Gene 342
Summary of Phenotypic Analysis

Changes related to genotype:

Behavior:  Homozygous mutant mice exhibited an increase in thermal response latency during hot plate testing.

There were no other significant differences detected in the homozygous mutant animals 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 homozygous mutant mice were produced by intercrossing F1N1 heterozygous males and females.  F2N2 homozygous mutant mice were produced by intercrossing F2N1 heterozygous mice.

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
• Aging studies
• Behavioral tests

Gene 342
Expression Summary

RT-PCR Summary:
Low levels of RNA transcripts are detectable in brain, cortex, subcortical region, cerebellum, brainstem, olfactory bulb, spinal cord, eye, salivary gland, testis and ovaries.

No RNA transcripts are detectable in Harderian glands, heart, lung, liver, pancreas, kidney, spleen, thymus, lymph nodes, bone marrow, skin, gallbladder, urinary bladder, pituitary gland, adrenal gland, skeletal muscle, tongue, stomach, small intestine, large intestine, cecum, epididymis, seminal vesicle, coagulating gland, prostate gland, uterus and white fat.

LacZ Summary:
LacZ (beta-galactosidase) expression is detectable in brain, spinal cord, retina, and testis.

Expression:
Brain
In wholemount staining strong lacZ expression is detectable in several regions of the brain including hippocampus, thalamus, cerebellum and parts of the brainstem.  Weaker lacZ expression is detectable in cortex.  On coronal sections of cerebrum very strong lacZ expression is detectable in many cells of the caudate putamen, thalamus and pyramidal cell layer of the hippocampus.  Less frequent staining is detectable in hippocampus and cortex.   Moderate lacZ expression is detectable in indusium griseum and a few cells in amygdala.  On coronal sections of cerebellum very strong lacZ expression is detectable in cerebellar nuclei.  Moderate to strong lacZ expression is also detectable in a few cells of the molecular and granular layers.  On coronal sections of brainstem very strong lacZ expression is detectable in many cells scattered throughout the brainstem.

Spinal cord
Very strong lacZ expression is detectable in many nuclei of ventral horns and central gray matter.  Moderate lacZ expression is detectable in a few nuclei of the dorsal horns.

Eyes
Moderate lacZ expression is detectable in some cells of the inner nuclear and ganglion cell layers of the retina.

Male Reproductive Systems
Testis
Moderate lacZ expression is detectable in spermatogenic cells of the seminiferous tubules.

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

Gene 342
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:
2 homozygous mutant females (214416, 214430)
4 homozygous mutant males (192046, 192049, 192050, 192053)
2 wild-type control females (211931, 214418)
2 wild-type control males (192047, 192099)

300 Day Cohort Mouse ID numbers are as follows:
2 homozygous mutant females (198847, 222742)
2 homozygous mutant males (235114, 235117)
1 wild-type control female (235111)

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. 

Certain gross changes, including black discoloration of the spleen, were present that occasionally occur spontaneously in mice of this age group.  This correlated with histopathologic splenic pigment deposition.  In this target, such lesions are present in 3 homozygous males (192049, 192050, 182053).  We are not reporting these findings as phenotypic changes, but we present them here for your consideration.  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:

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 342
Histopathology

There were no significant differences detected in the homozygous 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:
2 homozygous mutant females (214416, 214430)
4 homozygous mutant males (192046, 192049, 192050, 192053)
2 wild-type control females (211931, 214418)
2 wild-type control males (192047, 192099)

300 Day Cohort Mouse ID numbers are as follows:
1 homozygous mutant female (198847)
1 homozygous mutant male (235114)

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, 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, including pigment deposition of the spleen, are present in three homozygous males (192049, 192050, 182053) that correlated with gross black splenic discoloration and that could occur spontaneously in mice of this age group. Therefore, we are not reporting these findings as phenotypic changes, but we present them here for your consideration. 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, lesions due to procedural artifacts, and/or lesions of a nonspecific etiology. They are not considered to be genotype related.

Gene 342
Hematology

There were no significant differences 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:
2 homozygous mutant females (214416, 214430)
4 homozygous mutant males (192046, 192049, 192053, 194327)
2 wild-type control females (211931, 214428)
2 wild-type control males (192099, 209676)

90 Day Cohort Mouse ID numbers are as follows:
3 homozygous mutant females (198847, 222742, 222743)
4 homozygous mutant males (235114, 235117, 235118, 245462)
5 wild-type control females (198854, 235111, 235122, 235123, 235128)
4 wild-type control males (235132, 235135, 237310, 237311)

180 Day Cohort Mouse ID numbers are as follows:
3 homozygous mutant females (198847, 222742, 222743)
4 homozygous mutant males (235114, 235117, 235118, 245462)
6 wild-type control females (198854, 235111, 235113, 235122, 235123, 235128)
4 wild-type control males (235132, 235135, 237310, 237311)

300 Day Cohort Mouse ID numbers are as follows:
2 homozygous mutant females (198847, 222742)
2 homozygous mutant males (235114, 235117)
2 wild-type control females (235111, 235113)
1 wild-type control male (235135)

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

Gene 342
Clinical Chemistry

There were no significant differences in the homozygous mutant animals when compared with age- and gender-matched wild-type control mice. Pending additional mice.

