

We found sex-dependent changes in the frequencies of some leukocyte subpopulations, namely, an increased frequency
of NK cells and increased frequencies of CD44 high and CD11b expressing cells within the
NK cell compartment in male mutant mice, and in female mutants slightly increased frequencies of CD11c expressing
cells within the monocyte and NK cell compartments. In mutants of both sexes, the frequency
of CD11b expressing cells within the B cell compartment was slightly increased.



Discussion:

We found leukocyte subpopulations in the expected frequencies in peripheral blood from mice under baseline
conditions and found no evidence for a genotype related immune defect. However, we cannot exclude
that functional impairment of the immune cells occur in challenging conditions (infections e.g.). We saw,
however, an increased frequency of CD11b expressing cells within the B cell compartment and further
subtle partly sex-dependent differences between mutants and controls. When we  look
on the heatmap (see:101m_Rosa26aox_male_equal_1_65SD), showing normalized data of male mice (the ratio of the
measured value with the mean of the control group + - 1.65 SD), we can
see a high penetrance of the higher frequency of NK cells (third on the left,
colored in orange) and also changes in the frequencies of several CD4 and CD8 T
cell subpopulations in male mutants. In contrast, the heatmap of the data from female mice
(see: 101m_RosaAox_female_equal_1_65SD) does not hint towards changes in frequencies of subpopulation of the shown
compartments (Note: The B cell subpopulations of the heatmap do not include the CD11b expressing
population). Sex-dependent differences is a common finding in most mouse strains (Petkova et al. 2008).
We observe a lower T cell frequency in males compared to females in both groups
- mutants and controls -  (see table: 100t_2808-2809-2810-2811-2812-2813-2816-2817-2818_GMCTableMedianQuartileWilcoxon) which reflects at least partly the
known age-related thymus involution which occurs earlier in male mice (Gui et al., 2012). Also
the frequency of NK cells in the periphery declinces upon aging in mice and is
correlated to a loss of the maturation marker complement receptor CD11b (Beli, E. et al., 2013). We see that the frequency of NK cells
is lower in control males compared to females and male mutants (see: figure 100g_2812_boxplotWithStripchart).
One might therefore consider, that the higher frequency of NK cells and the higher frequency
of CD11b expressing NK cells in male mutants compared to male controls could reflect an
age-antagonizing effect of Aox on NK cells. On the other hand, we find a higher
frequency of CD44 positive cells within the NK compartment of mutant males (see figure: style="font-style:italic;">101m_NK_44
). CD44 is expressed in NK cells upon activation which can occur independ of NK
cell prolliferation (Sague, S. L., et al., 2004).

In
mutants from both sexes we found a slightly higher frequency of CD11b expressing cells within
the B cell compartment (see figure 109g_2822_boxplotWithStripchart). CD11b is described to be expressed on
a murine B cell subset denominated as B1- B cells. These B cells are found
only as a tiny subpopulation in peripheral blood, but constitute the majority of the B
cells in the peritoneal cavity
(Ghosn et al., 2008). Murine B1 B cells
co-express CD5
(Hardy, R. R., 2006). Our analysis of the frequency of
CD5 co-expressing B cells (denominated as B1 cells) did not reveal differences in the frequencies
of this subpopulation referring to all leukocytes (see table: 100t_2808-2809-2810-2811-2812-2813-2816-2817-2818_GMCTableMedianQuartileWilcoxon).
Please note
that murine B1-B cells do not exhibit the same functions as human analogs
(Garraud et al., 2012). Further studies are necessary to elucidate an effect of
Aox expression on immune cell function. As our observations might be influenced by unknown factors,
we recommend first a confirmation of our findings. 

Allergy

Summary:

The screening did not uncover an IgE phenotype or disturbances in the skin.



Discussion:

The analysis of basal total IgE levels in plasma revealed values within the range expected
for mice on C57BL/6 genetic background.

Steroid Metabolism

Summary:

The analysis of steroids was not performed for this mouse line.




Cardiovascular

Summary:

By recording ECGs, reduced hights of R and SR amplitudes were found in mutant mice
compared to controls.

By echocardiography, mild reductions in LV mass and LVPWd width were
found.

These alterations are probably by chance and do not represent a cardiovascular phenoytpe.




Discussion:

The alterations observed are probably by chance and do not represent a cardiovascular phenotype.

Lung Function

Summary:

Lung function tests were not performed for this mouse line.




Expression Profiling

Summary:

There was no expression profiling offered for this mouse line.




Pathology

Summary:

Histological examination using light microscopy did not reveal any pathological changes that could be attributed
to the genotype of the mice.



Discussion:

The mutant mice Rosa26 Aox express an alternative oxidases (AOX) to the mitochondrial cytochrome c
oxidase. Mitochondrial diseases can affect any organ of the body, most vulnerable are the organs
with high energy demand such as brain and heart. We found small mineral deposits distributed
throughout the myocardium in one of five mutant mice. Focal mineralization of myocardium could be
induced by cardiac damage but in some strain it is a common incidental finding (1).
Due to the low penetrance, this lesion was considered an incidental finding.

Bodyweight
Hmgn2Hmgn2Targeted mutation
Bone & Cartilage

Summary:

In the analysis for dysmorphology, bone and cartilage we found in the Hmgn2 mice decreased
body length and body size, as well as decreased bone mineral content, fat mass and
lean mass. Males were more affeceted than females for most findings. The found changes might
be confounded by body weight and body size.



Discussion:

In the Hmgn2 mice we found reduced body length and body weight as well as
bone mineral content, lean mass and fat mass. The findings were significant for males with
the same tendency in females. For body length also females differed significantly. The changes in
bone mineral content data might be secondary to the body weight and body length differences.
For lean mass and fat mass also the data from the Metabolism Screen should be
considered, as the DEXA technique in our screen is mainly focused in the bone data.
The minispec device has a higher accuracy for the measurement of fat and lean mass.


Behaviour

Summary:

There was increased time spent in the centre of the open field by the mutant
mice as well as decreased acoustic startle and prepulse inhibition.



Discussion:

In the open field, our test of spontaneous reactions to a novel environment, there were
no clear genotype effects on either total distance travelled or rearing activity. This suggests that,
in this test, locomotor and exploratory activity are normal in these mice. Nevertheless, there was
an increase in the amount of time spent in the centre by the mutant mice.
Such a pattern indicates that these animals exhibit a degree of decreased anxiety.

There
was also decreased startle reactivity in these mice. Impaired acoustic startle may be attributed to
reduced hearing ability and/or neuromuscular recruitment or decreased anxiety responding. There was decreased anxiety-related behaviour
in the open field that may tally with the decreased acoustic startle. Furthermore, a potential
muscle phenotype was detected in the Neurology screen, which may indicate that the decreased startle
response is secondary to this finding. It should also be borne in mind that this
decreased startle response may relate to the decrease body weight of these animals.

A
prepulse inhibition deficit was also revealed in this analysis. This effect was significant at the
67 dB intensity with a tendency globally. In theory, alterations in PPI indicates a change
in sensorimotor integration, hence alterations in brain function, which could be due to transformations in
several neurochemical systems (see Geyer et al., 2001 for review). PPI is largely regulated by
neuronal connections between the limbic cortex (including the entorhinal cortex, hippocampus and amygdala), ventral striatum,
ventral pallidum as well as the pontine tegmentum. Glutamatergic fibers from the limbic cortex converge
at the nucleus accumbens within the PPI regulatory circuitry. GABAergic projections, originating in the nucleus
accumbens, then project to the globus pallidus. Regulation of PPI is carried out by GABAergic
projections from the globus pallidus to the pedunculopontine nucleus. Increased PPI is associated with a
reduction of GABAergic projections from the globus pallidus while decreases produce the reverse effect (Geyer
and Dulawa, 2003; Swerdlow et al., 2001 for review). A similar effect was also evident
in the Hmgn1/2 double mutant mice.

Neurology

Summary:

Mutants show clearly reduced muscle force as well as slightly reduced transfer arousal. No differences
were observed at plasma lactate, rotarod performance or ABR.



Discussion:

Basic neurological function were not impaired in Hmgn2 mutants. However, muscle strength was clearly reduced
in addition to the effect on force by the reduced body mass. Transfer arousal was
not much changed and could be a secondary effect of the grip strenght reduction, although
general activity was not altered.

Since all other neurological tests were without a difference,
we suggest that the Hmgn2 show a specific muscle phenotype. This could be related to
the differences detected in energy metabolism.

 

 

Eye & Vision

Summary:

We did not find pathologic eye phenotypes for the Hmgn2 mutants.



Discussion:

The data from the primary eye screen indicates that eye development and visual properties are
not affected by the Hmgn2 knock-out deletion.

Nociception

Summary:

The mutation did not affect the pain sensitivity.



Discussion:

The mutation had no influence on the pain sensitivity of the animals.

Energy Metabolism

Summary:

Body mass was slightly but significantly decreased. Fat content was significantly lower when adjusted for
the body mass difference. During the indirect calorimetry trial energy expenditure was not clearly affected
but mice were hyperphagic and showed increased RER.



Discussion:

Metabolic phenotyping of Hmgn2 mutant mice revealed interesting effects of the mutation on energy turnover
and body composition. Despite the mild reduction in body mass and the slight shift in
body composition with increased lean content but decreased fat content both mutant males and females
showed a marked increase in food uptake that was not compensated by a clear up-regulation
of metabolic rate. The difference in daily food consumption between control and mutant mice results
in additional 5 to 6 kJ per day assuming that the apparent assimilation efficiency does
not differ between genotypes. So far, it remains unclear how the discrepancy in daily energy
regulation can be explained, and if the shift towards carbohydrate oxidation can be linked to
the detected hyperphagia.

Clinical Chemistry

Summary:

IpGTT: No evidence of genotype-related differences.

Clinical chemistry: Small sex-specific genotype-related differences in fasting
and fed plasma lipid and glucose levels, mainly in females. Slightly increased calcium, inorganic phosphate
and ALP activity in mutants compared to controls.

Hematology: Mildly increased red blood cell
and platelet counts in male mutants compared to controls, associated with a mild trend towards
smaller thrombocytes.

All findings are mild, and mostly sex-specific and therefore of unclear relevance.
Differences seen in plasma lipid levels can be related to changes in the regulation of
energy metabolism.



Discussion:

The genotype-related differences we observed in Hmgn2 knockout mice were mild to moderate and mainly
restricted to female mice. Especially after overnight food deprivation we saw a mild increase of
triglycerides, free fatty acids and glycerol in female mutants, compared to controls. And also in
the ad libitum fed state we saw trends towards higher triglyceride and cholesterol levels. Further
mild differences were observed for plasma protein levels (TP and albumin), urea and creatinine values,
phosphate calcium and ALP activity. Many of these parameters are physiologically connected, since e.g. albumin
represents the biggest proportion of total protein in plasma and binds physiologically inactive calcium in
plasma. Therefore these findings could be accidental and reproducibility in an independent cohort would be
required to confirm them being genotype-related. However, since the results of the Energy Metabolism Screen
suggest an effect on the regulation of energy metabolism indicated by effects on body composition
and a preferential usage of carbohydrates to cover energy demands, the alterations in plasma lipid
levels likely are symptoms of a differential regulation of energy metabolism. Although the effects in
males and females on body composition and in the calorimetry analysis are similar, we see
sex differences concerning plasma lipid levels. Taking into account that also strong sex-dependend differences in
the regulation of energy metabolism are a well-known fact, this might not be surprising.

Immunology

Summary:

Our analyses revealed sex-dependent subtle differences in the frequencies of some leukocyte subpopulations in blood.
Compared to controls, we found in female mutants a slightly lower frequency of Ly6C negative CD11c
positive cells within the monocyte compartment and a slightly higher frequency of CD44 high expressing
cells within the CD4 CD25 co-expressing T cell population, and a slightly lower frequency of
CD11c positive cells within the NK cell compartment. In male mutants we found a slightly
increased frequency of L-selectin (CD62L) expressing cells within the CD4 T cell compartment and further
subtle changes in small T cell subpopulations.



Discussion:

The immunophenotyping results do not give evidence for a different immune status in HMGN2 mutant
mice. Observed differences between mutants and controls are mostly found in tiny subpopulations and might
be accidental findings. Moreover, we found the expected differences between females and males concerning the
frequencies of T cells and corresponding subpopulations (see discussion in the HMGN1/2 report). We cannot, however,
exclude that the found subtle differences in the frequencies of subpopulations reflect changes in homeostatic
regulation or occure due to changes of unknown factors (stress, diet, etc.) that might be
influenced by the mutation. 

Allergy

Summary:

The screening did not uncover an IgE phenotype or disturbances in the skin.



Discussion:

The analysis of basal total IgE levels in plasma revealed values within the range expected
for mice on C57BL/6 genetic background.

Steroid Metabolism

Summary:

The analysis of steroids was not performed for this mouse line.




Cardiovascular

Summary:

By echocardiography, increased LVIDd, LVmass and SV were found in female mutants only. The findings
are probably by chance.

By electrocardiography, reduced HRV and QRS complex duration were found
for females. Increased QTdis and QTcdis were found for males. Alterations found for females are
very mild and probably by chance. Alterations found for males are of unclear relevance.



Discussion:

By electrocardiography, reduced HRV and QRS complex duration were found for female and increased QTdis
and QTcdis were found for male mutants. QT dispersion is defined as the difference between
the longest and the shortest QT intervals. Since the QT interval may vary according to
the heart rate, it was corrected for the heart rate (QTc). The QTdis is an
approximate measure of a general abnormality of repolarization. The alterations observed for males are of
unclear relevance so far, whearas alterations found for females are very mild and probably by
chance.

Lung Function

Summary:

The Lung screen will perform an Elastase Challenge.




Expression Profiling

Summary:

In this report we describe the results of transcript profiling of the Hmgn2 mutant mice.
Brain, liver, spleen and thymus were chosen for transcriptome analysis. Data analysis using various statistical
methods detected significant differences in gene expression patterns in liver and thymus; none in brain
and spleen despited strong down-regulation of Hmgn2. In liver down-regulated genes are associated with signal
transduction and response to stimulus and in thymus e.g. with oxidation-reduction process, metabolic process and
regulation of hydrolase activity.



Discussion:

Transcriptome profiling of brain, liver, spleen and thymus revealed significantly differentially expressed genes in liver
and thymus. All regulated genes displayed decreased expression levels. In each organ Hmgn2 was the
strongest down-regulated gene (fold change up to -173x). There is nearly no overlap of gene
regulation between brain and thymus. Hierarchical cluster analysis of all differentially expressed genes indicated tissue-specific
expression patterns.

