Gene: 1127	Name: Cacng4	Family: Channel	Subfamily: Calcium	Accession: NM_019431	GI: 9506454

Gene 1127
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 differentials.
• Clinical chemistry panels.
• Fertility.
• Densitometry. 
• Behavioral tests.
• Aging studies.

Gene 1127
Behavior

Changes related to genotype:

There were no significant differences noted between homozygous mutant and 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, Hot plate test, Startle/PPI, Tail flick test, and Metrazol test.

Mouse ID numbers are as follows:

11 homozygous mutant males (193576, 193575, 193573, 197398, 211560, 211570, 211639, 219451, 221108, 226712, 228237)
11 wild-type control males (193577, 197395, 211571, 211637, 219449, 219450, 221110, 226714, 226711, 226704, 228235)

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 differences noted between homozygous mutant and wild-type control mice for any other parameters evaluated during behavior testing.

Gene 1127
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.  For one mating (189711, 193569) no pups were recorded.

Mouse ID numbers are as follows:

3 homozygous mutant males (189710, 189711, 189713)

3 homozygous mutant females (219462, 193569, 193571)

 

Gene 1127
Expression Summary

RT-PCR Summary:
RNA transcripts are detectable whole brain, cortex, subcortical region, cerebellum, brainstem, olfactory bulb, spinal cord, eye, pancreas, kidney, thymus, skin, urinary bladder, pituitary gland, adrenal gland, salivary gland, skeletal muscle, tongue, stomach, small intestine, large intestine, testis, epididymis, coagulating gland, prostate gland, ovaries, uterus and white fat.

No RNA transcripts are detectable in Harderian glands, heart, lung, liver, spleen, lymph nodes, bone marrow, gallbladder, cecum and seminal vesicle.

LacZ Summary:
LacZ (beta-galactosidase) expression is detectable in brain, spinal cord, eyes, Harderian glands, lymph nodes, heart, lung, trachea, larynx, esophagus, pituitary gland, adrenal glands, salivary glands, tongue, skin, male and female reproductive systems.

Expression:
Brain
In wholemount staining very strong lacZ expression is detectable throughout the entire brain. On coronal sections very strong X-Gal staining is detectable in almost all nuclei throughout the brain. In hippocampus, corpus callosum and anterior commissure not all nuclei express lacZ. 

Spinal cord
Very strong lacZ expression is detectable throughout the spinal cord.

Eyes
Very strong lacZ expression is detectable in inner nuclear and ganglion cell layers and in cilliary body.

Harderian Glands
LacZ expression is detectable in distinct cells.

Lymph Nodes
Weak lacZ expression is detectable in very few cells.

Heart
Weak lacZ expression is detectable in ganglia.

Lung
LacZ expression is detectable in cartilage and smooth muscle of the main bronchi. 

Trachea
Strong lacZ expression is detectable in chondrocytes.  Weak lacZ expression is detectable in myocytes of the trachealis muscle.

Larynx
Strong lacZ expression is detectable in chondrocytes and ganglia.

Esophagus
LacZ expression is detectable in few cells of the muscle layer and in ganglion cells.

Pituitary Gland
Strong lacZ expression is detectable in pars nervosa.  Weak to moderate lacZ expression is detectable in pars distalis.

Adrenal Glands
Weak lacZ expression is detectable in the capsule.

Salivary Glands
Strong lacZ expression is detectable in ganglia.

Tongue
Strong lacZ expression is detectable in a few ganglion cells.

Male Reproductive Systems
Testis
Strong lacZ expression is detectable in some cells in the interstitium.  LacZ expression is detectable in some spermatogenic cells of the seminiferous tubules.

Female Reproductive Systems
Oviduct/Uterus
LacZ expression is detectable in smooth muscle cells of the uterine tubules.

Vagina/Cervix
Weak lacZ expression is detectable in some ganglion cells.

No Expression: 
LacZ expression is not detected in sciatic nerve, thymus, spleen, bone marrow, aorta, liver, gallbladder, pancreas, kidney, urinary bladder, thyroid gland, and skeletal muscle.

 

 

Gene 1127
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:
4 homozygous mutant females (164917, 164939, 172146, 172234)
3 homozygous mutant males (158300, 158301, 158303)
2 wild-type control females (158306, 164945)
2 wild-type control males (164942, 164943)

300 Day Cohort Mouse ID numbers are as follows:
2 homozygous mutant females (189717, 197401)
1 homozygous mutant male (211563)
2 wild-type control females (189715, 197400)
2 wild-type control males (199692, 211567)

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:

Certain densitometric differences between mice, including increased fat %, were present that occasionally occur spontaneously in this age group. In this target, such differences were present in three 49 day cohort homozygous mutant males (158300, 158301, 158303) when compared to age- and gender-matched contemporaneous but not whole population (historical) control mice. We have not reported these findings as phenotypic changes, but we have presented them here for your consideration.

