Gene: 1494	Name: Slc22a6	Family: Transporter	Subfamily: Organic cation	Accession: NM_008766	GI: 6679177

Gene 1494
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. F2 homozygous mutant mice were produced by intercrossing F1 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
• Behavior Tests
• Aging studies

 

Gene 1494
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 Test, Tail flick test, Hot plate test, and Metrazol test.

Mouse ID numbers are as follows for the N1 generation:
11 homozygous mutant males (214461, 253838, 268681, 278367, 281779, 281782, 281784, 285006, 285142, 289174, 289176)
10 wild-type control males (214465, 253836, 268682, 268683, 278364, 281791, 281793, 281794, 285003, 289175)

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 1494
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.

Mouse ID numbers are as follows:

3 homozygous mutant males (214206, 225541, 260010)

3 homozygous mutant females (214469, 214471, 254113)

 

 

Gene 1494
Expression Summary

RT-PCR Summary:
High levels of RNA transcripts are detectable kidney.

Very low levels of RNA transcripts are also detectable in brain, cortex, subcortical region, cerebellum, brainstem and olfactory bulb.

No RNA transcripts are detectable in spinal cord, eye, Harderian glands, heart, lung, liver, pancreas, 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 and kidney.

Expression:
Brain
On wholemount staining strong lacZ expression is detectable in meninges, ventricles and hippocampus.  On coronal sections, lacZ expression is detectable in meninges, ventricles, fasciola cinereum, habenular nuclei and hippocampus.

Kidney
Strong lacZ expression is detectable in tubules of the cortex.

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, urinary bladder, trachea, larynx, esophagus, thyroid gland, parathyroid gland, pituitary gland, adrenal glands, salivary glands, tongue, skeletal muscle, skin, male and female reproductive systems.

Gene 1494
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:
3 homozygous mutant females (184258, 189308, 191069)
3 homozygous mutant males (191065, 191068, 197853)
2 wild-type control females (184261, 191071)
2 wild-type control males (188266, 188267)

300 Day Cohort Mouse ID numbers are as follows:
2 homozygous mutant females (214207, 225285)
2 homozygous mutant males (230359, 230360)
2 wild-type control females (214210, 225286)
2 wild-type control males (235030, 235032)

Bone Mineral Density (BMD in g/cm² ), fat % (fat percentage expressed as a percentage of body soft tissue compartment), and R-value of soft tissue were calculated from Bone Mineral Content (BMC in g), bone and tissue areas (cm² ), and total tissue mass (g) generated by a PIXImus densitometer.

Densitometric Findings:

Incidental densitometric differences may have been present between some mice. For example, fat % was slightly increased in two 49 day cohort homozygous males (191065,191068). These findings are considered to represent background differences occasionally seen in this strain of mice, differences due to spontaneous disease, age-related differences, differences due to procedural artifacts and/or differences of a nonspecific etiology. They are not considered to be genotype related.

Gene 1494
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.

49 Day Cohort Mouse ID numbers are as follows:
3 homozygous mutant females (184258, 189308, 191069)
3 homozygous mutant males (191065, 191068, 197853)
2 wild-type control females (184261, 191071)
2 wild-type control males (188266, 188267)

300 Day Cohort Mouse ID numbers are as follows:
1 homozygous mutant female (214207)
1 homozygous mutant male (230360)

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, gallbladder, pancreas, spleen, kidneys, adrenal glands, 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 are present in some tissues. These findings are considered to represent background lesions occasionally seen in this strain of mice, lesions due to spontaneous disease, age-related lesions, lesions due to procedural artifacts, and/or lesions of a nonspecific etiology. They are not considered to be genotype related.

Gene 1494
Necropsy 

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 (184258, 189308, 191069)
3 homozygous mutant males (191065, 191068, 197853)
2 wild-type control females (184261, 191071)
2 wild-type control males (188266, 188267)

300 Day Cohort Mouse ID numbers are as follows:
2 homozygous mutant females (214207, 225285)
2 homozygous mutant males (230359, 230360)
1 wild-type control male (235032)

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 lesions, lesions due to procedural artifacts, and/or lesions of a nonspecific etiology.  They were not considered to be related to genotype.

Body and Organ Weight Findings:

When compared with age and gender matched wild type control mice, all homozygous mutant male mice had increased thymus and thymus to body weight ratio. All homozygous mutant female mice had slightly increased thymus to body weight (12%), on average.  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 1494
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 biochemistry panel.