In summary, the transcriptome profiling analysis of four organs of the knock
out mutant line Hmgn2 affects specific cellular function in a tissue-specific manner in brain and
thymus. However, no effects were observed in brain and spleen on molecular level.

Pathology

Summary:

The Hmgn2 mutant mice showed no pathological alterations that could be associated to the genotype.


Mutant and control mice revealed background and sporadic lesions.

 



Discussion:

The Hmgn2 mutant mice showed no pathological alterations that could be associated to the genotype.


Mutant and control mice reveal background and sporadic lesions.

Bodyweight
D014D11Nsun2Other
Bone & Cartilage

Summary:

In the primary Dysmorphology, Bone and Cartilage Screen we confirmed the smaller size of the
mutants. In the DXA analysis, we detected a significantly decreased BMC in males. Concurrently body
length, body mass and lean mass were decreased in males (same tendency in fat mass).
We observed similar trends in females (only six animals were measured as four mice had
a body mass of less than 18 g). The decreased BMC in males is probably
a secondary effect due to the big differences in body parameters.



Discussion:

The NSun2-D014D11 mutant mouse line was analyzed in the Dysmorphology, Bone and Cartilage module of
the German Mouse Clinic. In the morphological observation we confirmed that mutants were smaller than
their wild-type control littermates. In the DXA analysis, we detected a significantly decreased BMC in
males. Concurrently body length, body weight and lean mass were decreased in males (same tendency
in fat mass). We observed similar trends in females. The decreased BMC in males is
probably a secondary effect due to the big differences in body parameters.The sex differences we
observed are common in many mouse strains, and thus are not abnormal (unpublished data).

Behaviour

Summary:

There was decreased acoustic startle reactivity by the mutant mice



Discussion:

In the open field, our test for spontaneous reactions to a novel environment, there were
no major alterations. Nevertheless, the female mutant mice show a tendency to be more active
in this environment with the males showing the opposite. The female mutant mice also show
a tendency to spend more time in the centre of the open field arena. Generally,
such an increase in centre time could indicate less anxiety in this aversive situation. However,
this may also be secondary to the increased locomotor activity in these same mice where
increased centre time occurs as a consequence of increased movement. In any case, this effect
was subtle and a possible effect on anxiety responding would need to be confirmed with
other anxiety tests.

One test that can be used to index anxiety is the
acoustic startle response. We found that this was decreased in mutant mice, both males and
females. A decrease in acoustic startle could indicate decreased anxiety but could also be a
result of impaired neuromuscular recruitment or diminished hearing ability. There were no significant effects of
genotype on the clickbox test of the Dysmorphology screen to suggest that hearing ability is
markedly affected in these mice. Tests of muscular function were not performed in the Neurology
screen but these mice show impaired rotarod performance. This could indicate that they have a
problem with coordination and balance. Thus, to exclude that this acoustic startle effect is due
to altered muscular ability, further motor tests should be performed with these mice. Areas in
the brainstem at or below the pons, such as the caudal pontine reticular nucleus, mediate
the acoustic startle response (Koch, 1999). Given the widespread expression of NSUN2 in the brain
(Allen Brain Atlas: http://mouse.brain-map.org) it is also conceivable that this acoustic startle phenotype is a
direct effect of the mutation in the aforementioned brain regions.

NSun2 is a RNA
methyltransferase with substrate specificity towards tRNA and possibly other RNA polymerase III transcripts. NSun2 localizes
to the spindle in mitosis as an RNA-protein complex that includes 18S ribosomal RNA. The
presence of both RNA and Misu is required for correct spindle assembly. NSun2 may be
involved in a mechanism whereby c-Myc promotes proliferation by stabilizing the mitotic spindle in fast-dividing
cells. It is not clear if NSun2 is involved in adult neurogenesis and learning and
memory behaviour. We will test these mice further using cognitive tests to determine whether these
mutant mice exhibit social discrimination, object recognition and spatial memory alterations. We will also analyse
the brain tissue from these animals to determine whether adult neurogenesis is affected by the
mutation.

Neurology

Summary:

In the primary screen, we only performed the rotarod test. We detected more passive rotation
by mutants as well as impaired performance of male mutants.



Discussion:

Due to capacity reasons, the only neurological test performed in this study was the accelerating
rotarod. Here we detected an altered performance of mutant mice. The different strategy observed could
be attributed to behavioral / motivation changes and anxiety as well as to differences in
motor coordination or balance. Nsun2 is a RNA methyltransferase and has been associated in humans
with neurological deficits, e.g. movement coordination, and mental retardation (Khan et al., 2012) and knockout
mice have been described to have retarded body mass and e.g. reduced grip strength as
well as hyperactivity during the calorimetry measurement (Wellcome Trust Sanger Institute). Molecular phenotyping detected changes
of several genes involved in neurological diseases. We therefore suggest performing the complete primary neurological
screen also replicating the rotarod measur ment with the new cohort of mice entering the
GMC for secondary screening.

Eye & Vision

Summary:

There might be a significant effect of the mutation in NSun2 on the eye size
of male mice (not confirmed in a Secondary Eye Screen).



Discussion:

The results of the Primary Eye Screen point to an effect of the D014D011 mutation
in NSun2 on eye growth. However, further tests would be required to confirm significantly reduced
eye sizes in male mutants.

Nociception

Summary:

In the hote plate test we found no significant difference between the genotypes.



Discussion:

The mutation in NSun2 does not seem to have an influence on the nociceptive behavior.


Energy Metabolism

Summary:

Body mass was massively decreased especially in mutant males. The relation between body fat content
and body mass was also substantially (even though not significantly) shifted between control and mutant
mice indicating genotype effects on body composition. During indirect calorimetry we found a trend towards
hypermetabolism (daily energy expenditure as well as food intake) possibly due to hyperactivity especially rearing
behavior that was significantly increased. Infrared measurements of body surface temperature did not show differences
between control and mutant mice.



Discussion:

Nsun2-mutant mice showed interesting alterations in energy metabolism related variables. Body mass was markedly reduced
whereas body fat content was not drastically different but seemingly the mutation had an effect
on body composition. Energy turnover was also affected. Daily energy expenditure was increased when adjusted
for body mass. As resting metabolic rate, maximum oxygen uptake and body temperature were not
generally increased but locomotor and rearing behavior were, the hypermetabolism seemed to be linked to
hyperactivity. Surface temperatures, thus heat loss as detected by IR thermovision imaging was not different
between control and mutant mice.

Clinical Chemistry

Summary:

For reasons of animals' welfare we did not perform an IpGTT and reduced the duration
of food withdrawal before the clinical chemistry measures in pipeline 1 to five hours.


We did not see significant genotype differences in plasma lipid or glucose values, either after
food withdrawal or in ad libitum fed mice. In the latter group there was only
a trend towards higher triglyceride values. Calcium levels in contrast were clearly increased in mutant
mice. Additionally, mild but reproducible increases were seen in chloride and albumin concentrations. In female
mice only, iron concentrations were decreased and creatinine levels were elevated compared to controls


In the hematological analyses we found hypochromic erythrocytes in mutant mice of both sexes. This
was associated with microcytic anemia in female mutants, while microcytosis in males was seen in
the second test only.



Discussion:

Although mutant animals displayed markedly decreased body mass, we did not find significant differences in
energy metabolism-related parameters (plasma lipid and glucose values). However, differences found in albumin, chloride, calcium
and creatinine levels could be due to changes in renal function and/or acid-base balance, since
increased chloride levels can be a symptom of metabolic acidosis. Additionally, we saw some differences
in iron metabolism-related parameters in females hinting towards iron deficiency. These findings fit to the
changes we detected in hematological values: Female mutants showed hypochromic, microcytic anemia, as it can
be seen in iron deficiency. Hypochromic erythrocytes were also observed in male mutants but hemoglobin
levels were not reduced in these animals due to increased red blood cell counts. Therefore,
it is not clear, whether the hematological changes are secondary effects of changes in iron
metabolism or primary effects of the mutation. In female mutants, we saw additionally decreased platelet
counts and an increased mean platelet volume. This could be a results of increased peripheral
platelet destruction resulting in an increased proportion of immature platelets or might be due to
an impaired thrombopoiesis. 

Immunology

Summary:

FACS analysis of samples from peripheral blood revealed significant changes in the proportions of leukocyte
subsets. The frequency of CD8 T cells was increased, which went together with a slight
decrease in the frequency of CD4 T cells. Besides that, changes in B cell, T
cell and NK cell subsets were detected. Levels of IgG1 and IgG3 were decreased. The
phenotype was observed in mutants of both sexes.



Discussion:

We did not find a significant difference in the frequency of T cells between mutants
and controls but a change in the CD4/CD8 ratio - induced by a slightly increase
in the frequency of CD8+ T cells in mutants. A reduced CD4/CD8 ratio can be
seen under physiological conditions after peripheral, postthymic T cell proliferation (Thomas-Vaslin et al., 2008). However,
the T cell phenotype of the mutant mice does not characterize them as being activated:
we did not find differences in the frequency of CD44-expressing cells (constitutively expression) nor in
the frequency of Ly6C-expressing cells , which  was rather decreased within the CD8 T cell compartment  of mutants
(not shown). Ly6C is a surface molecule expressed upon activation or homeostatic proliferation (Hänninen et
al.,
2011). Interestingly,  the L-selectin expression on CD8+ T cells was slightly higher in mutants. In un-immunized
mice we expect a high proportion of L-selectin-expressing cells in the T cell compartment (>90%)
in peripheral blood. L-selectin is shedded upon activation. Additionally, due to erythrocyte lysis during sample
preparation, a notable part of the L-selectin is lost  (Stibenz and Bührer 1994, and own
observations), which might be induced by ATP and NAD released from lysed erythrocytes (Scheuplein et
al.,
2009). L-selectin is also lost in case of T cell apoptosis (Kern et al.,
2000). Therefore, our finding might be interpreted as a higher degree of CD8+ T cell
apoptosis in controls which results in a lower frequency of CD8+ T cells and a
preapoptotic loss of L-selectin in the living CD8+ T cells. However, the change in the
CD4/CD8 ratio might also have occured during the primary T cell production in the thymus,
where a slightly lower CD4 production is possibly compensated by a higher CD8 production. We propose
the analysis of the thymocyte subsets via flow cytometry to address these questions.

MCH
class II is constituvely expressed on B cells. The expression is increased upon acute activation,
for example upon LPS-stimulation (Barrachina et al., 1999). Interestingly, Schwab et al., (2005) could correlate
a reduction of the MHC class II expression on B cells from murine peripheral blood
to stress-induced immunosuppression. The phenotype in the mutant mice hints towards a less activated status
of the B cells.

We subdivided NK cells (Nk1.1./or and Nkp46+) by their expression
of CD11b and revealed a lower proportion of CD11b-expressing NK cells in mutants of both
sexes. NK cells are considered to be a relatively heterogenous population; CD11b expression is considered
to reflect a certain maturation state (Hayakawa et al., 2007). In this respect, the NK
cell compartment of mutants consists of more 'activated' NK cells. However, the frequency of total
NK cells is mostly lower in mutants. Thus, the higher proportion of CD11b-expressing NK cells
does not go together with a higher number of CD11b-expressing NK cells.

Under physiological
conditions, the composition of immunoglobulin isotypes reflects the adaption of the mouse to a particular
immune challenge. The IgG3 isotype is mostly associated with polysaccachid-antigens (->encapsulated bacteria) (McLay et al.,
2002). IgG3 (and IgG2b) can be produced during a Th1-biased immune response. Also a reduced
IgG1/IgG2b ratio is - under physiological conditions - indicative for a Th1-biased immune response (Toellner
et al.,1998), which is in contrast to the higher IgE levels (see Allergy Screen).


This contradiction supports the hypothesis of a NSun2-dependent immune-disturbance rather than an accidental physiological variation.
However, the observed immunoglobulin levels are low - as expected for non-immunized mice - in
both, controls and mutants. More pronounced differences might be expected under challenge conditions (immunization and/or
infection). 

Allergy

Summary:

The analysis of total plasma IgE provided hints for a possible phenotype of
increased IgE in mutant mice of both sexes.



Discussion:

The analysis of basal total IgE levels in plasma revealed elevated values in samples from
mutant mice. However, the values are within the normal range for healthy mice on C57BL6
genetic background. We suggest the testing of a second cohort for a confirmation of the
data.

Steroid Metabolism

Summary:

The data could not be evaluated due to technical failure of the equipment.




Cardiovascular

Summary:

The echocardiography had to be cancelled for reasons of animal's welfare because several mice died
during the testing.




Lung Function

Summary:

Pulmonary function testing did not reveal any genotype-related differences.



Discussion:

No significant differences in lung volumes or mechanical parameters were found between genotypes. Therefore, we
assume that the D014D11 mutation in NSun2 per se does not affect pulmonary function.

Expression Profiling

Summary:

In this report, we describe the results of transcript profiling of the Nsun2 mutant mouse
line. Brain was selected due to high expression levels of Nsun2 in brain and expected
changes in behavior. Experiments were performed for four control and four mutant animals of each
genotype at the age of 17 weeks (in total eight hybridizations).

Data analysis using
various statistical methods detected several significant differences in gene expression levels in brain of Nsun2
mutant mice compared to the controls. Regulated genes in brain are associated with axon guidance,
behavioral alterations, neurodegeneration and distinct neurological diseases e.g., epilepsy, AD or microcephaly.




Pathology

Summary:

The Phenotype 'testicular atrophy' could be confirmed in seven of ten male mice. Furthermore, we
noted differences of unclear relevance in organ weights, body weight and tibia length.



Discussion:

NSun2 is known to have an effect on the differentiation of stem cells. Spermatogenesis is
a process in which this differentiation of spermatogonia is essential since the testicular stem cells
have to undergo meiotic and mitotic divisions to mature. For the NSun2-mutant animals, the males
were  expected to be sterile due to this accumulation of spermatogonia which are unable to
differenciate. The pathology screen confirmed this phenotype in seven out of ten animals.

The
changes in organ weight are of unclear relevance and could not be linked to any
histological phenotype.

 

Bodyweight
Meis1Meis1Targeted mutation
Bone & Cartilage

Summary:

In the primary screening no genotype-specific differences were found between mutants and controls.



Discussion:

The Meis1a mutant mouse line was analyzed in the Dysmorphology, Bone and Cartilage module of
the German Mouse Clinic. No genotype-specific differences were found between mutants and controls. The sex
differences which were observed here are common in many mouse strains, and thus are not
abnormal (unpublished data).

Behaviour

Summary:

In the open field, the mutant mice were hyperactive. There was also decreased prepulse inhibition
responding in the mutant mice as well as decreased acoustic startle reactivity in the male
mutant mice with the opposite effect in the female mutant mice.



