Gene: 619	Name: Htr5a	Family: GPCR	Subfamily: Serotonin	Accession: Z18278	GI: 49758

Gene 619
Summary of Phenotypic Analysis


There were no genotype-related or biologically significant differences between mutant mice and gender-matched wild-type control mice

ES cells derived from the 129/SvJ x 129/Sv-CP 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 heterozygous and homozygous mutant mice were evaluated for the following examimations or tests:

· Physical examinations.
· Necropsy including body length, body weight, and organ weight measurements.
· Histological examination of tissues and organs.
· Bone marrow section evaluations.
· Complete blood counts and differentials.
· Clinical chemistry panels.
· Behavioral tests.
. Fertility evaluation.
. Aging studies.

Gene 619
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. However, see behavior findings below.

Homozygous mutant and wild-type control mice from both the No and N1 generation were evaluated for phenotypic changes by testing on six behavioral tasks: Open field test, Tail suspension test, Rotarod test, Hot plate test, Startle/PPI (for N1), and Metrazol test.

Mouse ID numbers for the N0 generation are as follows:

10 homozygous mutant males (59840, 59838, 59824, 58177, 58167, 58165, 54585, 54584, 53285)
17 wild-type control males (52523, 51716, 49907, 49908, 49679, 49674, 45789, 45781, 45216, 45217, 44453, 44455, 44457, 45782, 49673, 49676, 49689)

Mouse ID numbers for the N1 generation are as follows:

12 homozygous mutant males (82001, 82874, 82876, 82877, 86316, 89184, 89185, 89187, 93635, 93639, 93649, 93650)
9 wild-type control males (82000, 86315, 86317, 89171, 89183, 89186, 93632, 93637, 93651)

ES cells derived from the 129/SvJ x 129/Sv-+p+Tyr-c MgfSl-J/+ mouse substrain were used to generate chimeric mice. F1 N0 mice were generated by breeding with C57BL/6 females. F2 N0 homozygous mutant mice were produced by intercrossing F1 N0 heterozygous males and females. 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 mutant and wild-type control mice for any of the parameters evaluated during behavior testing. However, note that there were 2 wild-type mice from the N1 generation (mouse # 82000, 89183) which reached the 60 second time cutoff for the hot plate test. If these mice are included in an N1 generation analysis of the hot plate data then there is a statistically significant difference between homozygous mutants and wild-types.

Gene 619
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:

3 homozygous mutant males (59490, 46620, 46619)
2 homozygous mutant females (46630, 52518)

Gene 619
Expression Summary

Taqman Summary:
Low levels of RNA transcripts are detectable in brain, cortex, subcortical region, cerebellum, brainstem, olfactory bulb, spinal cord, eye, skin, pituitary gland, adrenal gland, ovary, and white fat.   

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

LacZ Summary:
LacZ (beta-galactosidase) expression is detectable in brain and esophagus. In brain, staining is restricted to the cerebellum with Purkinje cells and nuclei in the granular layer displaying expression.

Expression:
Brain
In wholemount staining, lacZ expression is detectable solely  in cerebellum. Coronal sections of the cerebellum reveal that X-Gal signals are restricted to the Purkinje cell layer. However, not every Purkinje cell stains. In addition, distinct nuclei adjacent to the Purkinje cells exhibit staining.

Esophagus
Strong X-gal signals are present in squamous epithelial cells.

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

Gene 619
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 mice listed below were evaluated histologically.

49 Day Cohort Mouse ID numbers are as follows:
3 homozygous mutant females (39610, 39613, 39614)
3 homozygous mutant males (39609, 42732, 45784)
1 heterozygous mutant female (39615)
1 heterozygous mutant male (39608)
2 wild-type control females (39616, 40345)
3 wild-type control males (39619, 39620, 42733)

300 Day Cohort Mouse ID numbers are as follows:
2 homozygous mutant females (40352, 42735)
2 homozygous mutant males (46624, 46626)
2 wild-type control females (40348, 40354)
2 wild-type control males (40349, 45099)

No Significant Abnormalities:

Tissues examined and considered histologically normal (no significant abnormality): brain, pituitary gland, salivary glands, 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, skin, mammary gland, vertebrae, spinal cord, bone (cranium, sternum, femur, tibia, and stifle joint), reproductive tract including gonads, eyes, and Harderian glands.

Bone marrow was evaluated in sections of sternum, vertebrae, and/or femur and tibia. Marrow cellularity, numbers of megakaryocytes, and myeloid: erythroid (M:E) ratio were within normal limits. Myeloid and erythroid precursors show a normal maturation sequence.

Incidental lesions may have been present in some tissues. These findings are considered to represent background lesions occasionally seen in this strain of mice, spontaneous disease, and/or procedural artifacts. They are not considered to be genotype related.

Gene 619
Necropsy


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

The mice listed below were necropsied. Organ weights were obtained and gross pathological findings were recorded.

