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Gene: 899	Name: Gabra4	Family: Channel	Subfamily: GABA Receptor	Accession: AK013727	GI: 12851204

Gene 899
Summary of Phenotypic Analysis

There were no significant differences detected in the homozygous mutant mice 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
Fertility tests
Behavioral tests
Aging studies

Gene 899
Behavior

There were no significant differences detected in the homozygous mutant animals 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:
10 homozygous mutant males (237435, 245754, 268770, 272262, 298881, 353726, 353727, 353728, 353729, 364030)
11 wild-type control males (237434, 245755, 245756, 245757, 245759, 268768, 272260, 298882, 298883, 353725, 364032)

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

Gene 899
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. The third pair (265554 and 256271) had no pups that were viable until weaning.

Mouse ID numbers are as follows:

3 homozygous mutant males (220782, 227640, 265554)

3 homozygous mutant females (240689, 230211, 256271)

Gene 899
Expression Summary

Taqman Summary:
The highest levels of RNA transcripts are detectable in brain, cortex, subcortical region, cerebellum, olfactory bulb, pancreas and pituitary gland.

Lower levels of RNA transcripts are also detectable in brainstem, spinal cord, eye, Harderian glands, lung, kidney, spleen, lymph nodes, skin, urinary bladder, adrenal gland, salivary gland, tongue, stomach, small intestine, testis, epididymis, seminal vesicle, coagulating gland, prostate gland, ovaries, uterus and white fat.

No RNA transcripts are detectable in heart, liver, thymus, bone marrow, gallbladder, skeletal muscle, large intestine and cecum.

LacZ Summary:
LacZ (beta-galactosidase) expression is detectable in the brain, spinal cord, pancreas, pituitary gland, salivary glands.

Expression:
Brain
In the wholemount brain, strong LacZ expression is detected in the thalamus and cortex of the cerebrum and in the purkinje layer of the cerebellum. Moderate LacZ expression is observed in focal areas of the olfactory bulb. Weak LacZ expression in a punctate pattern is found in the brainstem.

Examination of the coronal sections taken through the brain finds very strong to strong LacZ expression is detected in the following regions of the cerebrum and cerebellum: amygdala, cortex, caudate-putamen, dentate gyrus, lateral septal nucleus, lateral ventricles, pyramidal cell layer, thalamus, granular layer, purkinje cell layer, white matter and the brainstem. While moderate LacZ expression is detected in the anterior commissure, fornix, habenular nuclei and hypothalamus of the cerebrum and in the molecular layer of the cerebellum. Additional weak to very weak LacZ expression is detected in the CA fields of the hippocampus, corpus callosum, fornix, habenular nuclei, and the hypothalamus.

Spinal cord
Moderate X-Gal staining is localized in a few cells located in both the white and gray matter.

Pancreas
Moderate to strong LacZ expression is detected in many of the acinar cells.

Pituitary Gland
Faint to strong LacZ expression is observed in many cells of the pars distalis.

Salivary Glands
Moderate LacZ expression is found in some cells of the mucous gland in the sublingual gland.

No Expression:
LacZ expression is not detected in: sciatic nerve, eyes, Harderian glands, thymus, spleen, lymph nodes, bone marrow, aorta, heart, lung, liver, gallbladder, kidney, urinary bladder, trachea, larynx, esophagus, thyroid gland, parathyroid gland, adrenal glands, tongue, skeletal muscle, skin, male and female reproductive systems.

Gene 899
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.

Densitometric Findings:

Incidental densitometric differences may have been present between some mice. For example, bone mineral density was high in one 300 day homozygous mutant female (227634). 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 899
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.