Discussion:

In the open field, our test of spontaneous reactions to a novel environment, the mutant
mice were hyperactive, moving a greater distance over the course of the 20 minute test.
This increased movement was accompanied with an increased speed of movement in the mutant mice
as well as an increase in the number of entries into the central more aversive
zone. In general, an increase in centre entries can index a decrease in anxiety-related behaviour.
Nevertheless, this may also be secondary to the hyperactivity of these animals. This is consistent
with the hyperactivity observed by the Energy Metabolism screen in their calorimetry cages. Such behavior
corresponds to the activity in humans and can therefore be regarded as a part of
an RLS phenotype in mice. Furthermore, this is in line with the results in the
loss-of-function model of the RLS associated gene BTBD9.

The alteration in acoustic startle reactivity
is interesting given a study in restless legs syndrome patients showing that they had increased
ASR (Frauscher et al., 2007). As asserted by the authors, this change in ASR may
be due to loss of inhibitory control of hierarchical structures, for example, through a decreased
basal ganglia-induced inhibition of brainstem nuclei involved in the startle response. The nucleus reticularis pontis
caudalis receives both excitatory and inhibitory cholinergic projections from the pedunculopontine tegmental nucleus. This, in
turn, regulates the excitability by predominantly inhibiting startle-related structures of the pontine reticular formation. The
pedunculopontine tegmental nucleus then receives mostly inhibitory basal ganglia input, mainly from globus pallidus and
substantia nigra pars reticulata. An explanation for the opposing effects in the male versus the
female mutant mice is not clear at present. The effect of increased ASR in the
female mutant mice is more akin to the disinhibited ASR observed in the RLS patients.


Basal ganglia disease with substantia nigra pathology may result in disinhibition of the basal
ganglia output, thus contributing to net facilitation of ASRs. A potential role of dopamine in
ASR modification is supported by animal data; for example, in a rat model of Parkinson's
disease using 6-hydroxydopamine, neurons in the caudate nucleus and putamen become supersensitive to dopamine agonists
after denervation of the nigrostriatal pathway, and supersensitive dopamine D1 receptors have been suggested to
mediate enhanced ASRs. Recent neuropathologic studies in RLS have found impaired iron acquisition in neuromelanin
cells of the substantia nigra, implying that this brain structure may contribute to the pathogenesis
of RLS.

PPI has been shown to be affected by treatments that alter dopaminergic
transmission such as direct and indirect dopamine agonists; these effects can be blocked by administration
of antipsychotic drugs (for review, see Geyer et al. 2001). There is some evidence that
the effects of dopamine manipulations involve the nucleus accumbens and its associated neural circuits including
afferents originating from the mPFC. Thus, an altered dopaminergic system may be involved in the
decreased prepulse inhibition responding observed in these mutant mice.

A second cohort of mice
is currently in a secondary screen in the GMC. These mice were 8 months at
the start of testing and we were able to confirm the hyperactivity in the male
mutant mice. The female mutant mice are now showing a hypoactivity and this age. They
are currently in our voluntary running wheel system. Subsequent to this, they will be tested
for social discrimination memory due to the expression of MEIS1 in the olfactory bulbs and
reliance of this test on olfactory memory.

 

Neurology

Summary:

In the primary neurological screen the only altered parameter was the reduction in plasma lactate
level. Usual SHIRPA parameters tested were without differences between mutants and controls, but the observation
of the mice revealed a clicking noise of unclear origin in several mutant mice and
none of the controls.



Discussion:

Meis 1 mice did not show clear differences that would suggest increased activity/locomotor activity. However,
the activity tested in our screen is recorded at the first 30 seconds in the
viewing arena and does not cover larger time intervals with even undisturbed behavior. So a
hyperactivity caused by the urge to move as in RLS patients would not be covered
by the test conditions of the SHIRPA protocol. The slightly improved rotarod performance could also
be correlated to increased locomotor activity, but the effects were too mild to be considered
a phenotype.

Interestingly, several mutants made a clicking noise, shown in the already sent
movie file, an unusual observation with numbers too high to be a finding by chance.
Although it resembles human teeth chattering, the origin of this sound is unclear. If it
is a consequence of behavioral alterations or a more physiological effect remains to be elucidated.
We suggest analyzing nasopharyngeal structures as well as lung since the noise was associated with
sniffing in the arena.

Eye & Vision

Summary:

We found reduced axial eye lenghts, reduced retinal thicknesses, and reduced visual properties in the
Primary Eye Screen with Meis1.



Discussion:

Our data clearly indicate an effect of the Meis1 mutation on eye development and vision.
In particular, visual properties, total eye sizes, and total retinal thicknesses were significantly reduced in
both male and female mutants. In contrast, we did not find lens abnormalities. These observations
might be explained by irregularities during  retinal development. Previously published results indicated a role of
Meis1 and Meis2 during proliferation of early retinal progenitor cells in verterbates (Heine et al.,
2008). Consequently, there might be morphologic alterations in the adult retina of the Meis1 mutants
even though this was not detectable by OCT. Further studies by histology and Electroretinography might
confirm this assumption.

Nociception

Summary:

We did not detect any genotype effect on pain reaction in this line.



Discussion:

We did not find any effect on the pain reaction in this mutant line.

Energy Metabolism

Summary:

Mutant mice were slightly underweight but no shift in body composition could be detected. During
indirect calorimetry mutant mice showed highly significant hypermetabolism that was not clearly related to hyperactivity.




Discussion:

In Meis1aERT2 mutant mice we could show interesting effects on energy metabolism related parameters. Mice
were hyperactive and showed clearly increased energy expenditure. These effects could be confirmed in a
second cohort of mice (data were presented separately). It remains to be investigated in future
studies if hypermetabolism was just secondary to the behavioral phenotype or if basal metabolic functions
were also affected. To test this indirect calorimetry should be conducted at varying ambient temperatures
(especially basal metabolic rate and resting metabolic rate). It also needs to be evaluated if
very small movements of the mice resembling NEAT (non-exercise associated thermogenesis) also contributed to increased
energy expenditure. We suggest conducting energy expenditure measurements in parallel with the monitoring of micro-movements
using motion sensitive plates. This will also help to confirm slight effects on circadian rhythms
in activity we detected in the first cohort of mice. The findings regarding energy metabolism
and behavior provide specific read outs for future studies to test the effects of drug
treatment in rescue experiments in the mouse model for RLS.

Clinical Chemistry

Summary:

The clinical-chemical and hematological screen of Meis1a mice did not reveal major genotype related differences
in the parameters investigated. The only, clearly genotype-related difference was a decrease in plasma alpha-amylase
activity in mutants compared to controls. However, this effect was small and of unclear relevance.
Several other subtle changes might represent secondary effects of increased activity on metabolism.



Discussion:

Most of the genotype-related differences detected in the clinical-chemical screen were very mild, frequently not
reaching statistical significance. Additionally, several of these were sex-specific. This suggests, that these findings most
likely represent secondary effects, for example due to increased activity, rather than direct effects of
the mutation on metabolism. The only exception was the detection of decreased alpha-amylase activities in
mutant animals. Plasma alpha-amylase activity in mice depends on a variety of different factors. Alpha
amylase is produced in pancreas, parotid gland and liver. The pancreas isoform is rapidly eliminated
by the kidneys while other isoforms are eliminated mainly by proteolytic inactivation. Therefore functional changes
concerning protein metabolism or exocrine function, or structional damage of pancreas, parotid gland or liver
can affect amylase levels as well as effects on renal function or proteolytic activity could
have an influence on amylase activity in plasma. However, even this difference was small and
further investigations are necessary to clarify its relevance and underlying mechanisms.

Immunology

Summary:

We found a slightly lower frequency of T cells (CD4+ and CD8+) in female mutants
compared to controls together with a slightly higher frequency of granulocytes.



Discussion:

The slightly lower frequencies of T cells did not go together with changes of the
CD4:CD8 ratio, nor with changes in the T cell compartment concerning the expression of CD44
or CD62L. The CD25 co-expressing CD4+ T cell compartment (regulatory T cells) was also reduced.
This finding is of interest, as most often we observe a regulation of the CD25-coexpressing
CD4+ T cell compartment independently of the total CD4+ T cell population. The lower frequency
of T cells was negatively correlated to a higher frequency of granulocytes. Furthermore, in mutant
females the frequencies of B and T cells were both positively correlated, reflecting the negative
correlation of both lymphocyte subtypes to the frequency of granulocytes. In contrast, in female controls
the frequencies of B and T cells were rather negatively correlated. The female mutants showed
therefore a higher granulocyte:lymphocyte (Tcells+Bcells) ratio, and a lower Tcell: B cell ratio compared to
controls. Changes in the frequency of granulocytes in blood can occur rapidly upon stress or
other pathological conditions. In the case of Meis1 mice the differences are rather small. However,
we recommend to include the measurement of the frequencies of granulocytes and lymphocytes as markers
for yet unknown changes in these mice for further studies.

Allergy

Summary:

We observed significant elevated IgE levels in mutant mice, but median levels of IgE in
plasma were within the normal range for healthy mice on C57BL/6 genetic background.

 




Discussion:

Although significant differences were recorded, values of IgE in plasma are in the normal range
of healthy C57BL/6 WT mice and most probably the differences observed are not biologically relevant.


 

 

 

Steroid Metabolism

Summary:

The analysis of steroid hormones in plasma samples of Meis1aERT2-mice revealed differences between mutant and
control animals. Significant differences in the concentration of corticosterone have been found in male and
female mice.



Discussion:

Genotype-specific differences for corticosterone, concentrations in blood plasma were found. Therefore, the Meis1aERT2-mutation seems to
affect steroid metabolism, but the reasons for it are not known. We suppose that the
mutation disturbes mitochondrial functions or transport processes in the plasma membrane. Such disturbations can often
be displayed by the analysis of amino acids and lipids.

For further information please
contact:

Cornelia Prehn (+49-89-3187-3231) or Julia Scarpa (project manager, 3722), www.gac-munich.de

Cardiovascular

Summary:

The basal cardiovascular functions were investigated in the primary screen by awake echocardiography. The major
findings were:

- Decreased interventricular septum width in systole mainly in females

-
Decreased left ventricular inner diameter in systole mainly in females

- Slightly increased heart
rate and respiration rate

 



Discussion:

Only mild genotype-related differences without relevance for the cardiovascular system were found.

Lung Function

Summary:

Pulmonary function testing did not reveal any genotype-related differences.



Discussion:

No significant differences in lung volume or mechanical parameters were found between genotypes. Accordingly, we
claim that mutants Meis1aERT2 do not show an affected pulmonary function.

Expression Profiling

Summary:

In this report we describe the results of transcript profiling of the Meis1aERT2 mutant mouse
line. Brain was chosen for transcriptome analysis due to the association of Meis1 with RLS.
Experiments were performed for four mutant (mut) and four wild type (ct) mice as controls.
Data analysis using various statistical methods detected significant differences in gene expression patterns in brain.
Genes differentially expressed in brain are associated with cellular movement, neurological disease, nervous system development,
cancer and lipid metabolism.



Discussion:

Brain was chosen for the Meis1aERT2 mutant mouse line for expression profiling analysis and statistical
methods detected differential gene expression in this organ. Meis1 was down-regulated in the analysed heterozygote
male mice (fold change 1.4), however didn’t reach the level of significance. In this data
set overrepresented GO terms shown up annotations with neurological disease and neurological system development as
well as lipid metabolism. These findings might be in correlation with the expected phenotype regarding
RLS and the alterations found in the metabolism screen. Based on these results there is
an indication for a neurological phenotype in the Meis1aERT2 mutant mouse line on molecular level.


Pathology

Summary:

Meis1 is essential for megakaryocyte lineage and in humans it is an oncogene frequently activated
in leukemia. We found morphological changes in megakaryocytes of a Meis1aERT2- mutant mouse, so we
consider this finding as genotype-specific but with low penetrance (1/12 = 8% penetrance). All other
organs analyzed were normal.



Discussion:

According to the information from the mouse provider, Meis1 encodes a homeodomain transcription factor which
is essential for hematopoiesis and vascular patterning in the mouse embryo. Meis1 is essential for
megakaryocyte lineage development and in humans it is an oncogene frequently activated in leukemias. In
addition, genetic variants of Meis1 have been associated with the human Restless Legs Syndrome. The
described phenotypes have been observed in homozygous mice and there is the cause of intrauterine
death. Results of studies in humans suggest that heterozygosity may cause alterations in any of
the organs altered in the homozygous mice, albeit at lower penetrance or with milder phenotypes.


In the analysis performed by the pathology screen the adult Meis1aERT2-heterozygous mice did not
show any histopathological alteration in all organs analyzed, with exception of changes in the morphology
of the megakaryocytes in one mutant mouse. Therefore, we consider this finding as genotype-specific but
with low penetrance.  

 

Bodyweight
Adamts-7-KOAdamts7Targeted mutation
Bone & Cartilage

Summary:

In the primary screening no genotype-specific differences were found between mutants and controls.



Discussion:

The ADAMTS-7 mutant mouse line was analyzed in the Dysmorphology, Bone and Cartilage module of
the German Mouse Clinic. No genotype-specific differences were found between mutants and controls. The sex
differences which were observed here are common in many mouse strains, and thus are not
abnormal (unpublished data).

Behaviour

Summary:

The mutant mice spent more time in the centre of the open field and exhibited
increased acoustic startle reactivity.



Discussion:

In the open field, our test for spontaneous reactions to a novel environment, the mutant
mice exhibited normal exploratory and locomotor activity as indexed by total distance travelled and total
rearing activity. Nevertheless, the mutant mice were spending more time in the centre, entering into
this zone more and moving a greater distance suggesting a pattern of decreased anxiety. The
reason for this anxiety alteration is not clear at present.

While there was no
indication that sensorimotor gating was altered (normal prepulse inhibition responding), acoustic startle reactivity was increased
in the mutant mice. This could indicate that the mutant mice have enhanced hearing ability,
or increased anxiety or better neuromuscular recruitment. There were no changes in ABR or click
box responding in the Neurology screen to infer that hearing ability is altered in these
mice. The behavioural pattern in the open field suggests that, if anything, these mice are
exhibiting decreased anxiety. While the Neurology screen found decreased rotarod latencies in the female mutant
mice, this would go rather in the direction of impaired neuromuscular recruitment. A number of
brain areas are recruited for the acoustic startle response including areas in the brainstem at
or below the pons, such as the caudal pontine reticular nucleus (Koch, 1999). The expression
of ADAMTS-7 in the brain is low (http://mouse.brain-map.org/gene/show/72313). Thus, an explanation for this acoustic startle
effect is not clear at present.

