Gene: 647	Name: Galr1	Family: GPCR	Subfamily: Galanin	Accession: Y15004	GI: 2465113

Gene 647
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. 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.

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 test.
. Aging studies 

Gene 647
Behavior

Changes related to genotype:

• 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, Hot plate test, Startle/PPI, Tail flick test, and Metrazol test.

Mouse ID numbers are as follows:

10 homozygous mutant males (155337, 158063, 160206, 179820, 179824, 191609, 193966, 193967, 201156, 201159)

10 wild-type control males (155336, 155342, 158066, 160203, 160204, 179821, 191622, 193957, 201160, 201161)

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 647
Fertility

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

Three homozygous mutant male and six homozygous mutant female mice were set up in a fertility mating one on one with each other at seven to ten weeks of age.  One male (146364) was set up with three different females,  two of which were replacements each time a female died.  The number of pups born from three litters of the three matings was recorded.  Three weeks later, the live pups were counted and weaned.

Mouse ID numbers are as follows:

3 homozygous mutant males (146364, 146366, 158069)

5 homozygous mutant females (169810, 146548, 146389, 155348, 155343)

Gene 647
Expression Summary

RT-PCR Summary:
The strongest signals are detectable in pancreas.

Lower levels of RNA transcripts are also detectable in: brain, brainstem, spinal cord, pancreas pituitary gland, adrenal gland, salivary gland, tongue, stomach, small intestine, large intestine, cecum, testis and epididymis.

No RNA transcripts are detectable in: cortex, subcortical region, cerebellum, olfactory bulb, eye, Harderian gland, heart, lung, liver, kidney, spleen, thymus, lymph nodes, bone marrow, skin, gallbladder, urinary bladder, skeletal muscle, seminal vesicle, coagulating gland, prostate gland, ovary, uterus and white fat.

LacZ Summary:
LacZ (beta-galactosidase) expression is not detectable in any tissues or organs analyzed: brain, spinal cord, sciatic nerve, 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, male and female reproductive systems.

Gene 647
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.

20 Day Cohort Mouse ID numbers are as follows:
1 homozygous mutant female (203697)
1 homozygous mutant male (203696)

49 Day Cohort Mouse ID numbers are as follows:
3 homozygous mutant females (146380, 146382, 146386)
3 homozygous mutant males (146353, 146358, 146361)
2 wild-type control females (146378, 146379)
2 wild-type control males (146374, 146377)

300 Day Cohort Mouse ID numbers are as follows:
2 homozygous mutant females (146372, 158079)
2 homozygous mutant males (198829, 203673)
1 wild-type control female (146369)
2 wild-type control males (198830, 203682)

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

Densitometric Findings:

Incidental densitometric differences may have been present between some mice. These findings are considered to represent background differences occasionally seen in this strain of mice, differences due to spontaneous disease, age-related changes, and/or differences of a nonspecific etiology. They are not considered to be genotype related.

Gene 647
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.

20 Day Cohort Mouse ID numbers were as follows:
1 homozygous mutant female (203697)
1 homozygous mutant male (203696)

49 Day Cohort Mouse ID numbers were as follows:
3 homozygous mutant females (146380, 146382, 146386)
3 homozygous mutant males (146353, 146358, 146361)
2 wild-type control females (146378, 146379)
2 wild-type control males (146374, 146377)

300 Day Cohort Mouse ID numbers were as follows:
2 homozygous mutant females (146372, 158079)
2 homozygous mutant males (198829, 203673)
2 wild-type control females (146369, 158078)
2 wild-type control males (198830, 203682)

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 were present in some tissues such as otitis in two homozygous mice (203697, 203696), which correlated with the clininal symptoms of ataxia. 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 a nonspecific etiology. They were not considered to be genotype related.

Gene 647
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.

