Gene 302
Summary of Phenotypic Analysis

Changes related to genotype:

·  Embryonic Lethality: Homozygous mutant embryos possibly die at E10.5.

·  Lethality: Homozygous mice were not identified at weaning.

·  Behavior: Heterozygous mutants displayed significantly decreased levels of prepulse inhibition (PPI), during startle testing, when compared with age- and gender-matched wild-type control mice.

Weaned progeny from the heterozygous matings were genotyped. No homozygous mutant mice were identified by PCR, whereas wild-type and heterozygous mutant mice were present. Examination of embryonic development identified grossly normal homozygous mutant embryos at E8.5 detected by PCR.  However, Homozygous offspring detected at E10.5 are partially resorbed suggesting that homozygous mutant embryos die at ~ E10.5.

ES cells derived from the 129/OlaHsd  mouse substrain were used to generate chimeric mice. F1 mice were generated by breeding with C57BL/6 females. The resultant F1N0 heterozygotes were backcrossed to C57BL/6 mice to generate F1N1 heterozygotes.  F2N1 heterozygous mutant mice were produced by intercrossing F1N1 heterozygous males and females.

Wild-type control mice and heterozygous mutant mice were evaluated by the following examinations or tests:

• Physical examination
• Necropsy, including body length, body weight, and organ weight measurements
• Histological examination of tissues and organs
• Bone marrow section evaluation.
• Complete blood counts and differential cell counts.
• Clinical chemistry panels .
• Densitometry.
• Behavioral tests.
• Aging studies.

Gene 302
Expression Summary

Taqman Summary:
The highest levels of RNA transcripts are detectable in spleen and lymph nodes. 

Moderate levels of RNA transcripts are detectable in lung, thymus, bone marrow, gallbladder, pituitary gland, uterus and white fat. 

Lower levels of RNA transcripts are also detectable in brain, cortex, subcortical region, cerebellum, brainstem, olfactory bulb, spinal cord, Harderian glands, heart, liver, kidney, skin, urinary bladder, adrenal gland, salivary gland, skeletal muscle, tongue, stomach, small intestine, large intestine, cecum, testis, epididymis, coagulating gland, prostate gland and ovaries.

No RNA transcripts are detectable in eye, pancreas and seminal vesicle.

LacZ Summary:
Strong lacZ expression was detected in several spermatogenic cells in the seminiferous tubules of testis.

LacZ expression was detected in testis.

LacZ expression was not detected in:  brain, spinal cord, sciatic nerve, eyes, thymus, spleen, lymph nodes, bone marrow, aorta, heart, lung, liver, gallbladder, pancreas, kidney, urinary bladder, thyroid gland, pituitary gland, adrenal glands, skeletal muscle, skin, prostate gland, ovary, uterus cervix and vagina.

Gene 302
Necropsy 

There were no significant differences detected in the heterozygous mutant mice when compared with age- and gender-matched wild-type control mice.

The following mice were necropsied. Body weight, body length, and organ weights were obtained, and gross pathological findings were recorded. The data are compiled from the N0F2 and N1F2 generations.

49 Day Cohort Mouse ID numbers are as follows:
3 heterozygous mutant females (228661, 228662, 228663)
3 heterozygous mutant males (228657, 228658, 228659)
2 wild-type control females (238194, 238201)
2 wild-type control males (228656, 228660)

300 Day Cohort Mouse ID numbers are as follows:
2 heterozygous mutant females (239869, 239870)
2 heterozygous mutant males (239865, 239867)

Mice were examined for the following observables: adrenal glands, body length, body weight, bone marrow, bone - cranium, bone - femur, bone - sternum, bone - stifle joint, bone - vertebral column, brain, cecum, colon, duodenum, epididymis - seminal vesicle, esophagus, eyes, gallbladder, general appearance, Harderian glands, heart, heart weight, ileum, jejunum, kidney weight, kidneys, liver, liver weight, lungs, lymph nodes, mesentery, ovaries, pancreas, penis, salivary glands, sciatic nerve, scrotum, skeletal muscle, skin, skinned mouse, spleen, spleen weight, stomach, testes, testes - epididymis weight, thymus, thymus weight, tongue, trachea, urinary bladder, urine, uterus, and vagina. (Gender-specific observables apply to the appropriate gender.)

