Gene 717
Summary of Phenotypic Analysis

Behavior:  Homozygous mutant mice exhibited significantly decreased prepulse inhibition during startle testing, and an increase in thermal response latency during tail flick testing.

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 examinations.
· Necropsy including body length, body weight, and organ weight measurements.
· Histological examination of tissues and organs.
· Bone marrow section evaluations.
· Complete blood counts and differentials.
· Clinical chemistry panels.
· Densitometry.
· Fertility. 
· Aging Studies.
· Behavioral tests.

Certain findings, including scruffy haircoat and difficulty in skin removal, were present that occasionally occur spontaneously in mice of this sex and age group.  In this target, such findings were present in the homozygous males at 300 days, and scruffy haircoat was also present in a wild-type control male (206760).  We have not reported these findings as phenotypic changes, but we have presented them here for your consideration.

Gene 717
Expression Summary

RT-PCR Summary:
RNA transcripts are detectable in all tissues analyzed: brain, cortex, subcortical region, cerebellum, brainstem, olfactory bulb, spinal cord, eye, Harderian gland, heart, lung, liver, pancreas, kidney, spleen, thymus, lymph nodes, bone marrow, skin, gallbladder, urinary bladder, pituitary gland, adrenal gland, salivary gland, skeletal muscle, tongue, stomach, small intestine, large intestine, cecum, testis, epididymis, seminal vesicle, coagulating gland, prostate gland, ovary, uterus and white fat.

LacZ Summary:
LacZ (beta-galactosidase) expression is detectable in brain, spinal cord, lung, pancreas, esophagus, salivary glands, tongue, skin and male reproductive systems.  Most striking expression is observed in alveoli of the lung.

Expression:
Brain
In wholemount staining faint lacZ expression is detectable in choroid plexus, lateral ventricle, thalamus and hypothalamus. In coronal sections faint lacZ expression is detectable in cortex and lateral septal nuclei. Further, X-Gal signals are observed in blood vessels.

Spinal cord
In dorsal horns many nuclei express lacZ faint to moderately.

Lung
Strong lacZ expression is detectable in many cells of the alveoli. These cells are most likely pneumocytes.

Pancreas
Acinar cells express lacZ at varying levels, displaying pale to dark blue signals.

Esophagus
Few epithelial cells show faint to moderate lacZ expression.

Salivary Glands
Faint X-Gal signals are detectable.

Tongue
LacZ expression is detectable in blood vessels.

Skin
LacZ expression is detectable in hair follicles.

Male Reproductive Systems
Testis
Very faint X-Gal staining is detectable in few nuclei of the seminiferous tubules.

Coagulating Gland
LacZ expression is detectable in blood vessels.

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

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Gene 717
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 were as follows:
3 homozygous mutant females (143254, 145716, 153879)
3 homozygous mutant males (135264, 135266, 135272)
2 wild-type control females (133980, 133981)
2 wild-type control males (135265, 135269)

300 Day Cohort Mouse ID numbers were as follows:
2 homozygous mutant females (156204, 158098)
2 homozygous mutant males (180340, 180341)
2 wild-type control females (156208, 158097)
2 wild-type control males (206748, 206760)

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 appropriate gender).

Necropsy Findings:

Certain findings, including difficult skin removal, were present that occasionally occur spontaneously.  In this target, such a finding was present in both homozygous males at 300 days, but with no correlative histopathologic observations.  We have not reported these findings as phenotypic changes, but we have presented them here for your consideration.

There were no other genotype-related or biologically significant differences noted between mutant and wild-type control mice for any of the parameters evaluated at necropsy. Incidental lesions were present in some tissues. For example, a uterine mass in a wild-type control female (158097) was identified histologically as cystic endometrial hyperplasia, a non-neoplastic lesion.  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.

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 717
Histopathology

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

Tissues from the following mice were evaluated histologically.