Neurology

Summary:

In the primary neurological screen the only difference detected was a reduction of rotarod latencies
in female mutants compared to controls. SHIRPA parameters, grip strength, ABR and lactate levels were
without genotype effects.



Discussion:

The differences detected in our screen were the reduced rotarod latencies of female mutants and
the increased falling from the rod. 

Given the suggested role of ADAMTS-7 for vascular
remodeling and rheumatoid arthritis without known expression in neuronal tissue, a difference in motor performance
could be hypothesized to be a secondary effect. Only females were affected from the rotarod
impairment which was also seen as a slight tendency towards decreased rearing behavior in the
Open Field test. However, no difference in rearing had been detected during Indirect Calorimetry. On
the other hand, impaired lung functions could also have an impact on motor performance, but
locomotor activity was not altered in any of the measurements. So since there is no
supporting information about the link of ADAMTS-7 knockout, the rotarod finding is at the moment
of unclear relevance although the difference is quite pronounced (see size of difference plot, "General
results").

Eye & Vision

Summary:

We did not identify ocular irregularities in the primary Eye Screen with ADAMTS-7 KO.



Discussion:

Our data clearly demonstrate that eye development and visual properties are not affected by the
ADAMTS-7 KO mutation.

Nociception

Summary:

We did not find a significant genotype effect on the pain reaction in this line.



Discussion:

The mutation did not influence the pain reaction of the mutant animals.

Energy Metabolism

Summary:

Maximum oxygen consumption was increased especially in male mutants.



Discussion:

No clear effects of the mutation on energy metabolism related parameters could be detected even
though maximum oxygen consumption was increased especially in male mutants accompanied by small very specific
behavioral differences in rearing activity. As the behavioral screen could not detect any phenotypic alterations
that could be linked to this finding the relevance remains unclear so far.

Clinical Chemistry

Summary:

In the clinical-chemical screen we found only mild genotype-related differences of unclear relevance: Basal fasting
glucose values were slightly increased in a remarkable number of male and female mutant mice,
triglyceride values were slightly decreased in male mutants in the ad libitum fed state, and
some mutants of both sexes showed decreased red blood cell counts, hemoglobin and hematocrit values.
Further experiments are necessary to clarify whether these changes are really genotype-related effects.



Discussion:

All genotype-related differences found in clinical-chemical plasma parameters, hematological values and in the glucose tolerance
test were mild and/or sex-specific. Therefore results are so far of unclear relevance. Decreased plasma
triglyceride levels in mutant males might indicate effects of the mutation on energy metabolism. In
fact an increased maximum oxygen consumption was seen during indirect calorimetry done by the Metabolism
Screen in mutants males, which was associated with increased rearing behaviour during this test. In
the Behaviour Screen male mutants showed as a trend an increased forward locomotion in the
open field test as compared to male controls. Therefore it can not be excluded that
mild effects observed in parameters related to energy metabolism might be affected secondarily to behavioural
changes.

Immunology

Summary:

We analyzed the frequencies of leukocyte subsets in peripheral blood and found minor changes of
unclear relevance.



Discussion:

We found a tiny decrease in the frequency of T cells, and a subtle increase
in the frequency of NK cells (especially in males). These changes did not go together
with changes in the CD4:CD8 ratio, nor with changes in the frequencies of T cell
or B cell subsets. This finding might therefore be considered as a finding by chance,
reflecting physiological variation.

Allergy

Summary:

The screening did not uncover an IgE phenotype.



Discussion:

The analysis of basal total IgE levels in plasma revealed values within the range expected
for mice on C57BL/6 genetic background.

Steroid Metabolism

Summary:

The analysis of steroid hormones in plasma samples of ADAMTS-7 mice did not reveal differences
between mutant and control animals.



Discussion:

No genotype-specific differences in corticosterone, androstenedione and testosterone concentrations in blood plasma were found. Therefore,
the ADAMTS-7 mutation does not seem to affect steroid metabolism.

Cardiovascular

Summary:

No genotype-specific differences were found between mutants and controls.




Lung Function

Summary:

After performing the lung function tests we observed, that the mutations in ADAMTS-7 induced changes
in a number of the parameters we measured in the lung screen: FEV100, FVC, IC,
Cchord, VC, ERV, TLC, FRC, C, and TV were increased, and R was decreased. However,
though significant, the values are not highly relevant.



Discussion:

ADAMTS-7 KO animals showed changes in a number of our lung parameters tested. We recommend
further histological analyses.

Expression Profiling

Summary:

So far, no organs have been selected for RNA expression analysis.




Pathology

Summary:

We did not detect genotype-related morphological changes. A histological analysis of the aortic arch and
artery vessels did not reveal any wall thickening, accumulation of foam cells or atherosclerotic plaques.
Minor calcifications observed in myocardium occurred in similar incidence in mutant and control animals.



Discussion:

The ADAMTS-7 gene has been identified as a coronary artery disease risk locus. Atherosclerosis is
characterized by formation of plaques composed of cholesterols, lipids and inflammatory cells (Salter et al
2010). As a first morphological change in atherosclerosis (primary stage), macrophages migrate into the arterial
intima, take up lipoproteins and transform to lipid-laden foam cells. Histological analysis of endothelium of
the aorta and other vessels of ADAMTS-7 KO mice did not reveal indication of atherosclerosis,
as judged by the presence of such foamed cells or lipid depositions. Minor calcifications observed
in myocardium occurred in similar incidence in mutant and control mice. Therefore, we regard the
observed calcifications as not genotype-related.

ADAMTS-7 has been reported to be involved in the
degradation of extracellular matrix and loss of cartilage in rheumatoid arthritis (Lin et al 2010).
It is beyond the scope of the primary screen to investigate joints, synovium and cartilage,
but the pathology screen has conserved the relevant tissues for a possible histological investigation.

Bodyweight
Calcr-F6CalcrTargeted mutation
Bone & Cartilage

Summary:

No genotype-specific differences were found between mutants and controls. Histomorphometric analysis of spine sections performed
by the collaboration partner showed increased trabecular bone volume and trabecular number in 3 and
6 month old Calcr-deficient mice due to increased bone formation mediated by calcitonin receptor expression
in osteoclasts (unpublished results).



Discussion:

The Calcr-F6 mutant mouse line was analyzed in the Dysmorphology, Bone and Cartilage module of
the German Mouse Clinic. No genotype-specific differences were found between mutants and controls. Histomorphometric analysis
of spine sections performed by the collaboration partner showed increased trabecular bone volume and trabecular
number in 3 and 6 month old Calcr-deficient mice due to increased bone formation mediated
by calcitonin receptor expression in osteoclasts (unpublished results). This differences might not be detected by
our DXA analysis as the whole skeleton is measured and it is not possible to
separate the cortical from the trabecular bone. Also histomorphometric examination offers information on localized bone
turnover and remodeling, and structure on the cellular level unobtainable from other investigative means.

Behaviour

Summary:
Analogous to the hypoactivity observed by the Energy Metabolism screen in the homecage setting,
the mutant mice were also hypoactive in a novel environment: the open field. Furthermore, the
female mutant mice were less exploratory in this arena during the 20 minute test. They
also showed increased acoustic startle reactivity.


Discussion:

In the open field, our test for spontaneous reactions to a novel environment, the mutant
mice exhibited decreased total distance travelled over the course of the 20 minute test. This
indicates that these mutant mice were hypoactive under these conditions. Furthermore, the female mutant mice
showed decreased rearing activity suggesting that they were less exploratory. There were no apparent effects
of the mutation on anxiety-related behaviour. Reasons for this hypoactivity in these mutant mice are
not clear at present. Nevertheless, there was a similar decrease in home cage activity as
measured by the Energy Metabolism screen. This suggests that this is not a hypoactivity in
response to the aversiveness of the open field arena but rather a fundamental hypoactivity possibly
due to physical inabilities. As these mice are known to have changes in their bone
morphology, this may underlie this alteration.

The female mutant mice also exhibited increased acoustic
startle reactivity. Increased acoustic startle may be due to heightened anxiety, enhanced neuromuscular recruitment or
better hearing ability. There were no changes in acoustic brainstem responses in the Neurology screen
or in the Clickbox test of the Dysmorphology screen to indicate that hearing ability is
markedly different in these mutant mice. Furthermore, there were no changes in anxiety-related measures in
the open field test to suggest that anxiety responding was altered. As far as neuromuscular
recruitment is concerned, there were no pronouced differences in grip strength or rotarod performance to
imply that this could be enhanced in the female mutant mice. Thus, an explanation for
the acoustic startle changes in the female mutant  mice or why this is a sex-specific
effect is not clear at present. Areas in the brainstem at or below the pons,
such as the caudal pontine reticular nucleus, mediate the acoustic startle response (Koch, 1999). The
calcitonin receptor is mainly found in the hypothalamus in the brain (Allen Brain Atlas: http://mouse.brain-map.org/gene/show/12096).
This may make it improbable that the acoustic startle effect is due to primary effects
of the mutation in the brain areas involved in the ASR response. More research is
necessary to decipher the underlying cause of this alteration.

 

Neurology

Summary:

We did not detect any neurological abnormalities in Calcr-mutant mice. Basic neurolocical functions, grip strength,
rotarod performance and ABR were without genotype-related differences.



Discussion:

In our primary neurological screen, we did not detect any hints that the absense of
Calcr has an influence on neurological function at least at the age tested. Basic neurological
functions, movement, motor coordination, grip strength as well as hearing sensitivity were without effects of
the mutation.

The reduced  locomotor activity detected in behavior and metabolic testing was not
seen in our screen. This is most probably due to the fact that the experimental
conditions are very different and the locomotor activity  in our screen is measured by counting
the squares crossed within the first 30 second in the viewing arena, a very specific
and short time in a novel environment. There was no hint from our tests that
motor abilities are in any way impaired, so it is more likely that the hypoactivity
is a centrally regulated behavioral phenotype. We do not look for nociceptive phenotypes in our
screen, these experiments are performed by the nociceptive module (please see next chapter). However, if
there are differences in sensitivity towards mechanical stimuli we sometimes see differences also in grip
strength testing. This was not the case in the Calcr mutants.

Eye & Vision

Summary:

We did not identify ocular irregularities in the primary Eye Screen with Calcr.



Discussion:

The results of the primary Eye Screen clearly demonstrate that eye development is not affected
by the Calcr-F6 mutation.

Nociception

Summary:

We did not detect any significant genotype effect on the pain reaction




Energy Metabolism

Summary:

Mutant mice were hypoactive and energy turnover trended to be decreased. Body mass and body
composition were not affected.



Discussion:

The mutation had only mild effects on energy metabolism parameters. Differences in body mass and
body composition that indicate mid- or longterm effects on energy allocation could not be detected
between genotypes. However, distance travelled under home cage conditions was significantly reduced both in male
and female mutants. These findings support results of the behavioral screen showing hypoactivity in the
open field test. This reduction had a very mild effect on daily energy expenditure that
was reduced in mutant mice. So far this effect on energy turnover seems to be
secondary to the behavioural phenoytpe. Despite published evidence that salmon calcitonin had annorectic effects in
rodent models the results of the primary screen do not suggest strong effects of the
mutation on energy regulation.

Clinical Chemistry

Summary:

The only clearly genotype-related difference detected in the clinical chemistry screen was moderately increased plasma
ALP activity in Calcr-mutant mice.

Additionally, we saw a trend towards lower glucose values
after food deprivation in mutants in the Glucose Tolerance Test, and lower erythrocyte volumes in
the hematology results. Furthermore, few sex-specific differences between genotypes were noted, namely decreased iron and
increased creatinine in female mutants and increased platelet counts in male mutants. All these mostly
quite small differences are findings of unclear relevance.



Discussion:

The only, clearly genotype-related finding of the clinical-chemical screen was an increased activity of alkaline
phosphatase (ALP) in plasma of mutant mice. ALP in plasma can originate from different sources,
predominantly liver and bone. We did not see significant genotype affects on further parameters associated
with liver integrity or function (ALAT, ASAT, alpha-Amylase, albumin, lipid levels). Additionally, the Pathology Screen
did not recognize any morphological changes in the liver of mutant mice. Therefore, the liver
as source of elevated ALP activity seems unlikely. Investigations by the collaboration partner revealed an
increased bone formation activity in mutant animals resulting in structural changes of trabecular bone. ALP
activity in plasma is increased in both cases of increased bone formation and increased bone
resorption, with a more prominent effect in the latter situation.Therefore, the moderate increase of ALP
in mutant animals, which was more clearly seen in the female group, is most likely
due to changes in bone metabolism.

A few additional, mostly sex-specific findings were observed:


(1) In the glucose tolerance test, the fasting glucose values tended to be lower
in mutant mice. Similar observations have been made previously by the collaboration partner (unpublished results).
This could be a secondary effect of differences in overnight activity, which was observed by
the Metabolism Screen. Differences in activity result in different energy demands and might influence the
degree of gluconeogenesis activation.

(2) Only in the female mice we saw slightly increased
creatinine values and decreased iron values compared to controls. Since Calcr is expressed in the
kidney, one could expect potential effects on renal function. However, besides the small difference in
creatinine values in females we did not observe any other genotype-related difference in parameters associated
with kidney function (urea, electrolytes, plasma protein levels). Therefore, we did not see indications of
major effects on kidney function. Whether the small difference in females was an accidental finding
or an indicator of of a mild genotype effect could only be clarified by further
investigations.

(3) The slightly decreased iron values in females mutants and the small trend
towards smaller erythrocytes in mutant animals could indicate effects of the mutation on iron metabolism.
However, the differences are very small with all values still in the normal range of
C57BL/6J mice. Therefore, the relevance of these findings is not clear.

(4) Increased platelet
counts detected in male mutants compared to male controls are most likely an accidental finding.


Immunology

Summary:

We found sex-dependent effects of the mutation in Calcr: Female mutants showed higher levels of
immunoglobulines and changes in the frequencies of T cells (lower), NK cells (higher) and changes
in the frequencies of T and B cell supopulations. In male mutants we found  lower
levels of the immunglobulines IgG1 and IgM, and changes in the frequencies of T and
B cell subpopulations.



