Gene 623
Summary of Phenotypic Analysis

Changes related to genotype:

·         Behavior:  Homozygous mutant mice exhibited significantly decreased total distance traveled during open field testing when compared with age- and gender-matched wild-type control mice.

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 homozygous mutant mice were produced by intercrossing F1N1 heterozygous males and females.

Wild-type control mice and homozygous 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 
• Fertility
• Aging studies

Gene 623
Expression Summary

Taqman Summary:
The highest levels of RNA transcripts are detectable in pituitary gland and large intestine.

Lower levels of RNA transcripts are detectable in brain, cortex, subcortical region, cerebellum, brainstem, olfactory bulb, spinal cord, eye, Harderian gland, lung, spleen, lymph nodes, skin, gallbladder, urinary bladder, adrenal gland, stomach, small intestine, cecum, epididymis, prostate gland, ovaries, uterus and white fat.

No RNA transcripts are detectable in heart, liver, pancreas, kidney, thymus, bone marrow, salivary gland, skeletal muscle, tongue, testis, seminal vesicle and coagulating gland.

LacZ Summary:
LacZ (beta-galactosidase) expression is detectable in brain, trachea, larynx, pituitary gland, tongue, and male reproductive system.

Expression:
Brain
In wholemount staining moderate lacZ expression is detectable in many structures in the cerebrum including cortex, thalamus and hypothalamus.  Weak lacZ expression is detectable in cerebellum.  Moderate lacZ expression is also detectable in some areas of the brainstem.  On coronal sections of cerebrum moderate lacZ expression is detectable in some cells of cortex.  Weak lacZ expression is detectable in cells scattered throughout the amygdala, caudate putamen, corpus callosum, hippocampus, hypothalamus, thalamus and lateral septal nucleus. 

Trachea
Weak lacZ expression is detectable in some cells of the mucosal epithelium and in a few cartilage cells.

Larynx
Strong lacZ expression is detectable in many cells of the mucosal epithelium, submucosal glands and some cartilage cells.

Pituitary Gland
Strong to very strong lacZ expression is detectable in practically all cells of pars intermedia and in some cells of pars nervosa.  Moderate lacZ expression is detectable in some cells of pars distalis.

Tongue
Strong lacZ expression is detectable in some epithelial cells of the mucous glands.

Male Reproductive Systems
Testis
Weak lacZ expression is detectable in some spermatogenic cells of the seminiferous tubules.

Coagulating Gland
Moderate lacZ expression is detectable in some cells.

Prostate and Ampullary Gland
Moderate lacZ expression is detectable in some epithelial cells of the prostate gland.

No Expression: 
LacZ expression is not detected in: spinal cord, sciatic nerve, eyes, Harderian glands, thymus, spleen, lymph nodes, bone marrow, aorta, heart, lung, liver, gallbladder, pancreas, kidney, urinary bladder, esophagus, thyroid gland, adrenal glands, salivary glands, skeletal muscle, skin, and female reproductive system.

Gene 623
Necropsy 

There were no significant differences detected in the homozygous 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.

49 Day Cohort Mouse ID numbers are as follows:
3 homozygous mutant females (227094, 229384, 229385)
3 homozygous mutant males (236219, 236221, 239290)
2 wild-type control females (227095, 229380)
2 wild-type control males (227090, 227092)

300 Day Cohort Mouse ID numbers are as follows:
2 homozygous mutant females (229383, 239256)
2 homozygous mutant males (239285, 270697)

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

Body and Organ Weight Findings:

Liver weight, liver weight/body weight ratio, thymus weight and thymus weight/body weight ratio were high in one 300 day homozygous mutant male (239285). We have not reported these findings as phenotypic changes but we have presented them for your consideration.

Other differences in body length, body weight, organ weights, and/or organ weight to body weight ratios were present between individual mice. The variability between mice usually fell within our historical reference ranges and was not correlated with genotype.

Gene 623
Histopathology

There were no significant differences detected in the homozygous 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 homozygous mutant females (227094, 229384, 229385)
3 homozygous mutant males (236219, 236221, 239290)
2 wild-type control females (227095, 229380)
2 wild-type control males (227090, 227092)

300 Day Cohort Mouse ID numbers are as follows:
1 homozygous mutant female (229383)
1 homozygous mutant male (239285)

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, and/or lesions of a nonspecific etiology. They were not considered to be genotype related.

Gene 623
Hematology

There were no significant differences in the homozygous 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 homozygous mutant females (227094, 229384, 229385)
3 homozygous mutant males (236219, 236221, 250304)
2 wild-type control females (229380, 236227)
2 wild-type control males (227090, 227092)

90 Day Cohort Mouse ID numbers are as follows:
4 homozygous mutant females (229383, 239256, 239271, 250296)
4 homozygous mutant males (239285, 270697, 276981, 276982)
3 wild-type control females (236223, 236225, 239283)
4 wild-type control males (244758, 244759, 264712, 264716)

180 Day Cohort Mouse ID numbers are as follows:
4 homozygous mutant females (229383, 239256, 239271, 250296)
3 homozygous mutant males (239285, 276981, 276982)
4 wild-type control females (236223, 236225, 239282, 239283)
2 wild-type control males (244758, 244759)

300 Day Cohort Mouse ID numbers are as follows:
2 homozygous mutant females (229383, 239256)
2 homozygous mutant males (239285, 270697)
2 wild-type control females (236223, 236225)
2 wild-type control males (244758, 244759)

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 623
Clinical Chemistry

There were no significant differences in the homozygous 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.

