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Gene: 1688	Name: NHE-6 like	Family: Sodium-Hydrogen	Subfamily: Sodium-Hydrogen Exchan...	Accession: AK032326	GI: 26328150

Gene 1688
Summary of Phenotypic Analysis

Changes related to genotype:

Behavior: Hemizygous mutant male mice exhibited increased total distance traveled during open field testing and required a significantly lower dose of metrazol to reach various seizure stages.

There were no other significant differences detected in the homozygous mutant female or hemizygous mutant male mice when compared with age- and gender-matched heterozygous or 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 heterozygous or hemizygous mutant mice were backcrossed to C57BL/6 mice to generate F1N1 heterozygous or hemizygous mutant mice. F2N1 heterozygous or hemizygous mutant mice were produced by intercrossing F1N1 hemizygous males and heterozygous females.

As this gene is X-linked, the cohort consists of homozygous mutant females, hemizygous mutant males (-/0), heterozygous control females, and wild-type control males (+/0). No wild-type control females or heterozygous control males were analyzed for this target, as such mice cannot be generated from a male -/0 x female -/+ mating.

Female homozygous mutant mice, male hemizygous mutant mice, female heterozygous control mice, and male wild-type control 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
Aging studies
Behavioral tests

Gene 1688
Behavior

Hemizygous mutant male mice exhibited significantly increased total distance traveled during open field testing when compared with age- and gender-matched wild-type control mice.
Hemizygous mutant male mice required a significantly lower dose of metrazol to reach various seizure stages when compared with age- and gender-matched wild-type control mice.

Hemizygous mutant male 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:
9 hemizygous mutant males (291010, 291011, 291019, 291020, 291022, 293925, 293927, 300688, 305452)
9 wild-type control males (291009, 291017, 291018, 293926, 293928, 293929, 300689, 305454, 305455)

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 heterozygous or hemizygous mutant mice were backcrossed to C57BL/6 mice to generate F1N1 heterozygous or hemizygous mutant mice. F2N1 hemizygous mutant mice were produced by intercrossing F1N1 hemizygous males and heterozygous females.

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

When compared to age- and gender-matched wild-type control mice, hemizygous mutant males required a significantly lower dose of metrazol to reach various seizure stages, indicating a possible higher propensity towards seizure phenotype.

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

Gene 1688
Fertility

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

Three 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. One of the pairs (311539 and 311551) produced no viable pups.

Mouse ID numbers are as follows:

3 hemizygous mutant males (311540, 311541, 311539)

3 homozygous mutant females (311552, 311553, 311551)

Gene 1688
Expression Summary

Taqman Summary:
The highest levels of RNA transcripts are detectable in cerebellum, brainstem, adrenal gland and epididymis.

Moderate levels of RNA transcripts are detectable in whole brain, cortex, subcortical region, olfactory bulb, spinal cord, eye, Harderian glands, lung, liver, kidney, spleen, thymus, lymph nodes, skin, gallbladder, urinary bladder, pituitary gland, salivary gland, tongue, stomach, colon, testis, seminal vesicle, coagulating gland, prostate gland, ovary, uterus and white fat.

Lower levels of RNA transcripts are detectable in all other tissues analyzed: heart, pancreas, bone marrow, skeletal muscle, small intestine and cecum.

LacZ Summary:
LacZ expression was detected in all organs examined. Staining was observed in several tissue and cell types including, neurons, epithelium, smooth and cardiac muscle, adipose tissue, lymphoid tissue, cartilage and blood vessels.

As this gene is X-linked, the cohort consists of a hemizygous mutant male (-/0) and a heterozygous female (+/-).

LacZ expression was detected in: brain, spinal cord, sciatic nerve, eyes, thymus, spleen, lymph nodes, bone marrow, aorta, heart, lung, liver, pancreas, kidney, urinary bladder, thyroid gland, parathyroid gland, trachea, larynx, pituitary gland, adrenal glands, skeletal muscle, skin, adipose tissue, testis, prostate gland, ovary and uterus.

Gene 1688
Densitometry

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

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

Densitometric Findings:

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

Gene 1688
Histopathology

There were no significant differences detected in the homozygous mutant female or hemizygous mutant male mice when compared with age- and gender-matched heterozygous or 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.

Histopathological lesions, including moderate to severe atrophy of the purkinje cell layer in the cerebellum, were present in one hemizygous male (280536) and one homozygous female (286831) mice that could have occurred spontaneously. Therefore, we have not reported these findings as phenotypic changes, but we have presented them here for your consideration.