20 Day Cohort Mouse ID numbers were as follows:
1 homozygous mutant female (203697)
1 homozygous mutant male (203696)

49 Day Cohort Mouse ID numbers were as follows:
3 homozygous mutant females (146380, 146382, 146386)
3 homozygous mutant males (146353, 146358, 146361)
2 wild-type control females (146378, 146379)
2 wild-type control males (146374, 146377)

300 Day Cohort Mouse ID numbers were as follows:
2 homozygous mutant females (146372, 158079)
2 homozygous mutant males (198829, 203673)
2 wild-type control females (146369, 158078)
2 wild-type control males (198830, 203682)

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, 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 647
Clinical Chemistry

There were no significant differences detected 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 (146380, 146382, 146386)
3 homozygous mutant males (146353, 146358, 146361)
2 wild-type control females (146378, 146379)
2 wild-type control males (146374, 146377)

90 Day Cohort Mouse ID numbers are as follows:
3 homozygous mutant females (146372, 158081, 158082)
3 homozygous mutant males (198829, 203673, 214368)
3 wild-type control females (146369, 158083, 175278)
3 wild-type control males (198830, 203682, 203685)

180 Day Cohort Mouse ID numbers are as follows:
4 homozygous mutant females (146372, 158079, 158081, 158082)
4 homozygous mutant males (198829, 203673, 203674, 209726)
4 wild-type control females (158078, 158083, 164574, 169811)
4 wild-type control males (198830, 203682, 203685, 209735)

300 Day Cohort Mouse ID numbers are as follows:
2 homozygous mutant females (146372, 158079)
1 homozygous mutant male (203673)
2 wild-type control females (146369, 158078)
2 wild-type control males (198830, 203682)

Values for the various analytes evaluated were generally similar between homozygous 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 647
Hematology

There were no significant differences detected 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 (146382, 146386, 164575)
3 homozygous mutant males (146353, 146358, 146361)
2 wild-type control females (146378, 146379)
2 wild-type control males (146377, 160183)

90 Day Cohort Mouse ID numbers are as follows:
4 homozygous mutant females (146372, 158079, 158081, 158082)
4 homozygous mutant males (198829, 203673, 203674, 209726)
4 wild-type control females (146369, 158078, 158080, 158083)
4 wild-type control males (198830, 203682, 203685, 209735)

180 Day Cohort Mouse ID numbers are as follows:
4 homozygous mutant females (146372, 158079, 158081, 158082)
4 homozygous mutant males (198829, 203673, 203674, 209726)
4 wild-type control females (146369, 158078, 158083, 164574)
4 wild-type control males (198830, 203682, 203685, 209735)

300 Day Cohort Mouse ID numbers are as follows:
2 homozygous mutant females (146372, 158079)
2 homozygous mutant males (198829, 203673)
2 wild-type control females (146369, 158078)
2 wild-type control males (198830, 203682)

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

Gene 647
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.

20 Day Cohort Mouse ID numbers were as follows:
1 homozygous mutant female (203697)
1 homozygous mutant male (203696)

49 Day Cohort Mouse ID numbers were as follows:
3 homozygous mutant females (146380, 146382, 146386)
3 homozygous mutant males (146353, 146358, 146361)
2 wild-type control females (146378, 146379)
2 wild-type control males (146374, 146377)

300 Day Cohort Mouse ID numbers were as follows:
2 homozygous mutant females (146372, 158079)
2 homozygous mutant males (198829, 203673)
2 wild-type control females (146369, 158078)
2 wild-type control males (198830, 203682)

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 behavior comparedto wild-type control mice, such as ataxia in two homozygous mutants (203697, 203696), which correlated with the histological observation of unilateral otitis. 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 647
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 (146372, 158079, 158081, 158082)
4 homozygous mutant males (198829, 203673, 203674, 209726)
4 wild-type control females (146369, 158078, 158080, 158083)
4 wild-type control males (198830, 203682, 203685, 209735)

90 Day Cohort Mouse ID numbers are as follows:
4 homozygous mutant females (146372, 158079, 158081, 158082)
4 homozygous mutant males (198829, 203673, 203674, 209726)
4 wild-type control females (146369, 158078, 158080, 158083)
4 wild-type control males (198830, 203682, 203685, 209735)

180 Day Cohort Mouse ID numbers are as follows:
4 homozygous mutant females (146372, 158079, 158081, 158082)
4 homozygous mutant males (198829, 203673, 203674, 209726)
5 wild-type control females (146369, 158078, 158080, 158083, 164574)
4 wild-type control males (198830, 203682, 203685, 209735)

300 Day Cohort Mouse ID numbers are as follows:
2 homozygous mutant females (158081, 158082)
2 homozygous mutant males (203674, 209726)
2 wild-type control females (158080, 164574)
2 wild-type control males (203685, 209735)

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.