49 Day Cohort Mouse ID numbers are as follows:
3 homozygous mutant females (227094, 229384, 229385)
3 homozygous mutant males (236219, 236221, 250304)
2 wild-type control females (229380, 236227)
2 wild-type control males (227090, 227092)

90 Day Cohort Mouse ID numbers are as follows:
4 homozygous mutant females (229383, 239256, 239271, 250296)
4 homozygous mutant males (239285, 270697, 276981, 276982)
4 wild-type control females (236223, 273639, 273667, 273675)
4 wild-type control males (244758, 244759, 264712, 280507)

180 Day Cohort Mouse ID numbers are as follows:
2 homozygous mutant females (239256, 239271)
3 homozygous mutant males (270697, 276981, 276982)
2 wild-type control females (236225, 239283)
2 wild-type control males (244758, 244759)

300 Day Cohort Mouse ID numbers are as follows:
2 homozygous mutant females (229383, 239256)
2 homozygous mutant males (239285, 270697)
2 wild-type control females (236223, 236225)
2 wild-type control males (244758, 244759)

When compared to age- and gender-matched wild-type control mice, two 300 day homozygous mutant males (239285, 270697) and one of two 300-day homozygous mutant females (229383) had increased levels of cholesterol and high density lipoproteins. 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 homozygous 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 623
Densitometry

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

The following mice were evaluated by dual-energy x-ray absorptiometry.

49 Day Cohort Mouse ID numbers are as follows:
3 homozygous mutant females (227094, 229384, 229385)
3 homozygous mutant males (236219, 236221, 239290)
2 wild-type control females (227095, 229380)
2 wild-type control males (227090, 227092)

300 Day Cohort Mouse ID numbers are as follows:
1 homozygous mutant female (229383)
2 homozygous mutant males (239285, 270697)
2 wild-type control females (236223, 236225)
2 wild-type control males (244758, 244759)

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:

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

Gene 623
Physical Examination

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

The following mice were evaluated by physical examination.

49 Day Cohort Mouse ID numbers are as follows:
3 homozygous mutant females (227094, 229384, 229385)
3 homozygous mutant males (236219, 236221, 239290)
2 wild-type control females (227095, 229380)
2 wild-type control males (227090, 227092)

300 Day Cohort Mouse ID numbers are as follows:
2 homozygous mutant females (229383, 239256)
2 homozygous mutant males (239285, 270697)
2 wild-type control females (236223, 236225)
2 wild-type control males (244758, 244759)

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 homozygous 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, and/or findings of a nonspecific etiology. However, none of these differences was regarded as biologically significant or genotype related.

Gene 623
Aging Metrics

There were no significant differences detected in the homozygous 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 homozygous mutant females (236228, 239256, 239271, 250296)
4 homozygous mutant males (239285, 270697, 276981, 276982)
4 wild-type control females (236223, 236225, 239282, 239283)
4 wild-type control males (244758, 244759, 264712, 264716)

90 Day Cohort Mouse ID numbers are as follows:
4 homozygous mutant females (229383, 239256, 239271, 250296)
4 homozygous mutant males (239285, 270697, 276981, 276982)
4 wild-type control females (236223, 236225, 239282, 239283)
4 wild-type control males (244758, 244759, 264712, 264716)

180 Day Cohort Mouse ID numbers are as follows:
4 homozygous mutant females (229383, 239256, 239271, 250296)
1 homozygous mutant male (239285)
4 wild-type control females (236223, 236225, 239282, 239283)
2 wild-type control males (244758, 244759)

300 Day Cohort Mouse ID numbers are as follows:
2 homozygous mutant females (239271, 250296)
4 homozygous mutant males (239285, 270697, 276981, 276982)
4 wild-type control females (236223, 236225, 239282, 239283)
4 wild-type control males (244758, 244759, 264712, 264716)

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 623
Behavior

• Homozygous mutant mice exhibited significantly decreased total distance traveled during open field testing when compared with age- and gender-matched wild-type control mice.

Homozygous mutant and wild-type control mice were evaluated for phenotypic changes by testing on seven behavioral tasks: Open field test, Tail suspension test, Rotarod test, Startle response/PPI test, Tail flick test, Hot plate test, and Metrazol test.

Mouse ID numbers are as follows for the N1 generation:
11 homozygous mutant males (250169, 250171, 253169, 253174, 257944, 257948, 273606, 273609, 273654, 276980, 276983)
10 wild-type control males (250163, 250168, 250172, 253170, 255763, 257947, 273652, 273653, 273656, 276984)

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 homozygous mutant mice were produced by intercrossing F1N1 heterozygous males and females.

Behavior Findings:
When compared to age- and gender-matched wild-type control mice, homozygous mutant mice exhibited significantly decreased total distance traveled during open field testing, indicating a possible hypoactivity phenotype.

There were no other genotype-related differences noted between homozygous mutant and wild-type control mice for any other parameters evaluated during behavior testing.

Gene 623
Fertility

Both homozygous mutant males and females were fertile.  Their progeny were viable until weaning.

Three homozygous mutant mice of each gender were set up in a fertility mating one on one with each other at seven to ten weeks of age.  The number of pups born from three litters was recorded.  Three weeks later, the live pups were counted and weaned.

Mouse ID numbers are as follows:

3 homozygous mutant males (239243, 241945, 267479)

3 homozygous mutant females (239279, 239252, 273613)