Gene: 614	Name: Cnr2	Family: GPCR	Subfamily: Cannabinoid	Accession: X86405	GI: 791081

Gene 614
Summary of Phenotypic Analysis

There were no significant differences detected in the homozygous mutant and heterozygous 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. F2 homozygous mutant mice were produced by intercrossing F1 heterozygous males and females.

Wild-type control mice, as well as homozygous mutant and heterozygous 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.
· Behavioral testing.
· Fertility evaluation.
· Aging studies.

Gene 614
Behavior 

Changes related to genotype:

There were no significant differences detected in the homozygous 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 six behavioral tasks: Open field test, Tail suspension test, Rotarod test, Hot plate test, Startle/PPI, and Metrazol test.

Mouse ID numbers for the N0 generation are as follows:

10 homozygous mutant males (61502, 56069, 56067, 51651, 51652, 51649, 51647, 51644, 51642, 50487)
10 wild-type control males (50486, 49741, 49738, 49740, 49737, 46491, 45138, 45137, 46489, 49729)

Mouse ID numbers for the N1 generation are as follows:

11 homozygous mutant males (140464, 161355, 121399, 161352, 134186, 127302, 121409, 127304, 140468, 121397, 121415)
12 wild-type control males (121410, 121407, 121400, 127301, 121412, 140469, 134188, 127303, 161347, 161354, 140465, 127300)

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 614
Fertility


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

Two to three homozygous mutant mice of each gender were set up in a fertility mating with a wild-type mate at seven to eight weeks of age. The number of pups born from one to three litters were recorded. Three weeks later, the live pups were counted and weaned.

Mouse ID numbers are as follows:

2 homozygous mutant males (59505, 59504)
2 homozygous mutant females (49718, 45032)

Gene 614
Expression Summary

RT-PCR Summary:
RNA transcripts are detectable in lung, liver, thymus, spleen, lymph nodes, bone marrow, skin and testis.

No RNA transcripts are detectable in: brain, cortex, subcortical region, cerebellum, brainstem, olfactory bulb, eye, heart, pancreas, kidneys, gallbladder, urinary bladder, pituitary gland, adrenal gland, salivary gland, skeletal muscle, tongue, stomach, small intestine, large intestine, cecum, epididymis, seminal vesicle, coagulating gland, prostate gland, ovary and uterus.
 

Gene 614
Histopathology

There were no significant differences detected in the homozygous mutant and 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 (36789, 38259, 38260)
4 homozygous mutant males (36781, 42977, 45417, 60715)
1 heterozygous mutant female (36787)
1 heterozygous mutant male (38255)
2 wild-type control females (36790, 38261)
2 wild-type control males (36782, 36784)

300 Day Cohort Mouse ID numbers are as follows:
2 homozygous mutant females (31724, 42017)
2 homozygous mutant males (49715, 49719)
2 wild-type control females (42975, 42994)
2 wild-type control males (31722, 42992)

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, 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 614
Necropsy

There were no significant differences detected in the homozygous mutant and heterozygous 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 Mouse ID numbers are as follows:
3 homozygous mutant females (36789, 38259, 38260)
3 homozygous mutant males (42977, 45417, 60715)
1 heterozygous mutant female (36787)
1 heterozygous mutant male (38255)
2 wild-type control females (36790, 38261)
2 wild-type control males (36782, 36784)

300 Day Cohort Mouse ID numbers are as follows:
2 homozygous mutant females (31724, 42017)
2 homozygous mutant males (49715, 49719)
2 wild-type control females (42975, 42994)
2 wild-type control males (31772, 42992)

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 614
Clinical Chemistry

There were no significant differences detected in the homozygous mutant and heterozygous mutant animals 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 (38259, 73638, 73641)
3 homozygous mutant males (60712, 60715, 68116)
1 heterozygous mutant female (73639)
2 heterozygous mutant males (64392, 66396)
2 wild-type control females (68115, 73640)
3 wild-type control males (60714, 73631)

90 Day Cohort Mouse ID numbers are as follows:
4 homozygous mutant females (31724, 42017, 42983, 45031)
5 homozygous mutant males (49715, 49719, 59504, 64376, 64395)
5 wild-type control females (42973, 42975, 42976, 42994, 64394)
3 wild-type control males (31722, 42992, 43747)