Necropsy Findings:

There were no genotype-related or biologically significant differences noted between mutant and wild-type control mice for any of the parameters evaluated at necropsy. Incidental lesions may have been present in some tissues. For example, a 300 day heterozygous mutant male (239865) had a mesenteric nodule and two epididymal fat nodules for which there were no histopatholgical correlates. These findings were considered to represent background lesions occasionally seen in this strain of mice, lesions due to spontaneous disease, age-related lesions, lesions due to procedural artifacts, and/or lesions of a nonspecific etiology. They were not considered to be related to genotype.

Body and Organ Weight Findings:

Differences in body length, body weight, organ weights, and/or organ weight to body weight ratios were present between individual mice. For example, a 300 day heterozygous mutant male (239865) had a high body weight, that correlated with high total tissue mass at densitometry, and several high organ weights (but not organ weight/body weight ratios). The variability between mice usually fell within our historical reference ranges and was not correlated with genotype or were thought to be incidental findings.

Gene 302
Histopathology

There were no significant differences detected in the heterozygous mutant mice when compared with age- and gender-matched wild-type control mice.

Tissues from the following mice were evaluated histologically.

49 Day Cohort Mouse ID numbers are as follows:
3 heterozygous mutant females (228661, 228662, 228663)
3 heterozygous mutant males (228657, 228658, 228659)
2 wild-type control females (238194, 238201)
2 wild-type control males (228656, 228660)

300 Day Cohort Mouse ID numbers are as follows:
1 heterozygous mutant female (239870)
1 heterozygous mutant male (239867)

No Significant Abnormalities:

The following tissues were examined and considered to have no genotype-related abnormality: brain, pituitary gland, ears, nasal cavity, salivary glands, oral cavity, lymph nodes, aorta, lungs, liver, gallbladder, pancreas, spleen, kidneys, urinary bladder, stomach, small and large intestines, larynx, esophagus, trachea, thyroid gland, thymus gland, tongue, skeletal muscle, sciatic nerve, mammary glands, vertebrae, spinal cord, bone (skull, sternum, femur, tibia, and stifle joint), reproductive tract (including gonads), eyes, Harderian glands, integumentary system (skin and either clitoral or preputial glands), and bone marrow.

Bone marrow was examined in sections of sternum, vertebrae, and/or femur and tibia. Marrow cellularity, myeloid:erythroid (M:E) ratio, myeloid and erythroid maturation sequences, and numbers of megakaryocytes were evaluated.

Incidental lesions were present in some tissues. These findings were considered to represent background lesions occasionally seen in this strain of mice, lesions due to spontaneous disease, age-related lesions, lesions due to procedural artifacts, and/or lesions of a nonspecific etiology. They were not considered to be genotype related.

Gene 302
Hematology

There were no significant differences in the heterozygous mutant animals when compared with age- and gender-matched wild-type control mice.

Blood samples from the following mice were evaluated by a complete blood count and differential cell count.

49 Day Cohort Mouse ID numbers are as follows:
3 heterozygous mutant females (228663, 238198, 245204)
3 heterozygous mutant males (228657, 228658, 228659)
2 wild-type control females (260431, 263829)
2 wild-type control males (228660, 234797)

90 Day Cohort Mouse ID numbers are as follows:
4 heterozygous mutant females (239869, 239870, 240228, 240230)
4 heterozygous mutant males (239865, 239867, 245201, 245202)
4 wild-type control females (239868, 240227, 240229, 282522)
4 wild-type control males (240223, 240224, 240225, 240226)

180 Day Cohort Mouse ID numbers are as follows:
4 heterozygous mutant females (239869, 239870, 240228, 240230)
4 heterozygous mutant males (239865, 239867, 245201, 245202)
4 wild-type control females (239868, 240227, 240229, 282522)
4 wild-type control males (240223, 240224, 240225, 240226)

300 Day Cohort Mouse ID numbers are as follows:
2 heterozygous mutant females (239869, 239870)
2 heterozygous mutant males (239865, 239867)
2 wild-type control females (239868, 240227)
2 wild-type control males (240223, 240224)

Although minor variations of hematological values were present in some animals, these changes were not related to genotype and, thus, were not considered phenotypically relevant.

Gene 302
Clinical Chemistry

There were no significant differences in the heterozygous mutant animals when compared with age- and gender-matched wild-type control mice.

Serum samples from the following mice were evaluated by a clinical chemistry panel. The data are compiled from the N0F2 and N1F2 generations.