Other 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 1127
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:
4 homozygous mutant females (164917, 164939, 172146, 172234)
3 homozygous mutant males (158300, 158301, 158303)
2 wild-type control females (158306, 164945)
2 wild-type control males (164942, 164943)

300 Day Cohort Mouse ID numbers are as follows:
2 homozygous mutant females (189717, 197401)
2 homozygous mutant males (204448, 211563)

No Significant Abnormalities:
Tissues examined and considered to have no genotypically significant 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 may have been 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 1127
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 are as follows:
4 homozygous mutant females (164917, 164939, 172146, 172234)
3 homozygous mutant males (158300, 158301, 158303)
2 wild-type control females (158306, 164945)
2 wild-type control males (164942, 164943)

300 Day Cohort Mouse ID numbers are as follows:
2 homozygous mutant females (189717, 197401)
2 homozygous mutant males (204448, 211563)
2 wild-type control females (189715, 197400)
2 wild-type control males (199692, 211567)

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, feces, 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:
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 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 1127
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 (164917, 172232, 172234)
3 homozygous mutant males (158300, 158303, 172241)
2 wild-type control females (164945, 164946)
2 wild-type control males (164942, 164943)

90 Day Cohort Mouse ID numbers are as follows:
3 homozygous mutant females (189717, 197402, 197404)
4 homozygous mutant males (204448, 211563, 211565, 211566)
4 wild-type control females (189715, 197400, 197403, 204447)
4 wild-type control males (199692, 211567, 230644, 230646)

180 Day Cohort Mouse ID numbers are as follows:
4 homozygous mutant females (189717, 197401, 197402, 197404)
3 homozygous mutant males (211563, 211565, 211566)
4 wild-type control females (189715, 197400, 197403, 204447)
3 wild-type control males (199692, 211567, 230644)

300 Day Cohort Mouse ID numbers are as follows:
2 homozygous mutant females (189717, 197401)
2 homozygous mutant males (204448, 211563)
2 wild-type control females (189715, 197400)
2 wild-type control males (199692, 211567)

Values for the various analytes evaluated were generally similar between homozygous 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. Additional mice are pending.

Gene 1127
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:
4 homozygous mutant females (164917, 164939, 172146, 172234)
3 homozygous mutant males (158301, 158303, 164910)
2 wild-type control females (164945, 164947)
2 wild-type control males (164942, 164943)

90 Day Cohort Mouse ID numbers are as follows:
4 homozygous mutant females (189717, 197401, 197402, 197404)
4 homozygous mutant males (204448, 211563, 211565, 211566)
4 wild-type control females (189715, 197400, 197403, 204447)
4 wild-type control males (199692, 211567, 230644, 230646)

180 Day Cohort Mouse ID numbers are as follows:
4 homozygous mutant females (189717, 197401, 197402, 197404)
4 homozygous mutant males (204448, 211563, 211565, 211566)
4 wild-type control females (189715, 197400, 197403, 204447)
4 wild-type control males (199692, 211567, 230644, 230646)

300 Day Cohort Mouse ID numbers are as follows:
2 homozygous mutant females (189717, 197401)
2 homozygous mutant males (204448, 211563)
2 wild-type control females (189715, 197400)
2 wild-type control males (199692, 211567)

The percentages of neutrophils and monocytes are elevated, and the percentage of lymphocytes is decreased, in two homozygous mutant males (158301, 158303). These findings are not presented as a phenotype but are included for your consideration. Although minor variations of other hematological values were present in some mice, these changes were not related to genotype and, thus, were not considered phenotypically relevant.

Gene 1127
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:
4 homozygous mutant females (164917, 164939, 172146, 172234)
3 homozygous mutant males (158300, 158301, 158303)
2 wild-type control females (158306, 164945)
2 wild-type control males (164942, 164943)

300 Day Cohort Mouse ID numbers are as follows:
2 homozygous mutant females (189717, 197401)
2 homozygous mutant males (204448, 211563)
2 wild-type control females (189715, 197400)
2 wild-type control males (199692, 211567)

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

Gene 1127
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 (189717, 197401, 197402, 197404)
4 homozygous mutant males (204448, 211563, 211565, 211566)
4 wild-type control females (189715, 197400, 197403, 204447)
4 wild-type control males (199692, 211567, 230644, 230646)

90 Day Cohort Mouse ID numbers are as follows:
4 homozygous mutant females (189717, 197401, 197402, 197404)
4 homozygous mutant males (204448, 211563, 211565, 211566)
4 wild-type control females (189715, 197400, 197403, 204447)
4 wild-type control males (199692, 211567, 230644, 230646)

180 Day Cohort Mouse ID numbers are as follows:
4 homozygous mutant females (189717, 197401, 197402, 197404)
4 homozygous mutant males (204448, 211563, 211565, 211566)
4 wild-type control females (189715, 197400, 197403, 204447)
4 wild-type control males (199692, 211567, 230644, 230646)

300 Day Cohort Mouse ID numbers are as follows:
3 homozygous mutant females (197401, 197402, 197404)
3 wild-type control females (197400, 197403, 204447)
1 wild-type control male (230644)

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.