Discussion:

Sex-dependent differences in the frequencies of leukocyte subsets and in the levels of immunoglobulins are
often observed in mice and have been described in the literature. For example, Petkova et
al.
(2008) have presented data for several inbred strains that female mice show a higher
frequency of T cells compared to males, an observation we share, and which is also
seen in the control mice of this mouse line. The female mice showed higher levels
of all immunoglobulins. The immunoglobulins are physiologically produced in response to antigens. Typically, a pattern
of specific immunoglobulin isotypes is produced in response to specific antigens. This pattern also depends
on the timepoint of measurement within the timecourse of a given immune response. We rarely
observe an increase of the levels of all immunoglobuline isotypes in a mouse line as
seen in female Calcr mutants. We must therefore consider, whether specific conditions - as for
example dehydration - are causative for our finding. An immunization experiment or an in vivo
B cell stimulation might elucidate, whether Calcr-mutant female mice have a higher antibody response.


Male mutants showed lower levels of the immunoglobulins IgG1 and IgM; the IgG1/IgG2b ratio was
signficantly decreased. Whereas IgM are the first antibodies produced during a humoral immune response, IgG1
is produced later and IgG1 and IgE switching  can be triggered by IL-4 (Hasbold et
al.,
1998). In the mutants, besides reduced IgG1 levels also IgE levels tended to be
lower. However, the very low levels of total plasma IgE under baseline conditions did not
allow us to reveal significant changes (see results from Allergy Screen).  

In female
mutants, we found a higher frequency of NK cells. This heterogenous leukocyte population can be
further subdivided by CD11b, which is expressed upon maturation. The higher frequency of NK cells
does not go together with changes in the CD11b expression. NK cells are classified as
lymphocytes of the innate immune system, sharing properties with cytotoxic T cells. In adult mice,
the bone marrow is the main site of NK cell production, besides, there is thymus-dependent
pathway of NK cell development (Cheng et al., 2009). Under baseline conditions, NK cells represent
only a small proportion of the body's total NK cell pool in the peripheral blood.
However, acute stress can mobilize NK cells to the circulation, most likely from secondary lymphoid
tissue.  For example it has been described that the NK frequency in peripheral blood from
mice increases rapidly upon acute physiological activity, but starts to decrease already 30 minutes after
the physiological exercise (Nagao et al., 2000).

We found a lower frequency of IgD-expressing
cells within the  B cell cluster in mutants.  In peripheral blood, only a small proportion
of  immature B cells, especially transitional (T1) B cells, is present, which do not express
high levels of IgD (Carsetti, et al. 2004). However, memory B cells, or isotype-switched B cells
loose IgD (Nduati et al., 2010). Whereas MHC class II expression is upregulated on activated
B cells (Barrachina et al., 1999),  MHC class II is downregulated during plasma cell differentiation.
Both (IgD and MHC class II downregulation as a result of plasma cell differentiation) would
fit to the higher levels of immunoglobulins in female mutants. However, in male mutants we
found lower levels of IgG1 and IgM compared to controls. Interestingly, Schwab et al. (2005) could
correlate a reduction of the MHC class II expression on B cells from murine peripheral
blood to stress-induced immunosuppression.

In male mutants we found a higher activation status of
T cells, measured by the frequency of CD44 high-expressing cells within the T cell cluster.
This T cell marker is normally assigned to activated T cells but in non-infected mice
these cells might represent cells that arise via physiological homeostatic proliferation (Haluszczak et al., 2009). Also
CD25 is upregulated on T cells upon activation, especially triggered by IL-2 (Schuh et al.,
1998). CD25 is further constitutively expressed on regulatory T cells (Papiernik, 2001). The higher frequency
of CD25 co-expressing CD4+ T cells in males therefore can be either interpreted as a
higher actitvation status or a higher frequency of regulatory T cells.

We furthermore found
a lower frequency of L-selectin (CD62L)-expressing cells within the CD4 (male mutants) or CD8 (female
mutants) T cell cluster, respectively. In peripheral blood of naive mice, CD62L is expressed on
the mayority ( more than 90 percent) of T cells. However, it can be lost
(shedding) upon sample preparation, and is also lost on preapoptotic cells, and upon activation (Scheuplein
et al., 2009). In male mutant mice the lower frequency of L-selectin-expressing cells within the
CD4+ T cell cluster might reflect a higher activation status of the T cells, which
ist supported by the higher frequency of CD44 high-expressing cells.

Taken together, we found
a rather higher activation status of leukocytes in mutants. Further experiments are needed to clarify
the impact of Calcr on the immune function.

Allergy

Summary:

The mutation in Calcr did not
influence total plasma IgE levels.



Discussion:

The analysis of basal total IgE levels in plasma revealed values which were within the
normal range expected for healthy mice on C57BL/6 genetic background. 

Steroid Metabolism

Summary:

The analysis of steroid hormones in plasma samples of Calcitonin receptor -mice did not reveal
differences between mutant and control animals.



Discussion:

We assume that the mutation in Calcr does not affect steroid metabolism.

Cardiovascular

Summary:

In the cardiovascular Screen only  a mild genotype-related difference of unclear relevance was found:
echocardiography revealed decreased left ventricular inner diameter in diastole mainly in males.



Discussion:

From the functional data there was no clear hint for a physiological relevant constraint of
cardiovascular function due to the mutation.

Lung Function

Summary:

Pulmonary function testing did not reveal any genotype-related differences.



Discussion:

No significant genotype-specific differences in volumetric, flow or mechanical lung function parameters were found. Therefore,
the Calcr F6 deletion per se is suggested not to affect pulmonary function.

Expression Profiling

Summary:

No organ has been selected for analysis up to now.




Pathology

Summary:

The Calcr mutant mouse did not present any pathological alteration in target organs such as
thyroid, parathyroid, kidney and hypothalamus with the exception of subtle changes observed in the exocrine
pancreas. These data suggest that the major physiological role of the Calcr might be restricted
to bone.



Discussion:

High bone mass in Calcr-mutant mice due to increased bone formation mediated by calcitonin receptor
(CTR) expression in osteoclasts was demonstrated by the collaboration partner (unpublished results).  

An
interesting finding of the clinical chemistry screen was the moderate increased ALP activity in plasma.
ALP isoenzymes are produced either by bone or by other organs such as liver, bile
tubule, kidneys, epithelium of the intestine, and placenta (please see http://ahdc.vet.cornell.edu/clinpath/modules/chem/alkphos.htm). The complete histological
analysis of these organs performed by the pathology screen demonstrated that the moderate increased levels
of ALP do not derive from alterations in the organs named above and most probably
are due to the described alterations in bone.

In the morphological analysis performed by
the Dysmorphology module of the GMC, abnormal smelling and consistency of feces was observed in
a few Calcr-mutant mice. This is a clinical sign presented in cases of insufficiency of
exocrine pancreas. In fact, preliminary results show more obstructive changes of the pancreatic ducts of
Calcr-mutant mice.

In addition, the collaboration partner described a possible role of CTR in
kidney function and in the glucose metabolism since CTR is a common receptor for calcitonin
and amylin, a peptide co-secreted with insulin from pancreatic beta-cells. With exception of subtle changes
observed in the exocrine pancreas we did not find any pathological alterations in target organs
such as thyroid, parathyroid, kidney and hypothalamus. This data suggest that the major physiological role
of the CTR might be restricted to bone.

Bodyweight
Cip2aCip2AOther
Bone & Cartilage

Summary:

In the DXA analysis, we detected a significantly decreased BMC and bone content in male
mutants. Concurrently body weight, fat mass (males) and lean mass (females) were significantly reduced in
mutants.



Discussion:

The Cip2a mutant mouse line was analyzed in the Dysmorphology, Bone and Cartilage module of
the German Mouse Clinic. In the DXA analysis, we detected a significantly decreased BMC and
bone content in male mutants. Concurrently body weight, fat mass (males) and lean mass (females)
were significantly reduced in mutants. The sex differences we observed are common in many mouse
strains, and thus are not abnormal (unpublished data).

Behaviour

Summary:

The mutant mice show a slight increase in locomotor activity while spending more time in
the centre during the latter stages of the open field test. There were no significant
genotype effects on prepulse inhibition.



Discussion:

In the open field; our test of spontaneous reactions to a novel environment, there were
no major effects of genotype. The mutant mice showed slightly increased locomotor activity and decreased
exploratory activity but only during part of the test. The relevance of these subtle changes
are unclear at present. There was also a small increase in the time that the
mutant mice spent in the centre. This change was significant only during the middle of
test. While such an alteration may indicate a degree of reduced anxiety it may also
be secondary to the slightly increased locomotor activity in these animals. In all, these changes
are subtle and possibly secondary to other alterations in the model.

Neurology

Summary:

In the primary neurological screening we detected more tail elevation in Cip2a mutants in the
viewing arena. All other neurological parameters were without genotype-related differences.



Discussion:

A single significant finding in the Cip2a-mutant mice was less tail elevation. Since there are
no other changes hinting towards either more alertness or altered body tone this result as
stand-alone finding is too small to suggest further neurological screening.

However, tail elevation is
also regulated by spinal pathways involved in spinal nociceptive pathways and is also considered a
nociceptive behavior. Given the difference detected by the nociceptive screen the change in tail elevation
should be kept in mind during further analysis of these mice.

 

 


Eye & Vision

Summary:

In the primary Eye Screen, we did not find ocular abnormalities caused by the mutation
in Cip2a.



Discussion:

The data of the primary Eye Screen indicate that the Cip2a mutation does not affect
eye development.

Nociception

Summary:

We found a slight but significant difference in the first reaction: the mutant male mice
showed a week hypoalgesia. In addition, we found highly significant differences between the sexes. Therefore
we suggest the found phenotype not being a consequence of the mutation but rather caused
by the sex-difference in pain sensation.



Discussion:

We found a significant difference in pain reactivity between the control and mutant animals. The
mutant male mice showed a week hypoalgesia. The found hypoalgesia is subtle and could be
a consequence of the sex difference in pain sensitivity.

Energy Metabolism

Summary:
Cip2a-mutant mice had reduced body mass but significantly increased body fat content.
Lean mass was slightly but not significantly changed. During indirect calorimetry no genotype-related effects were
found apart from reduced body mass.


Discussion:

Overall, Cip2a-mutant mice had a reduced body mass. However, the regression between body fat content
and body mass was significantly different between control and mutant mice indicating a clear shift
in body composition with significantly increased body fat content. Lean mass was slightly but not
significantly changed. A change in body fat content suggests mid- or long-term changes in whole
body energy regulation. However, during indirect calorimetry no genotype-related effects were found apart from confirming
the reduced body mass. Therefore, no direct explanation for the shift in body composition could
be provided based on results of first-line phenotyping.

Clinical Chemistry

Summary:

Impaired glucose tolerance in male mutants together with altered body composition detected by the Metabolism
Screen indicate genotype effects on glucose and energy metabolism. An increased plasma chloride concentration in
mutant animals can be due to acidosis. Additional differences seen in cholesterol values of mice
after overnight food withdrawal, sodium and calcium concentrations, amylase activity as well as red cell
anisocytosis were small and of unclear relevance.



Discussion:

Our data indicate an impaired glucose tolerance in male mutants. Differences seen in cholesterol levels
of mice in the fasted state were mainly due to individual outliers, out of which
one male animal showed also unusually low glucose values during the IpGTT. Therefore, it is
not clear, whether this finding is a really genotype-related effect. In the Metabolism Screen, mutant
animals showed a decreased body mass but at the same time increased body fat content
was seen in male mutant mice. This is an unusual finding clearly indicating that at
least in males, the regulation of energy metabolism is affected by the mutation. In the
ad libitum fed mice we found mild differences in sodium and chloride values. A parallel
increase of sodium and chloride can occure in an altered water balance, either due to
increased water diuresis or due to inadequate water uptake. Since the differences in sodium were
mainly due to outlier values in thecontrol group, this might not have been a real
genotype-related effect.

An isolated increase of chloride values can be an indicator of an
altered acid-base balance. Higher cholride values occur in the case of decreased bicarbonate levels, associated
with an more acidotic blood pH. Acidosis can be caused by metabolic changes, impaired kidney
function or hyperventilation.

Additionally we saw small differences in alpha-amylase activity, calcium levels and
red cell anisocytosis. Since these differences were very small and not associated with other changes
that could explain these changes, further investigations are necessary to clarify their relevance.

Immunology

Summary:

We found a higher proportion of CD44-expressing cells within the T cell cluster (CD4+ and
CD8+ Tcells) in peripheral blood samples from mutants, especially in females, compared to controls. No
further differences in the frequencies of leukocyte subsets from peripheral blood or in the level
of immunoglobulins in the blood plasma between mutant mice and controls (primary screen) could be
obtained.

Listeria monocytogenes infection experiments revealed a specific immunological phenotype in mutant mice, namely
a reduced T cell response to Listeria, leading to an increased bacterial load in spleen
of infected mice.



Discussion:

Primary screen phenotype: The CD44 gene is located on chromosome 2 in the mouse (Colombatti
et al., 1982). Multiple isoforms of CD44 on different types on cells, result from post-translational
modification and alternative splicing (Hirano et al., 1994) Certain isoforms of CD44 are denominated as
'active or inactive', defined by binding to Hyaluronan (Lesley et al., 1993). The CD44 expression
on T cells is upregulated upon T cell activation, but  a proportion of T cells
from naïve mice show an intermediate surface expression of CD44. The level of CD44 expression
on T cells is a mouse strain specific trait (Haegel and Ceredig, 1991).

The
observed slight differences in the expression of CD44 on T cells might reflect differences in
the level of T cell activation (Seiter et al., 2000).

Allergy

Summary:

The primary screening could not uncover an influence of the mutation on IgE levels.



Discussion:

The analysis of basal total IgE levels in plasma revealed values within the range expected
for mice on C57BL/6 genetic background.

Steroid Metabolism

Summary:

Significant genotype effects in the concentration of corticosterone have been found in female mice.



Discussion:

Oncogenesis is known to be correlated with changes in lipid and amino acid metabolism. We
offer to analyse plasma and tissue of the mouse samples in the Metabolomic Platform of
the GAC.

We quantify amino acids, biogenic amines, acylcarnitines, glycerophospholipids, sphingolipids and hexoses (186
metabolites).

These analyses are not sponsored by NGFN and request cost coverage by mouse
provider.

For further information please contact:

Cornelia Prehn (+49-89-3187-3231) or Julia Scarpa (team
assistance, 3722)
www.gac-munich.de

Cardiovascular

Summary:

The experiment could not be performed due to technical difficulties.



Discussion:

If additional screening should be needed please contact Raffi Bekeredjian

(raffi.bekeredjian@med.uni-heidelberg.de).

Lung Function

Summary:

Whole body plethysmography did not reveal any genotype-related differences.



Discussion:

An absence of any difference in spontaneous breathing pattern between genotypes suggests that the Cip2a
mutation does not affect the respiratory system.

