Gene: 1217	Name: Scn9a	Family: Channel	Subfamily: Sodium	Accession: AI549833	GI: 4482196

Gene 1217
Summary of Phenotypic Analysis

Changes related to genotype:

• Embryonic lethality: Grossly normal homozygous embryos have been identified at E12.5 to E18.5.
• Behavior: Heterozygous mutant mice exhibited significantly increased prepulse inhibition 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. The genotypic ratio suggested a lethal phenotype. Embryonic studies have identified grossly normal homozygous embryos at E12.5 to E18.5, but not at later stages.

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. The resultant F1N0 heterozygotes were backcrossed to C57BL/6 mice to generate F1N1 heterozygotes.  F2N1 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 
• Behavior tests
• Aging studies

Gene 1217
Behavior

• Heterozygous mutant mice exhibited significantly increased prepulse inhibition 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, Startle response/PPI test, Tail flick test, Hot plate test, and Metrazol test.

Mouse ID numbers are as follows for the N1 generation:
10 heterozygous mutant males (259481, 259484, 259485, 259487, 271974, 271975, 275583, 275804, 276744, 298993)
10 wild-type control males (259482, 259483, 259486, 271973, 275572, 275584, 275800, 275803, 276742, 298985)

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:
When compared to age- and gender-matched wild-type control mice, Heterozygous mutant mice exhibited a significant increase in prepulse inhibition, indicating a stimulus-processing phenomenon opposite to that observed in some human schizophrenic patients.

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

Gene 1217
Expression Summary

Taqman Summary:
The highest levels of RNA transcripts are detectable in subcortical region, cerebellum and brainstem.

Moderate levels of RNA transcripts are detectable in whole brain, cortex, olfactory bulb, spinal cord, eye, pituitary gland, adrenal gland and prostate gland.

Lower levels of RNA transcripts are also detectable in Harderian glands, heart, pancreas, spleen, thymus, lymph nodes, skin, gallbladder, urinary bladder, salivary gland, skeletal muscle, tongue, stomach, small intestine, cecum, colon, testis, epididymis, seminal vesicle, coagulating gland, ovary and white fat.

No RNA transcripts are detectable in lung, liver, kidney, bone marrow and uterus.

LacZ Summary:
LacZ (beta-galactosidase) expression is detectable in brain, urinary bladder, trachea, larynx, pharynx, tongue and male reproductive system.  Urinary bladder, pharynx, tongue and prostate gland show staining in neuronal ganglia.

Expression:
Brain
In wholemount staining moderate to strong lacZ expression is detectable in various regions of the brain including, cortex, thalamus, hypothalamus, inferior colliculus, preoptic area and septum.  Further, expression is detectable in small regions of the olfactory bulb and brainstem.  On coronal sections of cerebrum very faint X-Gal staining is detectable in hypothalamus.  On coronal sections of brainstem faint lacZ expression is detectable in a few cells.

Urinary Bladder
Strong lacZ expression is detectable in ganglion cells.

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

Larynx
Weak lacZ expression is detectable in a few cells of the mucosal epithelium.

Pharynx
Strong lacZ expression is detectable in ganglion cells.  Weak lacZ expression is detectable in a few cells of the mucosal epithelium.

Tongue
Strong lacZ expression is detectable in ganglia.

Male Reproductive Systems
Prostate and Ampullary Gland
Very strong lacZ expression is detectable in ganglia.

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, esophagus, thyroid gland, pituitary gland, adrenal glands, salivary glands, skeletal muscle, skin, and female reproductive system.

Gene 1217
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.

49 Day Cohort Mouse ID numbers were as follows:
3 heterozygous mutant females (245578, 245579, 245581)
3 heterozygous mutant males (245575, 245576, 245577)
2 wild-type control females (245580, 260123)
2 wild-type control males (260058, 271969)

300 Day Cohort Mouse ID numbers were as follows:
2 heterozygous mutant females (259489, 259491)
2 heterozygous mutant males (245571, 245572)
2 wild-type control females (259490, 260066)
2 wild-type control males (263005, 263006)

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

Gene 1217
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 (245578, 245579, 245581)
3 heterozygous mutant males (245575, 245576, 245577)
2 wild-type control females (245580, 260123)
2 wild-type control males (260058, 271969)

300 Day Cohort Mouse ID numbers are as follows:
1 heterozygous mutant female (259491)
1 heterozygous mutant male (245571)

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 1217
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.

