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Gene 194
Summary of Phenotypic Analysis

Changes related to genotype:

Embryonic Lethality: Homozygous mutant embryos possibly die at E12.5.

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. Resorbing homozygous embryos were identified at E12.5 by PCR.

There were no significant differences detected in the heterozygous 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. F2 heterozygous mutant mice were produced by intercrossing F1 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
Embryonic development
Behavioral tests
Fertility evaluation
Aging studies

Gene 194
Behavior

There were no 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 seven behavioral tasks: Open field test, Tail suspension test, Rotarod test, Hot plate test, Startle/PPI, Tail flick test and Metrazol test.

Mouse ID numbers are as follows:

10 heterozygous mutant males (188951, 188952, 192150, 200579, 200578, 206555, 206557, 208772, 215772, 231129)
10 wild-type control males (188953, 192145, 192147, 192149, 200580, 206556, 206559, 208766, 215773, 231125)

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:

There were no 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 194
Fertility

Fertility on homozygous animals could not be determined, because of developmental lethality. However, both heterozygous males and females were fertile. Their progeny were viable until weaning, which suggests no abnormalities in the ability of the mutant females to nurture their pups.

Gene 194
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 glands, 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, ovaries, uterus and white fat.

LacZ Summary:
LacZ (beta-galactosidase) expression is detectable in brain, spinal cord, eyes, Harderian glands, thymus, spleen, lymph nodes, aorta, heart, lung, liver, gallbladder, pancreas, kidney, urinary bladder, trachea, larynx, esophagus, thyroid gland, pituitary gland, adrenal glands, salivary glands, tongue, skeletal muscle, skin, male and female reproductive systems. In many tissues lacZ is expressed in smooth muscle cells.

Expression:
Brain
In wholemount staining, weak lacZ expression is detectable in ventricles and blood vessels. In coronal sections faint X-Gal signals are present in choroid plexus, fourth ventricle and blood vessels. Very weak lacZ expression is detectable in few cells lateral to the third ventricle.

Spinal cord
Very few cells in dorsal horn and white matter express lacZ. Moderate to strong lacZ expression is detectable in all blood vessels.

Eyes
Very faint lacZ expression is detectable in distinct cells of the uvea.

Harderian Glands
LacZ expression is detectable in smooth muscle cells of blood vessels.

Thymus
LacZ expression is detectable in blood vessels.

Spleen
LacZ expression is detectable in blood vessels, capsule and trabeculae.

Lymph Nodes
LacZ expression is detectable in blood vessels.

Aorta
Strong lacZ expression is detectable in tunica media.

Heart
Moderate to strong lacZ expression is detectable in valves, aortic root and blood vessels.

Lung
LacZ expression is detectable in smooth muscle cells of bronchioli and blood vessels.

Liver
Strong lacZ expression is detectable in smooth muscle cells of bile ducts and blood vessels.

Gallbladder
LacZ expression is detectable in smooth muscle cells of the wall and blood vessels.

Pancreas
Strong lacZ expression is detectable in blood vessels.

Kidney
Strong lacZ expression is detectable in blood vessels, glomeruli, medulla, papilla and pelvis.

Urinary Bladder
LacZ expression is apparent in smooth muscle cells of muscularis and blood vessels. Weak X-Gal staining is detectable in lamina propria.

Trachea
Very strong lacZ expression is detectable in cartilage; weaker expression is detectable in trachealis muscle and blood vessels.

Larynx
Very strong lacZ expression is detectable in cartilage; strong expression is detectable in blood vessels.

Esophagus
Very faint lacZ expression is detectable in submucosa.

Thyroid Gland
LacZ expression is detectable in blood vessels.

Parathyroid Gland
Few cells express lacZ.

Pituitary Gland
Scattered, strong lacZ expression is detectable in pars nervosa.

Adrenal Glands
Strong lacZ expression is detectable in capsule and blood vessels. Weak lacZ expression is detectable in medulla.

Salivary Glands
LacZ expression is detectable in blood vessels.

Tongue
LacZ expression is detectable in blood vessels. Weak lacZ expression is detectable in lamina propria.