Expression Profiling

Summary:

In this report, we describe the results of the transcript profiling of the Cip2a mutant
mouse line. Spleen was chosen for transcriptome analysis. This organ was selected due to the
known strong expression of Cip2a in the spleen of adult mice. Therefore, an immunological phenotype
was expected in the mutant mice. Experiments were performed for four mutant (mut) and four
control (ct) mice as controls (in total eight hybridisations). Data analysis using various statistical methods
detected significant differences in gene expression patterns in spleen of Cip2a-mutant mice compared to the
control group. Differential gene expression in spleen is frequently annotated e.g. with immune response (e.g.,
Ccr5, CD22, CD96, Clec7a, Tank, Tff2, Vegfa), autoimmune disease (e.g., Adss, Clec4a, Dek, Tnfrsf193, Tram1,
Usp15
), cell surface receptor linked signal transduction (e.g., Ccr5, Nppa, Nsdhl, Stap1), and organization of
cytoskeleton (e.g., Csnk1d, Scydr, Elmo1, Myl2, Snx2).



Discussion:

Known expression of Cip2a in spleen of adult mice led to the analysis of this
organ on gene expression level. Data analysis using various statistical methods detected significant differences in
gene expression levels. The GO term analysis of regulated genes identified over-representation of several functional
annotations, e.g., immune response, tumorigenesis, metabolism of proteins, autoimmune disease, cellular homeostasis and organization of
cytoskeleton
. The more detailed research for functional gene annotations revealed genes differentially expressed in spleen
are annotated with NK-, B- and T-cell proliferation/activation/inhibition. The annotations of genes, wich were differentially
expressed in spleen, indicated alterations of the function of this organ in immune response on
the molecular level.

Pathology

Summary:

According to the information from the collaboration partner, staining of spleen and lymph nodes and
Payer's patches have shown that Cip2a is most probably expressed in germinal centers and testis.
Therefore, lymphoid tissue and testis was carefully examined but the analysis revealed no abnormalities.


Wholemount ß galactosidase assay showed LacZ-expression in testis of mutant Cip2a mouse which confirmed the
preliminary observations by the collaboration partner.

The histological analysis with standard stains (H&E) did
not reveal any genotype-specific morphological changes in the Cip2a mouse line.



Discussion:

According to the information from the mouse provider staining of spleen and lymph nodes and
Payer's patches have shown that Cip2a is most probably expressed in germinal centers and testis.
Therefore, lymphoid tissue and testis was carefully examined but revealed no abnormalities. The histological analysis
with standard stains (H&E) did not reveal any genotype-specific morphological changes in the Cip2a mouse
line.

Bodyweight
Zdhhc2Zdhhc2Targeted mutation
Bone & Cartilage

Summary:

In the primary screening no genotype-specific differences were found between mutants and controls. X-ray and
DXA analysis could not be performed due to problems with the anesthesia.




Behaviour

Summary:

There were no clear genotype effects on either open field behaviour or prepulse inhibition responding
other than a tendency to increased acoustic startle reactivity.



Discussion:

In the open field, there were no clear genotype effects. This indicates that locomotor and
exploratory activity as well as anxiety-related behaviour were ostensibly normal in these mice. There were
also no clear differences in prepulse inhibition suggesting that sensorimotor gating ability was intact in
these mice. Acoustic startle reactivity tended to be increased in these mice. This latter effect
was consonant with observations made previously of increased anxiety (made by the collaboration partner). Thus,
it may be worth performing additional anxiety-related behaviour tests such as the elevated plus maze,
light/dark box and the social interaction test. Nevertheless, the results of the open field test
do not indicate that anxiety-related behaviour is markedly affected under unchallenged conditions. Thus, exposing these
mice to acute psychological stress, such as acute restraint stress, may aid in determining whether
these mice exhibit altered stress responsivity when challenged.

Neurology

Summary:

No differences were detected at SHIRPA, grip strength, plasma lactate or rotarod performance. Interestingly, ABR
could not be performed due to increased anesthesia tolerance.



Discussion:

In the primary neurological screen we did not detect any alterations. Although we expected changes
due to alterations in synaptic transmission because of the involvement of Zdhh2c in vesicle cycling,
there were no genotype effects apparent. However, the Zdhhc2 mice could not easily be anesthesized.
Since ketamine/Xylazine (0.1mg/0.01mg per g body weight) was not sufficient to deeply anesthesize the mice,
ABR could not be performed.

Zdhhc2 had been reported to be involved in anchoring
postsynaptic density protein-95 (PSD-95), a scaffolding protein, which has been identified to attach NMDARs and
AMPARs to internal signaling molecules at neuronal synapses of the CNS (Tao and Johns 2014).
Excitatory NMDARs are targets for different anesthetics, - ketamine is a noncompetitive antagonist of NMDARs
-, and involved in chronic inflammatory pain (Tao and Johns 2008; 2010). NMDA receptor knockouts
are resistant to many intravenous and inhalational anesthetics (Petrenko et al., 2014). Thus the anesthesia
tolerance of Zdhhc2 mutants is presumably a direct effect of the mutation. PSD-95 also has
been reported to be downregulated after sevofluorane anesthesia in rats and ketamine in developing mice
(Zhang et al., 2014; Zhao et al., 2014). The observed ketamine tolerance could also be
augmented by earlier anesthesia (ketamin/xylazin in the eye screen of isofluorane for blood withdrawal), where
no abnormalities have been reported.

Eye & Vision

Summary:

We did not identify a genotype-related phenotype in the primary eye screen with the Zdhhc2
mutant line.



Discussion:

Our data indicate no effect of the Zdhhc2 mutation on the eye phenotype. Corneal opacities
of both control and mutant mice could possibly be due to injuries.

Nociception

Summary:

The mutation did not influence the pain sensitivity of these animals.



Discussion:

The mutant animals did not show any significant difference in the thermal pain reaction compared
to the wild type mice.

Energy Metabolism

Summary:

Body mass and body composition were not different between genotypes. However, mutant mice showed slightly
reduced food uptake and a consequent reduction in RER during the indirect calorimetry trial.



Discussion:

No major effects of the mutation on energy metabolism related parameters were detected. The slight
hypophagia was likely due to incompliance with the calorimetry set up. The relevance of this
finding remains unclear so far.

Clinical Chemistry

Summary:

Clinical Chemistry: Our clinical chemical data did not provide clear evidence of genotype-related effects. For
few parameters we found some mild sex-specific differences between genotypes representing subtle findings of unclear
relevance.

Hematology: Results did not provide evidence of genotype-related effects.

IpGTT: We did
not see differences between genotypes except mildly decreased basal fasting glucose values in mutants.



Discussion:

Although we found only subtle differences between Zdhhc2 mutant and control mice, some of the
findings could be associated with findings of other screens and might be a hint towards
a potential efffect of the mutation: The observation of a decreased food intake and RER
during the calorimetry in connection with the observed slightly decreased fasting glucose level in the
IpGTT and slightly increased fasting glycerol, could be hints towards an altered metabolic response to
fasting conditions, which might additionally be triggered by an altered feeding behaviour.

Immunology

Summary:

Beside a slightly decreased frequency of CD8 T cells within the T cell compartment in
mutant females, we did not find statistically significant differences between mutants and controls concerning the
frequencies of main leukocyte subsets (T cells, B cells, NK cells, NKT cells, granulocytes, monocytes)
nor in the frequencies of subsequentially characterized T cell (CD4, CD8, CD25, CD62L, CD44, Ly6C),
B cell (CD21, CD23, IgD, IgM), NK cell (CD11b, CD11c, Ly6C, CD44, CD62L) or monocyte
(Ly6C, CD11c) subpopulations. We found no significant different antibody levels in mutant mice compared to
controls. 



Discussion:

We found a slightly decreased frequency of CD8 T cells referring to the all T
cell compartment in female mutants. This finding might be a finding by chance, as we
do not see further differences within the CD8 T cell compartment, which would hint towards
a T cell specific change.

Allergy

Summary:

We observed a subtle IgE phenotype in the mutant mice, but controls were not in
the normal range of WT mice.

No TEWL phenotype was observed.

 



Discussion:

We found significant differences in the IgE levels in male mice, but the values of
control mice  were not between normal range of  C57BL/6 mice. For this reason the differences
observed probably were not biologically relevant.

Steroid Metabolism

Summary:

Analysis of steroids was not performed for this mouse line.




Cardiovascular

Summary:

By recording ECGs no alterations were found. By echocardiography, only a mild increase in SV
was found in male mutants compared to controls.

No significant genotype effects were found
on the cardiovascular function in this line.



Discussion:

The increased SV observed in male mutants was probably by chance.

Lung Function

Summary:

Analysis of lung function was not performed for this mouse line.




Expression Profiling

Summary:

There were no mice provided for organ withdrawl.




Pathology

Summary:

The Zdhhc2 mutant mice showed no pathological alterations that could be associated to the genotype.


Especially the brain displayed no histomorphologic changes.

 

 



Discussion:

The Zdhhc2 mutant mice showed no pathological alterations that could be associated to the genotype.


Especially the brain displayed no histomorphologic changes.

Bodyweight
Optn / EPD0116_2_A05-tm1aOptnTargeted mutation
Bone & Cartilage

Summary:

In the DXA analysis, we detected a significantly increased fat mass in males.



Discussion:

The  EPD0116 2 A05 mutant mouse line was analyzed in the Dysmorphology, Bone and Cartilage
module of the German Mouse Clinic. In the DXA analysis, we detected a significantly increased
fat mass in males. The sex differences we observed are common in many mouse strains,
and thus are not abnormal (unpublished data).

Behaviour

Summary:

We observed decreased locomotor activity during the last 10 minutes of the open field test
and increased prepulse inhibition responding in the female heterozygous mice.




Neurology

Summary:

We detected in Optn mutants more passive rotations on the rotarod with increased mean latencies.





Eye & Vision

Summary:

We did not find pathologic eye phenotypes.




Nociception

Summary:

We did not find any significant genotype effect on the pain reaction.




Energy Metabolism

Summary:

During the indirect calorimetry test we observed increased food intake and RER.




Clinical Chemistry

Summary:

We detected only sex specific genotype related differences in fasted mice of unclear relevance.




Immunology

Summary:

We observed no significant differences between mutants and controls.




Allergy

Summary:

The Allergy Screen detected no significant differences between mutants and controls.




Steroid Metabolism

Summary:

We did not find differences between mutant and control animals.



Discussion:

We do not recommend a secondary screening.

Cardiovascular

Summary:

The basal cardiovascular functions were investigated in the primary screen by awake echocardiography. We found
significantly increased Interventricular septum width in male mutant mice.




Lung Function

Summary:

The lung function tests were not performed.




Expression Profiling

Summary:

Expression analysis was not performed.




Pathology

Summary:

We detected LacZ staining in brain, cervical lymph nodes, colon and testes. Normalized heart weight
was significantly decreased in mutant males and females.




Bodyweight
Dis3 / HEPD0659_5_E08-tm1bDis3Targeted mutation
Bone & Cartilage

Summary:

Normal morphology and X-ray parameters in HET mice.




Behaviour

Summary:

There were no major genotype effects on prepulse inhibition and open field was not analysed
in these mice.




Neurology

Summary:

No difference at ABR.




Eye & Vision

Summary:

The Dis3 mutant mice show decreased retinal thickness. Lens development was not affected.




Energy Metabolism

Summary:

Body mass was reduced. No obvious effect on metabolic rate but mice were hyperactive.




Clinical Chemistry

Summary:

IpGTT: Slightly elevated basal glucose levels only in males and trend towards higher AUC values
predominantly in male mutants.

Insulin: No clear evidence of genotype-related differences.

Clinical Chemistry:
Slightly increased plasma ALP activity in both, female and male mutants, and decreased urea levels
in male mutants only.

Hematology: Small trends towards lower MCHC, MPV, PDW and P-LCR
in mutants.




Cardiovascular

Summary:

No alterations found by echocardiography.

ECG was performed and only minor changes of unclear
relevance were found. Increased QT dispersion and QTc dis in males.

No major effects
on the cardiovascular system could be detected.




Pathology

Summary:

LacZ expression was detected in brain except cerebellum and in the skin. Weak staining was
found in pancreas and kidney.

No statistically significant differences in absolute and normalized heart
weight between mutant and control mice.

Body weight is statistically significantly decreased in mutant
mice.

Histological examination using light microscopy did not reveal any pathological changes that could
be attributed to the genotype of the mice.



Discussion:

Histological examination using light microscopy did not reveal any pathological changes that could be attributed
to the genotype of the mice.

Bodyweight
Ifitm1_1F4Ifitm1Targeted mutation
Bone & Cartilage

Summary:

No genotype-specific differences were found between mutants and controls.



Discussion:

The Ifitm1_1F4 mutant mouse line was analyzed in the Dysmorphology, Bone and Cartilage module of
the German Mouse Clinic. No genotype-specific differences were found between mutants and controls. The sex
differences we observed are common in many mouse strains, and thus are not abnormal (unpublished
data).

Behaviour

Summary:

No significant genotype effects




Neurology

Summary:

There was a small but signficant reduction in rotarod latencies.



Discussion:

We detected a modest reduction in rotarod latencies in the Ifitm1_iF4 mice. This could hint
towards a motor coordination deficit but since the difference is quite small, this test should
be repeated with another cohort before drawing any conclusions, since the gene information is not
supporting a direct link between Ifitm1 and neurological disorders and our single finding is not
very strong evidence.

Eye & Vision

Summary:
We did not identify ocular irregularities in the primary Eye Screen with Ifitm1_1F4.

Discussion:
The results of the primary Eye Screen clearly demonstrate that eye development is not affected by
the Ifitm1\_1F4 mutation.
Nociception

Summary:

We found no significant difference in pain reactivity between mutant and control animals.



Discussion:

The mutant animals showed no significant difference in pain reaction compared to wild-type control mice.


Energy Metabolism

Summary:

No clear genotype related differences could be detected between wildtype and mutant mice. Oxygen consumption
trended to be higher in mutant females as well as RER. These differences are very
subtle and remain of unclear relevance.




Clinical Chemistry

Summary:

We did not find any significant genotype related differences in the IpGTT or clinical chemistry
parameters. In the hematological analyses female mutant Ifitm1 mice displayed somewhat higher MCV values, an
increased cellular hemoglobin content (MCH) and a trend towards higher cellular hemoglobin concentrations. However, these
differences were small and sex-specific and therefore of unclear relevance.



Discussion:

In our screen Ifitm1 mice showed almost no genotype related differences in comparison to the
corresponding controls. The only finding was a mild increase of MCV, MCH and MCHC values
in female mutant mice. This could be a hint towards sex-specific genotype effects on hematopoiesis.
However, also this difference was small and therefore of unclear relevance.