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 lesionspresent in some tissues. For example, one 300 day homozygous mutant female has an arteritis involving multiple 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 899
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 (217212, 217228, 220799)
3 homozygous mutant males (217206, 217210, 217226)
2 wild-type control females (217213, 217229)
2 wild-type control males (217211, 225485)

300 Day Cohort Mouse ID numbers are as follows:
2 homozygous mutant females (227634, 227635)
2 homozygous mutant males (227630, 245766)

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 homozygous mutant male (227630) was reported to have an epididymal nodule for which there was no histopathological correlate. 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:

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 899
Clinical Chemistry

There were no significant differences in the homozygous mutant mice 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 (217212, 217228, 220799)
3 homozygous mutant males (217206, 217226, 254230)
2 wild-type control females (217213, 217229)
2 wild-type control males (225485, 230209)

90 Day Cohort Mouse ID numbers are as follows:
4 homozygous mutant females (227634, 227635, 230227, 230231)
4 homozygous mutant males (227630, 245766, 259999, 260000)
4 wild-type control females (227633, 230232, 235220, 240692)
4 wild-type control males (227654, 245761, 263460, 263461)

180 Day Cohort Mouse ID numbers are as follows:
3 homozygous mutant females (227634, 230227, 230231)
2 homozygous mutant males (227630, 245766)
4 wild-type control females (227633, 227636, 230232, 235220)
4 wild-type control males (227653, 227654, 230222, 245761)

300 Day Cohort Mouse ID numbers are as follows:
2 homozygous mutant females (227634, 227635)
2 homozygous mutant males (227630, 245766)
2 wild-type control females (227633, 227636)
2 wild-type control males (227653, 227654)

When compared to age- and gender-matched wild-type control mice, two 90 day homozygous mutant males (227630, 245766) 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 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 899
Hematology

There were no significant differences in the homozygous mutant mice 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 (217212, 217228, 220799)
3 homozygous mutant males (217206, 254229, 263462)
2 wild-type control females (217229, 240687)
2 wild-type control males (225485, 240683)

90 Day Cohort Mouse ID numbers are as follows:
4 homozygous mutant females (227634, 227635, 230227, 230231)
4 homozygous mutant males (227630, 245766, 259999, 260000)
4 wild-type control females (227633, 227636, 230232, 235220)
4 wild-type control males (227654, 230217, 245761, 245764)

180 Day Cohort Mouse ID numbers are as follows:
4 homozygous mutant females (227634, 227635, 230227, 230231)
2 homozygous mutant males (227630, 245766)
2 wild-type control females (227636, 230232)
3 wild-type control males (227654, 230222, 245761)

300 Day Cohort Mouse ID numbers are as follows:
2 homozygous mutant females (227634, 227635)
3 homozygous mutant males (227630, 245766, 278121)
2 wild-type control females (227633, 227636)
1 wild-type control male (227653)

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

Gene 899
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 (217212, 217228, 220799)
3 homozygous mutant males (217206, 217210, 217226)
2 wild-type control females (217213, 217229)
2 wild-type control males (217211, 225485)

300 Day Cohort Mouse ID numbers are as follows:
2 homozygous mutant females (227634, 227635)
2 homozygous mutant males (227630, 245766)
2 wild-type control females (227633, 227636)
2 wild-type control males (227653, 227654)

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 899
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 (227634, 227635, 230227, 230231)
4 homozygous mutant males (227630, 245766, 259999, 260000)
4 wild-type control females (227633, 227636, 230232, 235220)
4 wild-type control males (227653, 227654, 227655, 230217)

90 Day Cohort Mouse ID numbers are as follows:
4 homozygous mutant females (227634, 227635, 230227, 230231)
4 homozygous mutant males (227630, 245766, 259999, 260000)
4 wild-type control females (227633, 227636, 230232, 235220)
4 wild-type control males (227653, 227654, 230217, 245761)

180 Day Cohort Mouse ID numbers are as follows:
4 homozygous mutant females (227634, 227635, 230227, 230231)
2 homozygous mutant males (227630, 245766)
4 wild-type control females (227633, 227636, 230232, 235220)
4 wild-type control males (227653, 227654, 230222, 245761)

300 Day Cohort Mouse ID numbers are as follows:
2 homozygous mutant females (230227, 230231)
3 homozygous mutant males (245766, 259999, 260000)
4 wild-type control females (227633, 227636, 230232, 235220)
4 wild-type control males (227653, 227654, 230222, 245761)

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.