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 1688
Necropsy

There were no significant differences detected in the homozygous mutant female or hemizygous mutant male mice when compared with age- and gender-matched heterozygous or 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 (280529, 280531, 280533)
2 heterozygous mutant females (273747, 273749)
3 hemizygous mutant males (273753, 273755, 273757)
2 wild-type control males (273751, 273752)

300 Day Cohort Mouse ID numbers are as follows:
2 homozygous mutant females (286831, 300693)
2 heterozygous mutant females (286830, 286833)
2 hemizygous mutant males (280536, 280544)

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 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 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 1688
Clinical Chemistry

There were no significant differences detected in the homozygous mutant female or hemizygous mutant male mice when compared with age- and gender-matched heterozygous or 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 (280529, 280531, 280533)
3 hemizygous mutant males (273753, 273755, 273757)
2 heterozygous mutant females (273747, 273749)
2 wild-type control males (273751, 273752)

90 Day Cohort Mouse ID numbers are as follows:
1 homozygous mutant female (280541)
2 hemizygous mutant males (280544, 286811)
1 heterozygous mutant female (283278)
2 wild-type control males (276931, 276932)

180 Day Cohort Mouse ID numbers are as follows:
1 homozygous mutant female (286831)
2 hemizygous mutant males (280536, 280544)
2 heterozygous mutant females (286830, 286833)
4 wild-type control males (276931, 276932, 286820, 286834)

300 Day Cohort Mouse ID numbers are as follows:
2 homozygous mutant females (300691, 300693)
2 hemizygous mutant males (280536, 280544)
2 heterozygous mutant females (286830, 286833)
2 wild-type control males (276931, 276932)

Compared to contemporary wild-type mice, but not to historical population controls, there was a tendency toward lower potassium levels in hemizygous males. We are not reporting this finding as a phenotypic change but are presenting it for your consideration.

Values for the other various analytes evaluated were generally similar between homozygous or hemizygous mutant mice and heterozygous or 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 1688
Hematology

There were no significant differences detected in the homozygous mutant female or hemizygous mutant male mice when compared with age- and gender-matched heterozygous or 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 (280529, 280531, 291014)
3 hemizygous mutant males (273753, 273755, 273757)
2 heterozygous mutant females (273747, 273749)
2 wild-type control males (273751, 273752)

90 Day Cohort Mouse ID numbers are as follows:
3 homozygous mutant females (280541, 283277, 286831)
4 hemizygous mutant males (280536, 280544, 286811, 286838)
4 heterozygous mutant females (283278, 283279, 286830, 286833)
4 wild-type control males (273754, 276931, 276932, 286834)

180 Day Cohort Mouse ID numbers are as follows:
2 homozygous mutant females (286831, 300693)
2 hemizygous mutant males (280536, 280544)
2 heterozygous mutant females (286830, 286833)
3 wild-type control males (276932, 286820, 286834)

300 Day Cohort Mouse ID numbers are as follows:
1 homozygous mutant female (300693)
2 hemizygous mutant males (280536, 280544)
1 heterozygous mutant female (286830)
2 wild-type control males (276931, 276932)

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 1688
Physical Examination

There were no significant differences detected in the homozygous mutant female or hemizygous mutant male mice when compared with age- and gender-matched heterozygous or 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 (280529, 280531, 280533)
2 heterozygous mutant females (273747, 273749)
3 hemizygous mutant males (273753, 273755, 273757)
2 wild-type control males (273751, 273752)

300 Day Cohort Mouse ID numbers are as follows:
2 homozygous mutant females (286831, 300693)
2 heterozygous mutant females (286830, 286833)
2 hemizygous mutant males (280536, 280544)
2 wild-type control males (276931, 276932)

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.)

When compared to age- and gender-matched wild-type control mice, one 300 day homozygous mutant female (286831) was reported to be hyperactive, exhibiting persistent jumping. We have not reported these findings as phenotypic changes, but we have presented them here for your consideration.

Individual heterozygous or hemizygous mutant mice had only occasional other 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 1688
Aging Metrics

There were no significant differences detected in the homozygous mutant female or hemizygous mutant male mice when compared with age- and gender-matched heterozygous or 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:
3 homozygous mutant females (283277, 286831, 286832)
4 heterozygous mutant females (283278, 283279, 286830, 286833)
1 hemizygous mutant male (286838)
4 wild-type control males (273754, 276931, 276932, 286834)

90 Day Cohort Mouse ID numbers are as follows:
2 homozygous mutant females (283277, 286831)
4 heterozygous mutant females (283278, 283279, 286830, 286833)
2 hemizygous mutant males (286811, 286838)
5 wild-type control males (273754, 276931, 276932, 286820, 286834)

180 Day Cohort Mouse ID numbers are as follows:
2 homozygous mutant females (286831, 300693)
2 heterozygous mutant females (286830, 286833)
2 wild-type control males (286820, 286834)

300 Day Cohort Mouse ID numbers are as follows:
1 hemizygous mutant male (280536)
4 wild-type control males (276931, 276932, 286820, 286834)

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.