Immunology

Summary:

We found subtle differences in the frequencies  of granulocytes and CD44 expressing T cells between
mutants (decreased) and controls, and a tendency of higher IgG1 levels in mutants.



Discussion:

We found a slightly increased frequency of granculocytes in control mice mainly in females. The
observation of a slightly lower frequency of granulocytes and CD44 expressing T cells might reflect
differences in leukocyte activation between mutants and controls.

Moreover, we see higher levels of
IgG1 in mutants of both sexes. Although statistical significance is not fullfilled, the finding is
in agreement with the finding from the Allergy screen of higher IgE levels in mutants.
The production of both IgG1 and IgE is controlled by IL-4 (Snapper et al.,
1988).

Allergy

Summary:

Strong phenotype, high IgE levels in male and female mutant mice



Discussion:

The analysis of total IgE levels in plasma (Median ± Stdev) of Ifitm1 mice did
revealed statistically significant differences between mutant female and control mice female, same tendency is observed
with the males (Table 25) Levels of total IgE are frequently higher in females than
in males for many mouse inbred strains (Allessandrini et al., 2001; Corteling et al., 2004; Seymour et al., 2002; Melgert et al., 2005)
such differences were observed in the mutant group.

 
Steroid Metabolism

Summary:

Significant differences in the concentration of testosterone have been found in male mice.



Discussion:

We do not recommend a secondary screening.

Cardiovascular

Summary:

We could not find any genotype specific differences



Discussion:

We do not recommend secondary screening

Lung Function

Summary:

Pulmonary function testing did not reveal any genotype-specific differences between mutants and control mice.




Pathology

Summary:

The macroscopical analysis showed increased liver weights in male mutants.



Discussion:

We found no pathological alterations that could be attribuited to the genotype .

secondary
screen is not recomended

Bodyweight
Setdb1 / EUC BL6-FP00042B07-tm1aSetdb1Targeted mutation
Bone & Cartilage

Summary:

In the primary Dysmorphology, Bone and Cartilage Screen no genotype-specific differences were observed between mutant
mice and controls.




Behaviour

Summary:

We observed a deficit in prepulse inhibition for the mutant mice of both sexes and
sex-specific hyperactivity (females) in the Open Field.




Neurology

Summary:

In the Neurology Screen we did not observe any genotype related differences.




Eye & Vision

Summary:

We did not identify ocular irregularities in the Eye Screen.




Nociception

Summary:

We did not find significant difference in pain reactivity between the mutant and control  animals.




Energy Metabolism

Summary:

The Energy Metabolism Screen detected no significant differences between mutants and controls.




Clinical Chemistry

Summary:

There were no genotype effects on either clinical chemistry or hematology parameters.




Immunology

Summary:

The Immunology Screen detected no significant differences between mutants and controls.




Allergy

Summary:

We measured  low levels of IgE for the mutant mice compared to controls.




Steroid Metabolism

Summary:

The analysis of steroid hormones in plasma samples did not reveal differences between mutant and
control animals.




Cardiovascular

Summary:

We found some sex-specific findings: Male mice showed Tachycardia and increased heart weights.




Lung Function

Summary:

The lung function of the mutant mice was slighty different, but of minor physiological relevance.





Expression Profiling

Summary:

Expression analysis was not performed.




Pathology

Summary:

Differences in the expression of ß galactosidase activity between mutant and control mice were observed
in brain and liver.




Bodyweight
Hmgn3Hmgn3Targeted mutation
Bone & Cartilage

Summary:

No genotype-specific differences were found between mutants and controls.



Discussion:

The Hmgn3 mutant mouse line was analyzed in the Dysmorphology, Bone and Cartilage module of
the German Mouse Clinic. No genotype-specific differences were found between mutants and controls. The sex
differences we observed are common in many mouse strains, and thus are not abnormal (unpublished
data).

Behaviour

Summary:

Decreased prepulse inhibition in the male mutant mice.




Neurology

Summary:

In the primary neurological screening Hmgn3 mutants showed reduced grip strength in both sexes,  more
pronounced in forelimbs. This reduction is not associated with a (not evident) reduction of body
mass.




Eye & Vision

Summary:

We did not find ocular irregularities in the Primary Eye Screen with Hmgn3.




Nociception

Summary:

The mutant animals showed hypoalgesia.



Discussion:

The mutant animals showed hypoalgesia.

Energy Metabolism

Summary:

No clear metabolic phenotype could be detected in Hmgn3 mice. There was an almost significantly
increased body fat content and maximum oxygen consumption rates were increased in mutants. However, differences
were small and remain of unclear relevance so far.




Clinical Chemistry

Summary:

We were not able to confirm the previously published phenotype of mild diabetes in Hmgn3
knockout mice. However, the glucose tolerance test showed a trend towards this direction in males.
Additionally we saw decreased NEFA and glycerol values predominantly in female mutant mice after overnight
fasting. This could indicate a genotype related effect on fasting induced lipolysis. No obviously genotype
related differences were detected in clinical chemistry parameters or hematological values measured in ad libitum
fed mice.




Immunology

Summary:

No phenotype, concerning leukocyte proportions in peripheral blood, nor plasma immunoglobulin levels.




Allergy

Summary:

No IgE phenotype




Steroid Metabolism

Summary:

Significant differences in the concentration of corticosterone have been found in female mice and in
male mice we found significant differences in the concentration of testosterone.



Discussion:

We offer to analyse plasma of the mice in the Metabolomic Platform of the GAC.
To check the affected metabolic pathways, we suggest to quantify amino acids, acylcarnitines, glyceropho-spholipids, sphingolipids
and hexose (Biocrates AbsoluteIDQ Kit) and additional steroids using an enhanced steroid panel (Biocrates SteroIDQ
Kit). The steroid assay request higher volumes of mouse plasma. We expect differences in the
lipidome and the sterome of the mice.

These analyses are not sponsored by NGFN
and request cost coverage by mouse provider.

For further information please contact:

           
Cornelia Prehn (+49-89-3187-3231) or

            Julia Scarpa (team assistance, 3722)

            www.gac-munich.de

Cardiovascular

Summary:

We could not find any genotype-specific differences.



Discussion:

We do not recommend secondary screening

Lung Function

Summary:

Pulmonary function testing did not reveal any genotype-specific differences between mutants and control mice.




Expression Profiling

Summary:

Brain and liver were chosen for analysis of 17 weeks old mice. Experiments were performed
for four control and four mutant animals of each genotype (in total 16 hybridizations). Data
analysis using various statistical methods detected several significant differences in gene expression levels in brain
and liver of Hmgn3 mutant mice compared to the controls. Regulated genes in brain are
associated with distinct nervous system functions and diseases. Among the differentially expressed genes in liver
associations with tumorigenesis were found beside distinct liver specific functions.




Pathology

Summary:

No pathological phenotype detected that could be attributed to the ganotype of the animals.



Discussion:

The pathology screen did not find histological changes that could be attributed to the genotype
of the mutant mice.

Bodyweight
Hmgn5Hmgn5Targeted mutation
Bone & Cartilage

Summary:

No genotype-specific differences were found between mutants and controls.



Discussion:

The Hmgn5 mutant mouse line was analyzed in the Dysmorphology, Bone and Cartilage module of
the German Mouse Clinic. No genotype-specific differences were found between mutants and controls. The sex
differences we observed are common in many mouse strains, and thus are not abnormal (unpublished
data).

Behaviour

Summary:

We observed decreased prepulse inhibition in the male mutant mice.




Neurology

Summary:

We did not find genotype-related differences in the primary neurological screening of Hmgn5 mutants. Basic
neurological functions were not affected by the mutation.




Eye & Vision

Summary:

We did not find ocular irregularities in the Primary Eye Screen with Hmgn5.




Nociception

Summary:

We did not find any genotype effect on pain reaction.



Discussion:

We found no significant difference in pain reactivity between mutant and control animals.

Energy Metabolism

Summary:

No metabolic phenotype could be detected in Hmgn5 mice. Mutant and control mice did not
differ in body mass or body composition. Decreased rearing in female mutants was the only
significant difference detected during the indirect calorimetry trial. This difference remains of unclear relevance so
far.




Clinical Chemistry

Summary:

Results of the IpGTT carried out in Hmgn5 mutant mice did not provide evidence of
genotype related effects on glucose metabolism. In overnight fasted mice, we found significantly higher triglyceride
values in mutant mice, suggesting that fat metabolism under challenge conditions might be affected by
the Hmgn5 knockout. In ad libitum fed mice plasma amylase activities were significantly reduced in
mice, which can be caused by alterations of the function of several organs: pancreas, salivary
gland or liver - as the amylase producing organs - or kidney function, since amylase
is partly cleared from blood by the kidneys. Mild increases in albumin, sodium and chloride
levels hint towards alterations of water balance resulting in mild hemoconcentration. Additionally we saw mild
microcytosis in Hmgn5-deficient animals with reduced cellular hemoglobin concentration in mutant animals, and a proportion
of mutant females with decreased platelet counts and a slightly increased mean platelet volume in
females knockout mice.



Discussion:

Our findings suggest that glucose metabolism, liver function and hematopoiesis might be affected by HMGN5
deficiency.

Immunology

Summary:

Our tests revealed a significant higher frequency of T cells, especially CD8+ T cells, and
a lower frequency of granulocytes.

We further found slightly higher levels of immunoglobulin A
in mutants.




Allergy

Summary:

All values of IgE are in the normal range of control mice, the low levels
of  females mutant mice and males, too.




Steroid Metabolism

Summary:

We did not find differences between mutant and control animals.




Expression Profiling

Summary:

Brain and liver were chosen for analysis of 17 weeks old mice. Experiments were performed
for four control and four mutant animals of each genotype (in total 16 hybridizations). Data
analysis using various statistical methods detected several significant differences in gene expression levels in brain
and liver of Hmgn5 mutant mice compared to the controls. Regulated genes in brain are
associated with neurodegeneration and cerebral angiogenesis. Among the differentially expressed genes in liver associations with
tumorigenesis, apoptosis, glucose metabolism and MAPK signaling pathway were found.




Pathology

Summary:

We did not find any genotype associated changes in the primary screening of Hmgn5 mutant
mice.



Discussion:

The pathology screen did not find histological changes that could be attributed to the genotype
of the mutant mice.

Bodyweight
Hmgn1Hmgn1Targeted mutation
Bone & Cartilage

Summary:

No genotype-specific differences were found between mutants and controls.



Discussion:

The Hmgn1 mutant mouse line was analyzed in the Dysmorphology, Bone and Cartilage module of
the German Mouse Clinic. No genotype-specific differences were found between mutants and controls. The sex
differences we observed are common in many mouse strains, and thus are not abnormal (unpublished
data).

Behaviour

Summary:

Decreased rearing activity by the mutant mice in the open field. Female mutant mice had
lower acoustic startle reactivity. No genotype effect on the behavioural response to acute psychological stress.





Neurology

Summary:

We did not find significant differences in HMGN1 mutants. Basic neurological functions appear to be
not affected by the mutation.




Eye & Vision

Summary:

We did not find ocular irregularities in the Primary Eye Screen with Hmgn1.




Nociception

Summary:

No significant genotype effect on the pain reaction.



Discussion:

We did not found any significant differences in pain reactivity between the wild type and
knockout animals.

Energy Metabolism

Summary:

No genotype related differences were found on body mass and body composition. No effects could
be detected on energy metabolism variables monitored during the indirect calorimetry trial.




Clinical Chemistry

Summary:

There were no significant genotype related differences detected for parameters analyzed by the clinical chemistry
screen, with the exception of a significant decrease in calculated non-HDL-cholesterol  values in mutant male
mice after overnight fasting. However, there were no significant differences detected in the directly measured
parameters total cholesterol and HDL-cholesterol, and no other hints on effects on energy or fat
metabolism were seen even in other screens. Therefore this difference is judged as a finding
without relevance.




Immunology

Summary:

We found a slightly lower CD4:CD8 ratio in mutant mice.




Allergy

Summary:

no IgE phenotype




Steroid Metabolism

Summary:

Significant differences in the concentration of corticosterone have been found in female mice.



Discussion:

We do not recommend a secondary screening.

Cardiovascular

Summary:

We could not find any genotype specific differences



Discussion:

We do not recommend secondary screening

Lung Function

Summary:

Pulmonary function testing did not show any genotype specific differences.




Expression Profiling

Summary:

Brain and liver were chosen for analysis of 17 weeks old mice. Experiments were performed
for four control and four mutant animals of each genotype (in total 16 hybridizations). Data
analysis using various statistical methods detected several significant differences in gene expression levels in brain
and liver of Hmgn1 mutant mice compared to the controls. Regulated genes in brain are
associated with neuroprotection, Notch signaling pathway, cancer and Down syndrome. Among the differentially expressed genes
in liver associations with tumorigenesis were prominent beside distinct liver specific functions.

 




Pathology

Summary:

No genotype associated histological findings.




Bodyweight
Hdac1 / EPD0028_5_G01-tm1aHdac1
Bone & Cartilage

Summary:

In the primary Dysmorphology, Bone and Cartilage Screen no genotype-specific differences were observed between mutant
mice and controls.




Behaviour

Summary:

We observed no significant genotype effects on either the open field behaviour or prepulse inhibition
responding.




Neurology

Summary:

In the primary neurological screening we detected more excitation/alertness (tail elevation). All other tests were
without genotype-specific differences.




Eye & Vision

Summary:

We did not identify ocular irregularities in the primary Eye Screen.




Nociception

Summary:

We did not find significant differences in pain reactivity between the mutant and control animals.




Energy Metabolism

Summary:

Especially female mutants were hypermetabolic and trended to have reduced fat mass.




Clinical Chemistry

Summary:

In the Clinical Chemistry Screen we observed subtle changes of unclear relevance.




Immunology

Summary:

We detected lower levels of IgM for both sexes.




Allergy

Summary:

In the Allergy Screen we did not observe any genotype related differences.




Steroid Metabolism

Summary:

The analysis of steroid hormones in plasma samples did not reveal differences between mutant and
control animals.




Cardiovascular

Summary:

We measured changes in parameters relevant for Bradycardia.




Lung Function

Summary:

The lung tests revealed differences of minor physiological relevance, specific TV and specific MV are
comparable.




Expression Profiling

Summary:

Expression analysis was not performed.




Pathology

Summary:

Differences in the expression of ß galactosidase activity between mutant and control mice were observed
in brain, testes and ovary.




