Gene: 2093	Name: Hcrtr1	Family: GPCR	Subfamily: Orphan GPCR	Accession: XM_131679	GI: 20845117

Gene 2093
Summary of Phenotypic Analysis

Behavior:  Homozygous mutant mice required a significantly lower dose of metrazol to reach various seizure stages when compared with age- and gender-matched wild-type control mice.

There were no other 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 differential cell counts
• Clinical chemistry panels
• Densitometry 
• Fertility 
• Behavioral tests

Gene 2093
Behavior

• Homozygous mutant mice required a significantly lower dose of metrazol to reach various seizure stages 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 N1 generation:
10 homozygous mutant males (320634, 335598, 335602, 335612, 335613, 342772, 342779, 348213, 350814, 362102)
25 wild-type control males (317170, 320288, 320473, 321525, 325099, 332836, 334042, 334720, 335601, 335803, 336216, 337325, 339150, 340804, 342270, 342767, 344731, 347796, 349916, 350123, 350584, 355106, 357191, 364153, 364473)

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:
When compared to age- and gender-matched wild-type control mice, homozygous mutant mice required a significantly lower dose of metrazol to reach various seizure stages, indicating a possible higher propensity towards seizure 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 2093
Fertility

Both homozygous mutant males and females had possible reduced fertility.  Their progeny that survived 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.  Two of the male and female pairs (304629 and 304636, 304630 and 304637) produced only one litter that was viable until weaning age. For the third pair (335609 and 335616) t he 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 (304629, 304630, 335609)

3 homozygous mutant females (304636, 304637, 335616)

 

 

Gene 2093
Expression Summary

Taqman Summary:
The highest levels of RNA transcripts are detectable in: whole brain, cortex, subcortical region, cerebellum, brainstem, spinal cord, pancreas, lymph nodes, skin, urinary bladder, pituitary gland, adrenal gland, small intestine and prostate gland.      

Lower levels of RNA transcripts are also detectable in: olfactory bulb, lung, liver, kidney, spleen, bone marrow, gallbladder, stomach, colon, testis, seminal vesicle, coagulating gland, ovary, uterus and white fat.

No RNA transcripts are detectable in: eye, Harderian glands, heart, thymus, salivary gland, skeletal muscle, tongue, cecum and epididymis.

LacZ Summary:
LacZ expression was detected in urinary bladder.  Faint to moderate staining was observed in a small number of cells in muscularis.

LacZ expression was not detected in:  brain, spinal cord, sciatic nerve, eyes, thymus, spleen, lymph nodes, bone marrow, aorta, heart, lung, liver, pancreas, kidney, thyroid gland, pituitary gland, adrenal glands, skeletal muscle, skin, testis, prostate gland, ovary, uterus and cervix.

Gene 2093
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:
3 homozygous mutant females (285626, 288466, 288467)
3 homozygous mutant males (294467, 316738, 316739)
2 wild-type control females (288462, 288463)
2 wild-type control males (285621, 285622)

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 2093
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:
3 homozygous mutant females (285626, 288466, 288467)
3 homozygous mutant males (294467, 316738, 316739)
2 wild-type control females (288462, 288463)
2 wild-type control males (285621, 285622)

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.

Incidental lesions were 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.

Gene 2093
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:
3 homozygous mutant females (285626, 288466, 288467)
3 homozygous mutant males (294467, 316738, 316739)
2 wild-type control females (288462, 288463)
2 wild-type control males (285621, 285622)

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. Incidental lesions were 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 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 2093

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 chemistry panel.

49 Day Cohort Mouse ID numbers are as follows:
3 homozygous mutant females (285626, 288466, 288467)
3 homozygous mutant males (294467, 316738, 316739)
2 wild-type control females (288462, 288463)
2 wild-type control males (285621, 285622)

When compared to age- and gender-matched wild-type control mice, one of three homozygous mutant females (288467) and one of three homozygous mutant males had increased levels of cholesterol (316739). Two homozygous mutant females (288466, 288467) had high level of tryglycerides. We are not reporting these findings as phenotypic changes, but we present them here for your consideration.

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 2093
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:
3 homozygous mutant females (285626, 288466, 288467)
3 homozygous mutant males (294467, 316738, 316739)
2 wild-type control females (288462, 288463)
2 wild-type control males (285621, 285622)

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

Gene 2093
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:
3 homozygous mutant females (285626, 288466, 288467)
3 homozygous mutant males (294467, 316738, 316739)
2 wild-type control females (288462, 288463)
2 wild-type control males (285621, 285622)

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.