49 Day Cohort Mouse ID numbers are as follows:
3 heterozygous mutant females (228663, 238193, 245204)
3 heterozygous mutant males (228657, 228658, 228659)
2 wild-type control females (238194, 245203)
2 wild-type control males (234794, 234798)

90 Day Cohort Mouse ID numbers are as follows:
3 heterozygous mutant females (239870, 240228, 240230)
4 heterozygous mutant males (239865, 239867, 245201, 245202)
3 wild-type control females (240227, 240229, 282522)
4 wild-type control males (240223, 240224, 240225, 240226)

180 Day Cohort Mouse ID numbers are as follows:
4 heterozygous mutant females (239869, 239870, 240228, 240230)
2 heterozygous mutant males (239865, 239867)
3 wild-type control females (240227, 240229, 282522)
4 wild-type control males (240223, 240224, 240225, 240226)

300 Day Cohort Mouse ID numbers are as follows:
2 heterozygous mutant females (239869, 239870)
2 heterozygous mutant males (239865, 239867)
2 wild-type control females (239868, 240227)
2 wild-type control males (240223, 240224)

When compared to age- and gender-matched wild-type control mice, two heterozygous mutant males (239865, 239867) and one of two heterozygous mutant females (239870) had increased levels of cholesterol and high density lipoprotein. We have not reported these findings as phenotypic changes, but we have presented them here for your consideration.

Values for the other various analytes evaluated were generally similar between heterozygous mutant and wild-type control mice. Although variations in clinical chemistry values were present in some mice, they were not related to genotype and, thus, were not considered phenotypically relevant.

Gene 302
Densitometry
 
There were no significant differences detected in the heterozygous mutant mice when compared with age- and gender-matched wild-type control mice.

The following mice were evaluated by dual-energy x-ray absorptiometry. The data are compiled from the N0F2 and N1F2 generations.

49 Day Cohort Mouse ID numbers are as follows:
3 heterozygous mutant females (228661, 228662, 228663)
3 heterozygous mutant males (228657, 228658, 228659)
2 wild-type control females (238194, 238201)
2 wild-type control males (228656, 228660)

300 Day Cohort Mouse ID numbers are as follows:
2 heterozygous mutant females (239869, 239870)
2 heterozygous mutant males (239865, 239867)
2 wild-type control females (239868, 240227)

Bone Mineral Density (BMD in g/cm² ), fat % (fat percentage expressed as a percentage of the soft tissue compartment), and R-value of soft tissue were calculated from Bone Mineral Content (BMC in g), bone and tissue areas (cm² ), total tissue mass (g) generated by a PIXImus densitometer.

Densitometric Findings:

Certain densitometric differences between mice, including slightly increased fat %, were present that occasionally occur spontaneously in this age group. In this target, such differences were present in two 49 day heterozygous female knockout mice. We have not reported these findings as phenotypic changes, but we have presented them here for your consideration.

Incidental densitometric differences may have been present between some mice. For example, one 300 day heterozygous mutant male (239865) had a high total tissue mass that correlated with high body weight at necropsy. These findings were considered to represent background differences occasionally seen in this strain of mice, differences due to spontaneous disease, age-related differences, differences due to procedural artifacts, and/or differences of a nonspecific etiology. They were not considered to be genotype related.

Gene 302
Physical Examination

There were no significant differences detected in the heterozygous mutant mice when compared with age- and gender-matched wild-type control mice.

The following mice were evaluated by physical examination. The data are compiled from the N0F2 and N1F2 generations.

49 Day Cohort Mouse ID numbers are as follows:
3 heterozygous mutant females (228661, 228662, 228663)
3 heterozygous mutant males (228657, 228658, 228659)
2 wild-type control females (238194, 238201)
2 wild-type control males (228656, 228660)

300 Day Cohort Mouse ID numbers are as follows:
2 heterozygous mutant females (239869, 239870)
2 heterozygous mutant males (239865, 239867)
2 wild-type control females (239868, 240227)
2 wild-type control males (240223, 240224)

Mice were examined in detail as follows: anus, behavior, body shape, claws, coat - fur, coat color - back, coat color - belly, ear - left, ear - right, eye - left, eye - right, eye color - left, eye color - right, feces, forelimb - left, forelimb - right, forelimb number of amputated digits - left, forelimb number of amputated digits - right, forelimb number of digits - left, forelimb number of digits - right, general appearance, genitals - female, genitals - male, hair type, head shape, hindlimb - left, hindlimb - right, hindlimb number of amputated digits - left, hindlimb number of amputated digits - right, hindlimb number of digits - left, hindlimb number of digits - right, injuries, lesions, limb shape, locomotor, lumps - masses, mammary glands, mice in cage, respiration, skin appearance, snout, swelling - joints, tail, teeth color, teeth length, urine, and whiskers. (Gender-specific observables apply to the appropriate gender.)