49 Day Cohort Mouse ID numbers were as follows:
3 homozygous mutant females (143254, 145716, 153879)
3 homozygous mutant males (135264, 135266, 135272)
2 wild-type control females (133980, 133981)
2 wild-type control males (135265, 135269)

300 Day Cohort Mouse ID numbers were as follows:
2 homozygous mutant females (156204, 158098)
2 homozygous mutant males (180340, 180341)
2 wild-type control females (156208, 158097)
2 wild-type control males (206748, 206760)

Histopathology Findings:

No Significant Abnormalities:
Tissues examined and considered to have no genotypically-significant abnormality except as specifically noted above: brain, pituitary gland, ears, nasal cavity, salivary glands, oral cavity, lymph nodes, aorta, lungs, 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. For example, at 49 days, two (135264, 135266) of three homozygous males had testicular hypospermatogenesis and epididymal oligospermia, both findings apparently reflecting testicular immaturity.  At 300 days, one homozygous male (180341) had a benign skin tumor, an adenoma, that correlated with an enlarged bulbourethral gland described at necropsy. 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 717
Hematology

There were no significant differences detected 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 (153876, 167107, 167119)
3 homozygous mutant males (133973, 135264, 145711)
2 wild-type control females (133980, 133981)
2 wild-type control males (135265, 135269)

90 Day Cohort Mouse ID numbers are as follows:
3 homozygous mutant females (158098, 299565, 299581)
3 homozygous mutant males (180340, 180341, 299585)
3 wild-type control females (158097, 220441, 235778)
5 wild-type control males (206748, 206760, 299571, 299572, 299573)

180 Day Cohort Mouse ID numbers are as follows:
2 homozygous mutant females (156204, 158098)
2 homozygous mutant males (180340, 180341)
4 wild-type control females (156208, 158097, 220441, 235778)
2 wild-type control males (206748, 206760)

300 Day Cohort Mouse ID numbers are as follows:
2 homozygous mutant females (156204, 158098)
2 homozygous mutant males (180340, 180341)
2 wild-type control females (156208, 158097)
2 wild-type control males (206748, 206760)

Certain variations, including increased percent neutrophils and decreased percent lymphocytes, were present in the homozygous males at 300 days.  Such differences may have occured spontaneously in mice of this age group.  Therefore, we have not reported these findings as phenotypic changes, but we have presented them here for your consideration.  Although other minor variations of hematological values were present in some mice, these changes were not consistent with genotype and thus were not considered phenotypically relevant. 

Gene 717
Clinical Chemistry

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

Serum samples from four homozygous mutant and four wild-type control mice were evaluated by a clinical biochemistry panel.

49 Day Cohort Mouse ID numbers are as follows:
3 homozygous mutant females (145716, 153879, 177300)
3 homozygous mutant males (133971, 135266, 135272)
2 wild-type control females (133978, 133981)
2 wild-type control males (135265, 135269)

90 Day Cohort Mouse ID numbers are as follows:
2 homozygous mutant females (156204, 158098)
2 homozygous mutant males (180340, 180341)
4 wild-type control females (156208, 158097, 220441, 235778)
2 wild-type control males (206748, 206760)

180 Day Cohort Mouse ID numbers are as follows:
2 homozygous mutant females (156204, 158098)
1 homozygous mutant male (180341)
4 wild-type control females (156208, 158097, 220441, 235778)
2 wild-type control males (206748, 206760)

300 Day Cohort Mouse ID numbers are as follows:
2 homozygous mutant females (156204, 158098)
2 homozygous mutant males (180340, 180341)
2 wild-type control females (156208, 158097)
2 wild-type control males (206748, 206760)

Values for the various analytes evaluated were generally similar between homozygous mutant and wild-type control mice. Variations in clinical chemistry values, if present, were not consistent with genotype and thus were not considered phenotypically relevant.