Serum samples from the following mice were evaluated by a clinical biochemistry panel.

49 Day Cohort Mouse ID numbers are as follows:
2 homozygous mutant females (214416, 214430)
4 homozygous mutant males (192046, 192050, 192053, 194327)
2 wild-type control females (211931, 214419)
2 wild-type control males (192047, 192099)

90 Day Cohort Mouse ID numbers are as follows:
2 homozygous mutant females (222742, 222743)
4 homozygous mutant males (235114, 235117, 235118, 245462)
5 wild-type control females (235111, 235113, 235122, 235123, 235128)
4 wild-type control males (235132, 235135, 237310, 237311)

180 Day Cohort Mouse ID numbers are as follows:
1 homozygous mutant female (222742)
4 homozygous mutant males (235114, 235117, 235118, 245462)
5 wild-type control females (235111, 235113, 235122, 235123, 235128)
4 wild-type control males (235132, 235135, 237310, 237311)

300 Day Cohort Mouse ID numbers are as follows:
2 homozygous mutant females (198847, 222743)
2 homozygous mutant males (235114, 235117)
2 wild-type control females (235111, 235113)
2 wild-type control males (235132, 235135)

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.

Gene 342
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:
2 homozygous mutant females (214416, 214430)
4 homozygous mutant males (192046, 192049, 192050, 192053)
2 wild-type control females (211931, 214418)
2 wild-type control males (192047, 192099)

300 Day Cohort Mouse ID numbers are as follows:
2 homozygous mutant females (198847, 222742)
2 homozygous mutant males (235114, 235117)
2 wild-type control females (235111, 235113)
2 wild-type control males (235132, 235135)

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 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 342
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:
2 homozygous mutant females (214416, 214430)
4 homozygous mutant males (192046, 192049, 192050, 192053)
2 wild-type control females (211931, 214418)
2 wild-type control males (192047, 192099)

300 Day Cohort Mouse ID numbers are as follows:
2 homozygous mutant females (198847, 222742)
2 homozygous mutant males (235114, 235117)
2 wild-type control females (235111, 235113)
2 wild-type control males (235132, 235135)

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 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 were regarded as biologically significant or genotype related.

Gene 342
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:
3 homozygous mutant females (198847, 222742, 222743)
4 homozygous mutant males (235114, 235117, 235118, 245462)
6 wild-type control females (198854, 235111, 235113, 235122, 235123, 235128)
4 wild-type control males (235132, 235135, 237310, 237311)

90 Day Cohort Mouse ID numbers are as follows:
3 homozygous mutant females (198847, 222742, 222743)
4 homozygous mutant males (235114, 235117, 235118, 245462)
6 wild-type control females (198854, 235111, 235113, 235122, 235123, 235128)
4 wild-type control males (235132, 235135, 237310, 237311)

180 Day Cohort Mouse ID numbers are as follows:
3 homozygous mutant females (198847, 222742, 222743)
4 homozygous mutant males (235114, 235117, 235118, 245462)
6 wild-type control females (198854, 235111, 235113, 235122, 235123, 235128)
4 wild-type control males (235132, 235135, 237310, 237311)

300 Day Cohort Mouse ID numbers are as follows:
1 homozygous mutant female (198847)
1 homozygous mutant male (235118)
4 wild-type control females (235113, 235122, 235123, 235128)
4 wild-type control males (235132, 235135, 237310, 237311)

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 342
Behavior

• Homozygous mutant mice exhibited a significant increase in thermal response latency during hot plate testing 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 response/PPI test, Tail flick test, Hot plate test, and Metrazol test.

Mouse ID numbers are as follows for the N2 generation:
10 homozygous mutant males (360524, 370456, 370464, 370465, 370467, 377814, 380706, 390045, 405294, 413629)
8 wild-type control males (360523, 370462, 370463, 377813, 380704, 390047, 405293, 413655)

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.  F2N2 homozygous mutant mice were produced by intercrossing F2N1 heterozygous mice.

Behavior Findings:
When compared to age- and gender-matched wild-type control mice, homozygous mutant mice exhibited a significantly increased latency to lick a hindpaw or jump during hot plate testing, indicating a possible increased pain threshold phenotype.

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

Gene 342
Fertility

Both homozygous mutant males and females showed reduced fertility. 

Two 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.  One mating pair (232402 x 224639) had no pups.  The number of pups born from three litters of the second mating pair was recorded.  Three weeks later, the live pups were counted and weaned.

Mouse ID numbers are as follows:

2 homozygous mutant males (232402, 198846)

2 homozygous mutant females (224639, 198836)