Gene: 705	Name: Capn5	Family: Protease	Subfamily: Cysteine	Accession: Y10656	GI: 2065241

Gene 705
Summary of Phenotypic Analysis

There were no significant differences detected in the homozygous mutant animals 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, whereas, wild-type control and heterozygous mutant mice were present. Data suggests that homozygous mutant progeny die in utero at or before E3.5.

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 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.
• Densitometry.
• Fertility evaluation.
• Embryonic development.
• Behavioral tests.
• Fertility tests.

Gene 705
Behavior


Changes related to genotype:
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 six behavioral tasks: Open field test, Tail suspension test, Rotarod test, Hot plate test, Startle/PPI, and Metrazol test.

Mouse ID numbers are as follows:

9 heterozygous mutant males (106834, 106838, 106839, 106842, 106844, 112701, 112702, 114127, 114130)
10 wild-type control males (106830, 106835, 106837, 106841, 106843, 112276, 112699, 112700, 114128, 114129)

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 705
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 (335554, 350635, 359598)

3 homozygous mutant females ()335561, 350638, 379281

 

 

Gene 705
Expression Summary

RT-PCR Summary:
The highest levels of RNA transcripts are detectable in salivary gland.

Lower levels of RNA transcripts are detectable in subcortical region, cerebellum, brainstem, olfactory bulb, spinal cord, eye, Harderian glands, lung, liver, pancreas, kidney, spleen, thymus, bone marrow, skin, gallbladder, urinary bladder, stomach, large intestine, cecum, epididymis, seminal vesicle, coagulating gland, prostate gland, ovaries and uterus. 

No RNA transcripts are detectable in: brain, cortex, subcortical region, cerebellum, brainstem, olfactory bulb, spinal cord, eye, Harderian glands, heart, lymph nodes, pituitary gland, adrenal gland, skeletal muscle, tongue, small intestine, testis and white fat.

LacZ Summary:
Striking lacZ (beta-galactosidase) expression is detectable in brain, spinal cord and ganglia. Other tissues displaying X-Gal staining are kidney, urinary bladder, salivary glands, and male reproductive system.

Expression:
Brain
In wholemount staining, the whole brain stains deeply blue. On frozen sections, all regions of forebrain and midbrain show strong lacZ expression. In cerebellum, the strongest expression is seen in Purkinje cells with X-Gal signals being present in all layers. Strong X-Gal signals are detectable throughout the brainstem.

Spinal cord
LacZ expression is detectable throughout the spinal cord, in the white matter, gray matter and central canal. Motor neurons stain strongly.

Kidney
Weak lacZ expression is detectable in tubular cells of cortex and medulla. Strong lacZ signals are detectable in the papilla.

Urinary Bladder
Weak X-Gal signals are detectable in the epithelium.

Salivary Glands
Scattered X-Gal staining is seen in few acinar cells of the submandibular salivary glands.

Male Reproductive Systems
Coagulating Gland
Several epithelial cells show weak to moderate X-Gal staining.

Penis
Many nerve cells stain show weak to moderate X-Gal staining. Many fibroblast-like cells throughout the body display X-Gal signals.

Female Reproductive Systems
Vagina/Cervix
Ganglia express lacZ strongly.

No Expression:
LacZ expression is not detected in: sciatic nerve, eye, Harderian glands, thymus, spleen, lymph nodes, bone marrow, aorta, heart, lung, liver, gall bladder, pancreas, trachea, larynx, esophagus, thyroid gland, parathyroid gland, adrenal glands, pituitary gland, tongue, skeletal muscle, and skin.

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Gene 705
Histopathology

There were no significant differences detected in the heterozygous mutant animals 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 (335682, 335684, 336141)
3 homozygous mutant males (335695, 335696, 335698)
2 wild-type control females (106807, 106814)
2 wild-type control males (106801, 106802)

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.

Certain histopathological lesions are present, including a jejunal adenocarcinoma in a wild-type male (118343) and splenic and hepatic lymphoma in another wild-type male (118345), that can occur spontaneously in mice of this age group. 

Other 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, and/or lesions of nonspecific etiology. They are not considered to be genotype related.

Gene 705
Necropsy

There were no significant differences detected in the homozygous mutant animals 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:
3 homozygous mutant females (335682, 335684, 336141)
3 homozygous mutant males (335695, 335696, 335698)
2 wild-type control females (106807, 106814)
2 wild-type control males (106801, 106802)

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 heterozygous 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, and/or lesions of 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. Certain incidental findings were present, including at 300 days, increased liver and heart weights, and increased heart weight to body weight ratio, in one heterozygous male (106828). These differences did not correlate with any histopathologic alterations.

Gene 705
Clinical Chemistry

There were no significant differences detected in the homozygous mutant animals 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 homozygous mutant females (335682, 335684, 336141)
3 homozygous mutant males (335695, 335696, 335698)
2 wild-type control females (106807, 106814)
2 wild-type control males (106801, 106802)

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 705
Hematology

There were no significant differences detected in the homozygous mutant animals 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 (335684, 336141, 350622)
3 homozygous mutant males (335695, 335696, 346123)
2 wild-type control females (106807, 106814)
2 wild-type control males (106801, 106802)

Although minor variations of hematological values were present in some animals, these changes were not consistent with genotype and, thus, were not considered phenotypically relevant.

Gene 705
Physical Examination

There were no significant differences detected in the homozygous mutant animals 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 (335682, 335684, 336141)
3 homozygous mutant males (335695, 335696, 335698)
2 wild-type control females (106807, 106814)
2 wild-type control males (106801, 106802)

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

Gene 705
Summary of Embryonic Development

Data suggests that homozygous mutant embryos die at or before E3.5

Embryos were isolated at 8.5 to 14.5 days post coitum. No homozygous offspring were detected by conventional PCR at any stage. To examine earlier development, blastocysts (E3.5) were also isolated and either genotyped directly or grown in tissue culture for 6 days, the resulting colonies of cells were then genotyped.  No homozygous blastocysts were detected by PCR.

Offspring were isolated at E3.5 to E14.5.
Nine litters were examined comprising of 81 blastocysts, blastocyst outgrowths, resorptions and partial resorptions, of which 72 were successfully genotyped.

Litter	Embryonic stage	+/+	+/-	-/-	complete resorption/unknown
1	3.5+6 days in culture	0	0	0	4
2	3.5+6 days in culture	1	4	0	2
3	3.5+6 days in culture	1	2	0	1
4	3.5+6 days in culture	3	8	0	0
5	3.5+6 days in culture	0	7	0	2
6	7.5	2	7	0	0
7	8.5	3	9	0	0
8	12.5	2	10	0	0
9	14.5	4	9	0	0