Skeletal Muscle
LacZ expression is detectable in blood vessels.

Skin
LacZ expression is detectable in the dermis.

Skin of the Ear
Strong lacZ expression is detectable in all chondrocytes. Faint X-Gal signals are detectable in dermis.

Male Reproductive Systems
Testis
Strong lacZ expression is detectable in Sertoli cells and blood vessels. X-Gal signals are also detectable in myofibroblasts of the seminiferous and in tunica albuginea.

Penis
LacZ expression is detectable in fibroblasts and smooth muscle cells throughout.

Seminal Vesicles
Strong lacZ expression is detectable in myocytes of the capsule and blood vessels.

Coagulating Gland
Strong lacZ expression is detectable in myocytes of the capsule and blood vessels.

Prostate and Ampullary Gland
Strong lacZ expression is detectable in myocytes of the capsule and blood vessels.

Female Reproductive Systems
Ovary
Weak lacZ expression is detectable in thecal cells and corpus lutea.

Oviduct/Uterus
LacZ expression is detectable in myometrium and blood vessels.

Vagina/Cervix
Weak lacZ expression is detectable in lamina propria, muscularis and blood vessels.

No Expression:
LacZ expression is not detected in sciatic nerve and bone marrow.

Gene 194
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 were compiled from N0F2 and N1F2 generations.

49 Day Cohort Mouse ID numbers are as follows:
4 heterozygous mutant females (164953, 177931, 177932, 183666)
3 heterozygous mutant males (158354, 177934, 177936)
2 wild-type control females (152724, 192315)
2 wild-type control males (142063, 145706)

300 Day Cohort Mouse ID numbers are as follows:
2 heterozygous mutant females (187193, 197140)
2 heterozygous mutant males (183270, 183271)
2 wild-type control females (197141, 200585)
2 wild-type control males (183273, 197139)

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 are 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 are not considered to be genotype related.

Gene 194
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. The data were compiled from N0F2 and N1F2 generations.

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, 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.

Certain microscopic lesions, including spinal cord epidermal inclusion cysts, were present in two heterozygous female mice (164953, 177931) and in one heterozygous male mouse (177936) that could occur spontaneously in mice of this age group. Therefore, we have not reported these findings as phenotypic changes, but we presented them here for your consideration. Other 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 changes, and/or lesions of a nonspecific etiology. They were not considered to be genotype related.

Gene 194
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 were compiled from N0F2 and N1F2 generations.

49 Day Cohort Mouse ID numbers were as follows:
4 heterozygous mutant females (164953, 177931, 177932, 183666)
3 heterozygous mutant males (158354, 177934, 177936)
2 wild-type control females (152724, 192315)
2 wild-type control males (142063, 145706)

300 Day Cohort Mouse ID numbers were as follows:
2 heterozygous mutant females (187193, 197140)
2 heterozygous mutant males (183270, 183271)
2 wild-type control females (197141, 200585)
2 wild-type control males (183273, 197139)

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 changes, 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 194
Clinical Chemistry

There were no significant differences detected in the heterozygous 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:
4 heterozygous mutant females (164953, 177931, 177932, 183659)
3 heterozygous mutant males (158354, 177936, 183669)
2 wild-type control females (152724, 192314)
2 wild-type control males (142063, 145704)

90 Day Cohort Mouse ID numbers are as follows:
4 heterozygous mutant females (187193, 197140, 197142, 200587)
4 heterozygous mutant males (183270, 183271, 209189, 255511)
4 wild-type control females (197141, 200585, 200586, 209192)
4 wild-type control males (183273, 197139, 225555, 225560)

180 Day Cohort Mouse ID numbers are as follows:
4 heterozygous mutant females (187193, 197140, 200581, 200587)
3 heterozygous mutant males (183270, 209189, 248649)
4 wild-type control females (197141, 200585, 200586, 209192)
4 wild-type control males (183273, 197139, 225560, 239860)

300 Day Cohort Mouse ID numbers are as follows:
2 heterozygous mutant females (197140, 200582)
2 heterozygous mutant males (183270, 183271)
2 wild-type control females (197141, 200585)
2 wild-type control males (183273, 197139)