Bodyweight
Ino80etm1a KOIno80eTargeted mutation
Bone & Cartilage

Summary:

In the DXA analysis, we detected a significantly increased BMD in females compared to controls.
Concurrently fat mass was significantly increased in males, and body weight was increased by trend.
In the Clickbox test (test for hearing ability of mice), we observed a decreased hearing
reaction in female mutants. As no differences in hearing ability were detected in the Behavior
and Neurology Screen, the biological relevance is unclear.




Behaviour

Summary:

We observed tendency to decreased locomotor activity in the female mutant mice in the open
field with the opposite effect in the males and no significant genotype effect on prepulse
inhibition.




Neurology

Summary:

We observed a tendency towards decreased locomotor activity and additionally we found reduced plasma lactate
levels.




Eye & Vision

Summary:

We did not identify ocular irregularities in the primary Eye Screen.




Nociception

Summary:

We did not find any significant genotype effect on the pain reaction.




Energy Metabolism

Summary:

Mutant mice showed increased body mass, increased body fat content and trended to be hypometabolic.





Clinical Chemistry

Summary:

Mutant animals were slightly overweight and lost less body mass due to overnight food withdrawal
than corresponding controls. Genotype-related differences in clinical-chemical parameters were mainly seen in males in blood
lipid and protein concentrations. In both sexes, mutant animals had slightly decreased creatinine concentrations compared
to controls. These results hint towards effects on energy metabolism, especially muscle energy metabolism and
fat metabolism.




Immunology

Summary:

The Immunology Screen detected no significant differences between mutants and controls.




Allergy

Summary:

The Allergy Screen detected no significant differences between mutants and controls.




Steroid Metabolism

Summary:

The analysis of steroid hormones in plasma samples of Ino80etm1a-mice did not reveal differences between
mutant and control animals.



Discussion:

No genotype-specific differences in corticosterone, androstenedione and testosterone concentrations in blood plasma were found. Therefore,
the Ino80etm1a-mutation does not seem to affect steroid metabolism. A secondary screening is not recommended.


Cardiovascular

Summary:

In the blood pressure analysis no genotype specific differences could be obtained.




Lung Function

Summary:

We found lower respiratory rates in mutant males at rest, abolished at activity; lower respiratory
rates in female mutants at activity (4% lower), differences are physiologically not relevant.




Expression Profiling

Summary:

Expression analysis was not performed.




Pathology

Summary:

We detected LacZ staining in brain, salivary gland, kidney, uterus and testis.




Bodyweight
Spryd3 / HEPD0539_9_C10-tm1aSpryd3
Bone & Cartilage

Summary:

No radiological abnormalities were observed. In the DXA analysis, we detected a significantly decreased body
length, body weight, and lean mass in males compared to controls.




Behaviour

Summary:

In the open field we observed decreased locomotor activity by the female mutant mice with
the opposite effect in the males and decreased rearing activity by the female mutant mice
and increased centre time by the mutant mice. Additionally the prepulse inhibition response was decreased
in the mutant mice, significant at the 69 dB intensity.




Neurology

Summary:

Mutants showed increased locomotor activity at SHIRPA. No difference at grip strength or rotarod performance.





Eye & Vision

Summary:

We observed reduced eye size in the mutant mice compared to controls.




Nociception

Summary:

We did not find any significant genotype effect on the pain reaction.




Energy Metabolism

Summary:

Male mutants were significantly underweight confounding both body composition analysis and indirect calorimetry results.




Clinical Chemistry

Summary:

Several genotype-related differences in plasma enzyme activities hint towards possible effects of the mutation on
liver and/or muscle metabolism.




Immunology

Summary:

We observed no significant differences between mutants and controls.




Allergy

Summary:

The Allergy Screen detected no significant differences between mutants and controls.




Steroid Metabolism

Summary:

The analysis of steroid hormones in plasma samples of Spryd3-mice did not reveal differences between
mutant and control animals.



Discussion:

No genotype-specific differences in corticosterone, androstenedione and testosterone concentrations in blood plasma were found. Therefore,
the Spryd3-mutation does not seem to affect steroid metabolism. A secondary screening is not recommended.


Cardiovascular

Summary:

In the blood pressure analysis no genotype specific differences could be obtained.




Lung Function

Summary:

The lung function tests were not performed.




Expression Profiling

Summary:

Expression analysis was not performed.




Pathology

Summary:

Heart weight normalized to tibia length was decreased in mutant males and normalized to body
weight it was also decreased in mutant females. Normalized spleen weights were decreased in females
no matter if normalized to tibia length or to body weight. In males, spleen weight
was significantly decreased if normalized to tibia length and liver weights were decreased no matter
if normalized to tibia length or to body weight.

LacZ expression was observed in
brain, atria, pericardial vessels, pancreas, uterus, ovary, oviducts, bladder, testis, kidney, lung and stomach.




Bodyweight
SetmarSetmarTargeted mutation
Bone & Cartilage

Summary:

No genotype-specific differences were found between mutants and controls.




Behaviour

Summary:

We observed a clear increase in PPI in the mutant mice and no significant genotype
effects on open field behaviour.




Neurology

Summary:

We detected no genotype effects during SHIRPA, on grip strength or rotarod performance. There was
a subtle decrease in lactate levels of male mutants.




Eye & Vision

Summary:

We did not identify ocular irregularities in the primary Eye Screen.




Nociception

Summary:

We did not find any significant genotype effect on the pain reaction.




Energy Metabolism

Summary:

No clear metabolic phenotype could be detected.




Clinical Chemistry

Summary:

Mutant animals showed significantly decreased plasma glycerol levels after overnight food-withdrawal and increased ALP activities
in plasma of ad libitum fed mice.




Immunology

Summary:

We found slightly lower frequency of CD4+ T cells together with lower frequency of CD62L
expressing cells. No relevant differences in the immunoglobulin levels in blood plasma were found (but
small number of samples).



Discussion:

Slightly lower frequency of CD4+ T cells together with lower frequency of CD62L expressing cells.
The correlation of these frequencies is an oftenly seen phenomenom; and is a phenotype linke
to apoptosis, occuring during sample preparation. Red cell lysis induces L-selectin shedding in pre-apoptotic T
cells, especially CD4+ T cells. There is evidence that the occurrence of these effects is
genetically regulated. However, the relevance of this phenotype within one cohort has to be interpreted
cautiously.

Allergy

Summary:

In the Allergy Screen we detected no genotype related differences between mutants and controls.




Steroid Metabolism

Summary:

We did not find differences between mutant and control animals.



Discussion:

We do not recommend a secondary screening.

Cardiovascular

Summary:

Echocardiography did not reveal any genotype-specific differences between mutant and control mice.




Lung Function

Summary:

The lung function tests were not performed.




Expression Profiling

Summary:

Expression analysis was not performed.




Pathology

Summary:

In the mutant animals we detected LacZ staining in brain and kidney.




Bodyweight
Hprt1 / HEPD0543_8_G03-tm1aHprtTargeted mutation
Bone & Cartilage

Summary:

No genotype-specific differences were found between mutants and controls.




Behaviour

Summary:

We observed increased rearing activity by the mutant mice in the open field.




Neurology

Summary:

We detected decreased locomotor activity during the modified SHIRPA analysis but no differences in grip
strength or rotarod performance.




Eye & Vision

Summary:

We did not identify ocular irregularities in the primary Eye Screen.




Nociception

Summary:

Nociception was not analysed.




Energy Metabolism

Summary:

We detected increased body fat content in male mutants. Indirect calorimetry was not performed.




Clinical Chemistry

Summary:

Mutant mice showed pronounced macrocytic and erythropenic anemia with increased anisocytosis, as well as subtle
genotype-related differences in IpGTT results and clinical chemistry values, suggesting mild possibly secondary effects on
energy metabolism and water or electrolyte balance.




Immunology

Summary:

We found lower CD4/CD8 ratio and lower T cell frequency together with a lower  freq.
of CD62L expressing T cells in the mutant mice compared to controls.



Discussion:

The observed combination of changes is a typically seen phenotype:  The lower CD4/CD8 ratio, together
with the lower frequency of CD62L expressing cells within the T cell cluster hinds towards
a apoptosis dependent change, as CD4+ T cells are more susceptible to preparation-induced cell death,
and L-selectin loss occurs can be considered as preapoptotic marker. The phenotype thus might have
been induced by erythrocyte lysis during preparation.

Allergy

Summary:

The Allergy Screen detected no significant differences between mutants and controls.




Steroid Metabolism

Summary:

The analysis of steroid hormones in plasma samples of Hprt1-mice did not reveal differences between
mutant and control animals.



Discussion:

No genotype-specific differences in corticosterone, androstenedione and testosterone concentrations in blood plasma were found. Therefore,
the Hprt1-mutation does not seem to affect steroid metabolism. A secondary screening is not recommended.


Cardiovascular

Summary:

The cardiovascular tests were not performed.




Lung Function

Summary:

The lung tests were not performed.




Expression Profiling

Summary:

Expression analysis was not performed.




Pathology

Summary:

We detected LacZ staining in brain, atria, stomach, colon, brain, liver, kidney, ovary and
uterus.




Bodyweight
Mtmr4 / EPD0059_2_D02-tm1aMtmr4Targeted mutation
Bone & Cartilage

Summary:

In the primary Dysmorphology, Bone and Cartilage Screen no genotype-specific differences were observed betwen mutant
mice and controls.




Behaviour

Summary:

In the open field the mutant mice showed an increased locomotor activity and no significant
genotype effects on prepulse inhibition responding were observed.




Neurology

Summary:

In male mutant mice the lactate level was reduced. We observed no differences at SHIRPA,
grip strength or rotarod.




Eye & Vision

Summary:

We did not identify ocular irregularities in the primary Eye Screen.




Nociception

Summary:

We did not find significant difference in pain reactivity between the mutant and control animals.




Energy Metabolism

Summary:

For male mutant mice we observed decreased minimum VO2 and increased RER in the Indirect
calorimetry and no clear shift in body composition (Minispec NMR).




Clinical Chemistry

Summary:

The Clinical Chemical Screen observed several changes in the mutant mice, that hint to the
glucose and energy metabolism.

Fasted mice: Glucose was decreased in mutants of both sexes
(genotype ANOVA p=0.008).

Fed mice: Urea was slightly increased (genotype ANOVA p=0.048), Glucose decreased
in males and increased in females (genotype:sex ANOVA p=0.023) and AP increased (genotype ANOVA p=0.017).


IpGTT: We found increased AUC values in female mutants (AUC 0-30 and AUC 30-120,
p=0.01).

The hematological tests revealed small differences in MCH and MCV of unclear relevance.





Immunology

Summary:

The Bioplex assay revealed higher IgA levels in the male mutant mice.




Allergy

Summary:

In the Allergy Screen we did not observe any genotype related differences.




Steroid Metabolism

Summary:

The analysis of steroid hormones in plasma samples did not reveal differences between mutant and
control animals.




Cardiovascular

Summary:

In the Cardiovascular Screen we detected lower pulse rate, that  might be physiologically relevant. In
addition the normalized heart weight was increased in mutant females and males.




Lung Function

Summary:

We observed slightly higher respiratory rates in mutant females at activity, but we think there
is no physiological relevance.




Expression Profiling

Summary:

Expression analysis was not performed.




Pathology

Summary:

Differences in the expression of ß galactosidase activity between mutant and control mice were observed
in ovary, tube, uterus, skin, liver, brain.




Bodyweight
Aldh2 / EPD0089_4_F11-tm1aAldh2Targeted mutation
Bone & Cartilage

Summary:

In the primary Dysmorphology, Bone and Cartilage Screen no genotype-specific differences were observed between mutant
mice and controls.




Behaviour

Summary:

We observed sex-specific alterations regarding activity in the open field test.




Neurology

Summary:

Aldh2 mutants did not show abnormalities in our primary neurological screen.




Eye & Vision

Summary:

We did not identify ocular irregularities in the primary Eye Screen.




Nociception

Summary:

We did not find significant differences in pain reactivity between the mutant and control animals.




Energy Metabolism

Summary:

The Energy Metabolism Screen was not performed.




Clinical Chemistry

Summary:

We observed decreased triglyceride levels and mild macrocytosis in the mutant animals.




Immunology

Summary:

The Immunology Screen detected no significant differences between mutants and controls.




Allergy

Summary:

In the Allergy Screen we did not observe any genotype related differences.




Steroid Metabolism

Summary:

The male mutant mice had slightly decreased DHEA concentrations.




Cardiovascular

Summary:

We observed an increased heart weight as well as signs of Tachycardia.




Lung Function

Summary:

The lung function tests of the mutant mice revealed no genotype-related differences.




Expression Profiling

Summary:

Expression analysis was not performed.




Pathology

Summary:

We observed ß galactosidase activity in the following organs of mutant mice: brain, heart, liver,
lung, spleen, kidney, thyroid, skin, testes and ovaries. Additionally we found 2 of 4 mutant
males with mild testicular tubular degeneration.




Bodyweight
Nfya / EUCJ004_F10-tm1aNfyaTargeted mutation
Bone & Cartilage

Summary:

In the primary Dysmorphology, Bone and Cartilage Screen we observed a smaller head, decreased body
length, body weight, and fat mass in male mutants.




Behaviour

Summary:

There were no genotype effects on either open field behaviour or prepulse inhibition responding.




Neurology

Summary:

In the primary neurological screening we detected hind limb shaking upon tail suspension and reduced
grip strength.




Eye & Vision

Summary:

There was a significant difference of absolute axial eye lengths.




Nociception

Summary:

We did not find significant difference in pain reactivity between the mutant and control animals.




Energy Metabolism

Summary:

We observed reduced body mass and fat mass in male mutant mice.




Clinical Chemistry

Summary:

We detected small effects on potassium, phosphorus, creatinine, glucose and iron.




Immunology

Summary:

We observed the following sex-dependent phenotype: Only in female mutants we observed higher freq. of
T cells and an inverse trend in B cells and granulocytes. T cells showed a
higher expression of CD62L and a lower expression of Ly6C (CD8+), corresponding to a more
‘naive’ phenotype.




Allergy

Summary:

In the Allergy Screen we did not observe any genotype related differences.




Steroid Metabolism

Summary:

The analysis of steroid hormones in plasma samples did not reveal differences between mutant and
control animals.




Cardiovascular

Summary:

We obseved decreased pulse rate, mainly in male mutant mice, slightly decreased heart weight to
tibia length in combination with decreased body weight and increased tibia length  (not significant trend
to decreased heart weigth). These findings are probably secondary to body weight differences.




Lung Function

Summary:

The lung function tests of the mutant mice revealed no genotype-related differences.




Expression Profiling

Summary:

Expression analysis was not performed.




Pathology

Summary:

Nfya mutants did not show abnormalities in our Pathology Screen.




Bodyweight