49 Day Cohort Mouse ID numbers were as follows:
3 heterozygous mutant females (245578, 245579, 245581)
3 heterozygous mutant males (245575, 245576, 245577)
2 wild-type control females (245580, 260123)
2 wild-type control males (260058, 271969)

300 Day Cohort Mouse ID numbers were as follows:
2 heterozygous mutant females (259489, 259491)
2 heterozygous mutant males (245571, 245572)

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 (245571) had an abdominal fat mass that had 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 1217
Clinical Chemistry

There were no significant differences 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:
3 heterozygous mutant females (245579, 245581, 245582)
3 heterozygous mutant males (245575, 245576, 245577)
2 wild-type control females (245580, 260123)
2 wild-type control males (260058, 273175)

90 Day Cohort Mouse ID numbers are as follows:
3 heterozygous mutant females (259489, 259491, 260069)
4 heterozygous mutant males (245571, 245572, 245573, 245574)
3 wild-type control females (259490, 260066, 263048)
4 wild-type control males (263005, 263006, 263013, 263015)

180 Day Cohort Mouse ID numbers are as follows:
4 heterozygous mutant males (245571, 245572, 245573, 245574)

300 Day Cohort Mouse ID numbers are as follows:
2 heterozygous mutant females (259489, 259491)
2 heterozygous mutant males (245571, 245572)
2 wild-type control females (259490, 260066)
2 wild-type control males (263005, 263006)

Values for the 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 1217

Hematology

There were no significant differences 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:
3 heterozygous mutant females (245578, 245579, 245581)
3 heterozygous mutant males (245575, 245577, 259480)
2 wild-type control females (259488, 260123)
2 wild-type control males (260058, 271969)

90 Day Cohort Mouse ID numbers are as follows:
4 heterozygous mutant females (259489, 259491, 260067, 260069)
4 heterozygous mutant males (245571, 245572, 245573, 245574)
4 wild-type control females (259490, 260066, 260068, 263048)
4 wild-type control males (263005, 263006, 263013, 263015)

180 Day Cohort Mouse ID numbers are as follows:
3 heterozygous mutant males (245572, 245573, 245574)

300 Day Cohort Mouse ID numbers are as follows:
2 heterozygous mutant females (259489, 259491)
2 heterozygous mutant males (245571, 245572)
2 wild-type control females (259490, 260066)
2 wild-type control males (263005, 263006)

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 1217
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.

49 Day Cohort Mouse ID numbers were as follows:
3 heterozygous mutant females (245578, 245579, 245581)
3 heterozygous mutant males (245575, 245576, 245577)
3 wild-type control females (237160, 245580, 260123)
3 wild-type control males (237151, 260058, 271969)

300 Day Cohort Mouse ID numbers were as follows:
2 heterozygous mutant females (259489, 259491)
2 heterozygous mutant males (245571, 245572)
2 wild-type control females (259490, 260066)
2 wild-type control males (263005, 263006)

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

Gene 1217
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 (259489, 259491, 260067, 260069)
4 heterozygous mutant males (245571, 245572, 245573, 245574)
4 wild-type control females (259490, 260066, 260068, 263048)
4 wild-type control males (263005, 263006, 263013, 263015)

90 Day Cohort Mouse ID numbers are as follows:
4 heterozygous mutant females (259489, 259491, 260067, 260069)
4 heterozygous mutant males (245571, 245572, 245573, 245574)
4 wild-type control females (259490, 260066, 260068, 263048)
4 wild-type control males (263005, 263006, 263013, 263015)

180 Day Cohort Mouse ID numbers are as follows:
4 heterozygous mutant males (245571, 245572, 245573, 245574)

300 Day Cohort Mouse ID numbers are as follows:
4 heterozygous mutant females (259489, 259491, 260067, 260069)
4 heterozygous mutant males (245571, 245572, 245573, 245574)
4 wild-type control females (259490, 260066, 260068, 263048)
4 wild-type control males (263005, 263006, 263013, 263015)

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  1217
Summary of Embryonic Development

 

Grossly normal homozygous embryos have been identified at E18.5

Embryos were isolated at  10.5 to 18.5 days post coitum and as 0-48hr neonates. Grossly normal homozygous offspring were detected by PCR at E18.5. However, homozyous offspring have not been detected at later stages .

Offspring were isolated at  E10.5 to 0-48hrs post birth.
Five litters were examined comprising of 35 neonates, embryos , resorptions and partial resorptions, of which 32 were successfully genotyped.

Litter	Embryonic stage	+/+	+/-	-/-	complete resorption or unknown
1	10.5	2	4	0	1
2	12.5	2	5	1	0
3	15.5	1	3	1	1
4	18.5	2	4	3	1
5	0-48hr	1	3	0	0