Individual heterozygous mutant mice had only occasional minor differences in observed physical features compared to wild-type control mice. These findings were considered to represent individual variability, background features occasionally seen in this strain of mice, findings due to spontaneous disease, age-related findings, procedural artifacts, and/or findings of a nonspecific etiology. However, none of these differences was regarded as biologically significant or genotype related.

Gene 302
Aging Metrics

There were no significant differences detected in the heterozygous mutant mice when compared with age- and gender-matched wild-type control mice.

Body weights and body lengths were measured for mice at 49, 90, 180, and 300 days of age.

49 Day Cohort Mouse ID numbers are as follows:
4 heterozygous mutant females (239869, 239870, 240228, 240230)
4 heterozygous mutant males (239865, 239867, 245201, 245202)
5 wild-type control females (239868, 240227, 240229, 245608, 282522)
4 wild-type control males (240223, 240224, 240225, 240226)

90 Day Cohort Mouse ID numbers are as follows:
4 heterozygous mutant females (239869, 239870, 240228, 240230)
4 heterozygous mutant males (239865, 239867, 245201, 245202)
4 wild-type control females (239868, 240227, 240229, 282522)
4 wild-type control males (240223, 240224, 240225, 240226)

180 Day Cohort Mouse ID numbers are as follows:
4 heterozygous mutant females (239869, 239870, 240228, 240230)
4 heterozygous mutant males (239865, 239867, 245201, 245202)
4 wild-type control females (239868, 240227, 240229, 282522)
4 wild-type control males (240223, 240224, 240225, 240226)

300 Day Cohort Mouse ID numbers are as follows:
2 heterozygous mutant females (240228, 240230)
2 heterozygous mutant males (245201, 245202)
4 wild-type control females (239868, 240227, 240229, 282522)
4 wild-type control males (240223, 240224, 240225, 240226)

Body Weight and Length Findings:

Differences in body length and body weight were present between individual mice. The variability between mice usually fell within our historical reference ranges and was not correlated with genotype.

Gene 302
Behavior

Heterozygous mutants displayed significantly decreased levels of prepulse inhibition (PPI), during startle testing, when compared with age- and gender-matched wild-type control mice.
There were no other significant differences detected in the heterozygous mutant animals when compared with age- and gender-matched wild-type control mice.

Heterozygous mutant and wild-type control mice were evaluated for phenotypic changes by testing on six behavioral tasks: Open field test, Tail suspension test, Rotarod test, Tail flick test, Hot plate test, and Metrazol test.

Mouse ID numbers are as follows for the N1 generation:
10 heterozygous mutant males (238178, 238196, 241205, 241207, 241208, 245205, 245606, 250638, 250640, 250643)
10 wild-type control males (238190, 238197, 241202, 241203, 241204, 245206, 245603, 250639, 250641, 250642)

ES cells derived from the 129/OlaHsd mouse substrain were used to generate chimeric mice.  F1 mice were generated by breeding with C57BL/6 females. The resultant F1N0 heterozygotes were backcrossed to C57BL/6 mice to generate F1N1 heterozygotes.  F2N1 heterozygous mutant mice were produced by intercrossing F1N1 heterozygous males and females.

Behavior Findings:
Heterozygous mutants displayed significantly decreased levels of prepulse inhibition (PPI), during startle testing, when compared with age- and gender-matched wild-type control mice.  Specifically, heterozygous mice had lower average PPI scores when a 120 db startling stimulus was preceded by a 90 db prepulse.

There were no other genotype-related or biologically significant differences noted between heterozygous mutant and wild-type control mice for any of the parameters evaluated during behavior testing.

Gene  302
Summary of Embryonic Development

Homozygous mutant embryos die at ~ E10.5

Embryos were isolated at 8 .5 to 10.5 days post coitum. Homozygous offspring were detected by PCR at E8.5 and E10.5, but not at later stages.   One grossly normal homozygous embryo was detected at 8.5, but at E10.5 all the homozygous embryos are partially resorbed.

Embryos were isolated at E8.5 to 12.5.
Four litters were examined comprising of 36 embryos, resorptions and partial resorptions, of which 33 were successfully genotyped.