Gene 717
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:
1 homozygous mutant female (143254)
3 homozygous mutant males (135264, 135266, 135272)
2 wild-type control females (133980, 133981)
2 wild-type control males (135265, 135269)

300 Day Cohort Mouse ID numbers are as follows:
2 homozygous mutant females (156204, 158098)
2 homozygous mutant males (180340, 180341)
2 wild-type control females (156208, 158097)
2 wild-type control males (206748, 206760)

No Significant Abnormalities:
Evaluations of densitometric data included Bone Mineral Density (BMD presented as g/cm2), Bone Mineral Content (BMC in g), bone and tissue area, total tissue mass, and fat as a percent of body soft tissue (presented as fat %). No genotypically-significant differences between mice were observed.

Gene 717
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 were as follows:
3 homozygous mutant females (143254, 145716, 153879)
3 homozygous mutant males (135264, 135266, 135272)
2 wild-type control females (133980, 133981)
2 wild-type control males (135265, 135269)

300 Day Cohort Mouse ID numbers were as follows:
2 homozygous mutant females (156204, 158098)
2 homozygous mutant males (180340, 180341)
2 wild-type control females (156208, 158097)
2 wild-type control males (206748, 206760)

Mice were examined for the following observables: 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, feces color, feces exam, 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 exam, mice in cage, respiration, skin appearance, snout, swelling - joints, tail, teeth color, teeth length, urine, urine color, urine exam and whiskers (gender specific observables apply to appropriate gender).

Certain differences at physical examination, including scruffy haircoat, were present that occasionally occur spontaneously.  In this target at 300 days, such differences were present in one wild-type control male (206760) and in both homozygous males.  One of the homozygous males (180341) also had abnormal body shape and externalized penis.  We have not reported these findings as phenotypic changes, but we have presented them here for your consideration. 

Individual homozygous mutant mice had other occasional minor differences in observed physical features comparedto 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 717
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 (156204, 158098, 299565, 299581)
4 homozygous mutant males (180340, 180341, 235776, 299585)
4 wild-type control females (156208, 158097, 220441, 235778)
4 wild-type control males (206748, 206760, 299572, 299573)

90 Day Cohort Mouse ID numbers are as follows:
4 homozygous mutant females (156204, 158098, 299565, 299581)
3 homozygous mutant males (180340, 180341, 299585)
4 wild-type control females (156208, 158097, 220441, 235778)
4 wild-type control males (206748, 206760, 299572, 299573)

180 Day Cohort Mouse ID numbers are as follows:
2 homozygous mutant females (156204, 158098)
2 homozygous mutant males (180340, 180341)
4 wild-type control females (156208, 158097, 220441, 235778)
2 wild-type control males (206748, 206760)

300 Day Cohort Mouse ID numbers are as follows:
2 homozygous mutant females (156204, 158098)
2 wild-type control females (156208, 158097)
1 wild-type control male (206760)

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 717
Behavior

• Homozygous mutant mice exhibited significantly decreased prepulse inhibition when compared with age- and gender-matched wild-type control mice.
• Homozygous mutant mice exhibited a significant increase in thermal response latency during tail flick 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:
7 homozygous mutant males (136524, 136533, 143242, 143243, 167115, 197246, 197247)
9 wild-type control males (136517, 136522, 136527, 143239, 143244, 143247, 167113, 167114, 197245)

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 a significant decrease in prepulse inhibition, indicating a stimulus processing deficit similar to that observed in some human schizophrenic patients.

When compared to age- and gender-matched wild-type control mice, homozygous mutant mice exhibited a significant increase in thermal response latency during tail flick testing, indicating a possible increased pain threshold 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 717
Fertility

Both homozygous mutant males and females had reduced fertility.  Their progeny that survived 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.  Two of the male and female pairs (145714 and 206758, 156201 and 206759) produced only one litter that survived until weaning.  For the third pair (156202 and 158099) 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 (145714, 156201, 156202)

3 homozygous mutant females (206758, 206759, 158099)