Values for the various analytes evaluated were generally similar between heterozygous 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 194
Hematology

There were no significant differences detected in the heterozygous 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:
4 heterozygous mutant females (164953, 177931, 177932, 183666)
3 heterozygous mutant males (177934, 177936, 183667)
2 wild-type control females (164952, 192315)
2 wild-type control males (142063, 145704)

90 Day Cohort Mouse ID numbers are as follows:
4 heterozygous mutant females (187193, 197140, 200587, 209194)
4 heterozygous mutant males (183270, 183271, 209189, 255511)
4 wild-type control females (197141, 200585, 200586, 209192)
4 wild-type control males (183273, 197139, 225555, 225560)

180 Day Cohort Mouse ID numbers are as follows:
4 heterozygous mutant females (187193, 200581, 200587, 206553)
4 heterozygous mutant males (183270, 183271, 209189, 255511)
4 wild-type control females (197141, 200585, 200586, 209192)
4 wild-type control males (183273, 197139, 225560, 239860)

300 Day Cohort Mouse ID numbers are as follows:
2 heterozygous mutant females (187193, 197140)
2 heterozygous mutant males (183270, 183271)
2 wild-type control females (197141, 200585)
2 wild-type control males (183273, 197139)

Although 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 194
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 were compiled from N0F2 and N1F2 generations.

49 Day Cohort Mouse ID numbers were as follows:
4 heterozygous mutant females (164953, 177931, 177932, 183666)
3 heterozygous mutant males (158354, 177934, 177936)
2 wild-type control females (152724, 192315)
2 wild-type control males (142063, 145706)

300 Day Cohort Mouse ID numbers were as follows:
2 heterozygous mutant females (187193, 197140)
2 heterozygous mutant males (183270, 183271)
2 wild-type control females (197141, 200585)
2 wild-type control males (183273, 197139)

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, 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, and whiskers. (Gender-specific observables apply to appropriate gender.)

Individual heterozygous mutant mice had only 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 were regarded as biologically significant or genotype related.

Gene 194
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. The data were compiled from N0F2 and N1F2 generations.

49 Day Cohort Mouse ID numbers are as follows:
4 heterozygous mutant females (187193, 197140, 197142, 200587)
4 heterozygous mutant males (183270, 183271, 209189, 255511)
5 wild-type control females (187194, 197141, 200585, 200586, 209192)
4 wild-type control males (183273, 197139, 225555, 225560)

90 Day Cohort Mouse ID numbers are as follows:
4 heterozygous mutant females (187193, 197140, 197142, 200587)
4 heterozygous mutant males (183270, 183271, 209189, 255511)
4 wild-type control females (197141, 200585, 200586, 209192)
4 wild-type control males (183273, 197139, 225555, 225560)

180 Day Cohort Mouse ID numbers are as follows:
4 heterozygous mutant females (187193, 197140, 200581, 200587)
3 heterozygous mutant males (183270, 183271, 209189)
4 wild-type control females (197141, 200585, 200586, 209192)
4 wild-type control males (183273, 197139, 225560, 239860)

300 Day Cohort Mouse ID numbers are as follows:
3 heterozygous mutant females (187193, 200581, 200587)
1 heterozygous mutant male (209189)
3 wild-type control females (200585, 200586, 209192)

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 194
Summary of Embryonic Development

Resorbing homozygous mutant embryos have been Identified at E12.5.

Embryos were isolated at 8.5 to 14.5 days post coitum. Homozygous offspring were only detected by PCR at E12.5. One homozygous embryo appeared normal, then the next two showed abnormal heart and abdomen, and the fourth embryo was partially resorbed. These preliminary data suggests that death is around day 12.5 of embryonic development.

Embryos were isolated at E8.5 to E14.5
Four litters were examined comprising of 37 embryos, resorptions and partial resorptions, of which 30 were successfully genotyped.

Litter	Embryonic stage	+/+	+/-	-/-	complete resorption or unknown
1	8.5	4	3	0	0
2	11.5	5	5	0	2
3	12.5	0	3	4	0
4	14.5	0	6	0	5