49 Day Cohort Mouse ID numbers are as follows:
3 homozygous mutant females (184258, 189308, 189309)
3 homozygous mutant males (188269, 197853, 201044)
2 wild-type control females (184261, 191071)
2 wild-type control males (188267, 188270)

90 Day Cohort Mouse ID numbers are as follows:
3 homozygous mutant females (214207, 240682, 260013)
3 homozygous mutant males (230360, 235029, 268650)
5 wild-type control females (214210, 240678, 240679, 242166, 260015)
3 wild-type control males (235030, 235032, 240636)

180 Day Cohort Mouse ID numbers are as follows:
3 homozygous mutant females (214207, 225285, 240682)
4 homozygous mutant males (230359, 230360, 235029, 235033)
4 wild-type control females (214210, 225286, 240678, 240679)
4 wild-type control males (235030, 235032, 240636, 289160)

300 Day Cohort Mouse ID numbers are as follows:
2 homozygous mutant females (214207, 225285)
2 homozygous mutant males (230359, 230360)
2 wild-type control females (214210, 225286)
2 wild-type control males (235030, 235032)

When compared to age- and gender-matched wild-type control mice, one of two 300-day homozygous mutant males (230359) had increased levels of cholesterol and high density lipoprotein. We are not reporting these findings as phenotypic changes, but we present them here for your consideration.
Values for the other various analytes evaluated were generally similar between homozygous or 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 1494
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 (189308, 189314, 191069)
3 homozygous mutant males (191065, 191068, 197853)
2 wild-type control females (184261, 189318)
2 wild-type control males (188266, 188267)

90 Day Cohort Mouse ID numbers are as follows:
3 homozygous mutant females (225285, 240682, 260013)
3 homozygous mutant males (230359, 230360, 235029)
4 wild-type control females (214210, 225286, 240678, 240679)
4 wild-type control males (235030, 235032, 240636, 289160)

180 Day Cohort Mouse ID numbers are as follows:
3 homozygous mutant females (214207, 225285, 240682)
4 homozygous mutant males (230359, 230360, 235029, 235033)
4 wild-type control females (214210, 225286, 240678, 240679)
4 wild-type control males (235030, 235032, 240636, 289160)

300 Day Cohort Mouse ID numbers are as follows:
2 homozygous mutant males (230359, 230360)
2 wild-type control females (214210, 225286)
2 wild-type control males (235030, 235032)

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 1494
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 (184258, 189308, 191069)
3 homozygous mutant males (191065, 191068, 197853)
2 wild-type control females (184261, 191071)
2 wild-type control males (188266, 188267)

300 Day Cohort Mouse ID numbers are as follows:
2 homozygous mutant females (214207, 225285)
2 homozygous mutant males (230359, 230360)
2 wild-type control females (214210, 225286)
2 wild-type control males (235030, 235032)

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, 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, 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 the appropriate gender.)

Individual homozygous mutant mice had only occasional minor differences in observed physical features compared to wild-type control mice  These findings are considered to represent individual variability, background features occasionally seen in this strain of mice, findings due to spontaneous disease, age-related findings, procedural artifacts, and/or findings of a nonspecific etiology. However, none of these differences was regarded as biologically significant or genotype related.

 

Gene 1494
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 (214207, 225285, 240682, 260013)
4 homozygous mutant males (230359, 230360, 235029, 235033)
4 wild-type control females (214210, 225286, 240678, 240679)
5 wild-type control males (235030, 235032, 240636, 240644, 289160)

90 Day Cohort Mouse ID numbers are as follows:
4 homozygous mutant females (214207, 225285, 240682, 260013)
3 homozygous mutant males (230359, 230360, 235029)
4 wild-type control females (214210, 225286, 240678, 240679)
4 wild-type control males (235030, 235032, 240636, 289160)

180 Day Cohort Mouse ID numbers are as follows:
3 homozygous mutant females (214207, 225285, 240682)
4 homozygous mutant males (230359, 230360, 235029, 235033)
4 wild-type control females (214210, 225286, 240678, 240679)
4 wild-type control males (235030, 235032, 240636, 289160)

300 Day Cohort Mouse ID numbers are as follows:
1 homozygous mutant female (240682)
2 homozygous mutant males (235029, 235033)
4 wild-type control females (214210, 225286, 240678, 240679)
2 wild-type control males (235030, 240636)

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.