180 Day Cohort Mouse ID numbers are as follows:
3 homozygous mutant females (31724, 42017, 45031)
3 homozygous mutant males (49715, 49719, 64376)
5 wild-type control females (42973, 42975, 42976, 42994, 64394)
4 wild-type control males (31722, 42992, 43747, 66372)

300 Day Cohort Mouse ID numbers are as follows:
2 homozygous mutant females (31724, 42017)
2 homozygous mutant males (49715, 49719)
2 wild-type control females (42975, 42994)
2 wild-type control males (31722, 42992)

Values for the various analytes evaluated were generally similar between homozygous mutant, heterozygous mutant, and wild-type control mice. Variations in clinical chemistry values, if present, were not consistent with genotype and, thus, were not considered phenotypically relevant.

Gene 614
Hematology

There were no significant differences detected in the homozygous mutant and heterozygous 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. The data were compiled from N0F2 and N1F2 generations.

49 Day Cohort Mouse ID numbers are as follows:
3 homozygous mutant females (38260, 73638, 73641)
3 homozygous mutant males (60715, 66376, 68116)
1 heterozygous mutant female (73639)
1 heterozygous mutant male (73630)
2 wild-type control females (60728, 73640)
2 wild-type control males (60716, 71500)

90 Day Cohort Mouse ID numbers are as follows:
3 homozygous mutant females (42017, 42983, 45031)
5 homozygous mutant males (49697, 49715, 49719, 64376, 64395)
5 wild-type control females (42973, 42975, 42976, 42994, 64394)
2 wild-type control males (42992, 43747)

180 Day Cohort Mouse ID numbers are as follows:
3 homozygous mutant females (31724, 42983, 45031)
4 homozygous mutant males (49715, 49719, 119881, 119882)
4 wild-type control females (42973, 42975, 42976, 42994)
4 wild-type control males (31722, 42992, 43747, 66372)

300 Day Cohort Mouse ID numbers are as follows:
2 homozygous mutant females (31724, 42017)
2 homozygous mutant males (49719, 66383)
2 wild-type control females (42975, 42994)
3 wild-type control males (31722, 42992, 66386)

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 614
Physical Examination

There were no significant differences detected in the homozygous mutant and heterozygous 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 (36789, 38259, 38260)
4 homozygous mutant males (36781, 42977, 45417, 60715)
1 heterozygous mutant female (36787)
1 heterozygous mutant male (38255)
2 wild-type control females (36790, 38261)
2 wild-type control males (36782, 36784)

300 Day Cohort Mouse ID numbers are as follows:
2 homozygous mutant females (31724, 42017)
2 homozygous mutant males (49715, 49719)
2 wild-type control females (42975, 42994)
2 wild-type control males (31772, 42992)

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.)

Certain physical findings, including alopecia, are present in one female homozygote (42017) and two male homozygotes (49714, 49719) 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.

Individual homozygous mutant and heterozygous mutant mice had only occasional other 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 614
Aging Metrics

There were no significant differences detected in the homozygous mutant animals 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 (42017, 42983, 45031)
4 homozygous mutant males (49697, 49715, 49719, 64395)
5 wild-type control females (42973, 42975, 42976, 42994, 64394)
2 wild-type control males (42992, 43747)

90 Day Cohort Mouse ID numbers are as follows:
4 homozygous mutant females (31724, 42017, 42983, 45031)
4 homozygous mutant males (49697, 49715, 49719, 64395)
4 wild-type control females (42973, 42975, 42976, 42994)
3 wild-type control males (31722, 42992, 43747)

180 Day Cohort Mouse ID numbers are as follows:
4 homozygous mutant females (31724, 42017, 42983, 45031)
4 homozygous mutant males (49715, 49719, 64376, 64395)
5 wild-type control females (42973, 42975, 42976, 42994, 64378)
4 wild-type control males (31722, 42992, 43747, 66372)

300 Day Cohort Mouse ID numbers are as follows:
4 homozygous mutant females (31724, 42017, 42983, 45031)
5 homozygous mutant males (49715, 49719, 64374, 64376, 64395)
5 wild-type control females (42973, 42975, 42976, 42994, 64378)
3 wild-type control males (31722, 42992, 66372)


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.