49 Day Cohort Mouse ID numbers are as follows:
3 homozygous mutant females (39610, 39613, 39614)
3 homozygous mutant males (39609, 42732, 45784)
1 heterozygous mutant female (39615)
1 heterozygous mutant male (39608)
2 wild-type control females (39616, 40345)
3 wild-type control males (39619, 39620, 42733)

300 Day Cohort Mouse ID numbers are as follows:
2 homozygous mutant females (40352, 42735)
2 homozygous mutant males (46624, 46626)
2 wild-type control females (40348, 40354)
2 wild-type control males (40349, 45099)

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 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 nonspecific etiology sometimes seen in genetically-manipulated mice. They were not considered to be genotype related.

Organ Weight Findings:

Differences in organ weights and/or organ weight to body weight ratios were present between individual mice. The variability between mice fell within our historical reference ranges and was not correlated with genotype.

Gene 619
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 mice listed below were evaluated by a clinical biochemistry panel.

49 Day Cohort Mouse ID numbers are as follows:
3 homozygous mutant females (81991, 81997, 88070)
3 homozygous mutant males (45784, 84198, 86320)
1 heterozygous mutant female (81995)
1 heterozygous mutant male (39608)
2 wild-type control females (81994, 81996)
2 wild-type control males (82014, 84212)

90 Day Cohort Mouse ID numbers are as follows:
(Some mice are older than 110 days)
3 homozygous mutant females (40352, 42735, 45214)
3 homozygous mutant males (46617, 46624, 46716)
2 wild-type control females (40354, 40351)
2 wild-type control males (40349, 39605)

180 Day Cohort Mouse ID numbers are as follows:
(Some mice are older than 200 days)
3 homozygous mutant females (40352, 45214, 45221)
3 homozygous mutant males (46624, 46626, 46716)
3 wild-type control females (40348, 40351, 40354)
1 wild-type control male (40349)

300 Day Cohort Mouse ID numbers are as follows:
3 homozygous mutant females (42735, 53277, 53287)
2 homozygous mutant males (46624, 70181)
2 wild-type control females (40354, 40348)
2 wild-type control males (40349, 45099)

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 619
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 mice listed below were evaluated by a complete blood count and differential cell count. The data are compiled from the N0F2 and N1F2 generations.

49 Day Cohort Mouse ID numbers are as follows:
3 homozygous mutant females (81997, 81998, 88070)
3 homozygous mutant males (42732, 84198, 93048)
1 heterozygous mutant female (81995)
1 heterozygous mutant male (82002)
2 wild-type control females (81996, 81994)
2 wild-type control males (82014, 84212)

90 Day Cohort Mouse ID numbers are as follows:
3 homozygous mutant females (40352, 45214, 45221)
4 wild-type control females (40354, 40348, 42754, 44458)
4 wild-type control males (40349, 39605, 45099, 44448)

180 Day Cohort Mouse ID numbers are as follows:
3 homozygous mutant females (40352, 45214, 45221)
2 wild-type control females (40354, 40351)
2 wild-type control males (40349, 39605)

300 Day Cohort Mouse ID numbers are as follows:
1 homozygous mutant female (42735)
2 homozygous mutant males (46626, 46624)
2 wild-type control females (40354, 54581)
2 wild-type control males (40349, 45099)

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 619
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 mice listed below were evaluated by physical examination.

49 Day Cohort Mouse ID numbers are as follows:
3 homozygous mutant females (39610, 39613, 39614)
3 homozygous mutant males (39609, 42732, 45784)
1 heterozygous mutant female (39615)
1 heterozygous mutant male (39608)
2 wild-type control females (39616, 40345)
3 wild-type control males (39619, 39620, 42733)

300 Day Cohort Mouse ID numbers are as follows:
2 homozygous mutant females (40352, 42735)
2 homozygous mutant males (46624, 46626)
2 wild-type control females (40348, 40354)
2 wild-type control males (40349, 45099)

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, feces color, feces exam, 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 exam, mice in cage, respiration, skin appearance, snout, swelling - joints, tail, teeth color, teeth length, urine, urine color, urine exam and whiskers (gender specific observables apply to 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, procedural artifacts, and/or findings of nonspecific etiology. However, none of these differences was regarded as biologically significant or genotype related.

Gene 619
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:
4 homozygous mutant females (40352, 42735, 45214, 45221)
4 homozygous mutant males (46617, 46624, 46626, 46716)
4 wild-type control females (40348, 40351, 40354, 44458)
4 wild-type control males (39605, 40349, 44448, 45099)

90 Day Cohort Mouse ID numbers are as follows:
4 homozygous mutant females (40352, 42735, 45214, 45221)
4 homozygous mutant males (46617, 46624, 46626, 46716)
4 wild-type control females (40348, 40351, 40354, 44458)
4 wild-type control males (39605, 40349, 44448, 45099)

180 Day Cohort Mouse ID numbers are as follows:
2 homozygous mutant females (40352, 42735)
4 homozygous mutant males (46617, 46624, 46626, 46716)
4 wild-type control females (40348, 40351, 40354, 44458)
4 wild-type control males (39605, 40349, 44448, 45099)

300 Day Cohort Mouse ID numbers are as follows:
4 homozygous mutant females (40352, 42735, 45214, 45221)
3 homozygous mutant males (46624, 46626, 46716)
3 wild-type control females (40348, 40354, 44458)
3 wild-type control males (40349, 44448, 45099)


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.