Gene: 1521	Name: Adcy4	Family: Cyclase	Subfamily: Adenylate	Accession: NM_080435	GI: 17978249

Gene 1521
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. 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 tests

Gene 1521
Fertility

Both homozygous mutant males and females were fertile.  Their progeny 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.  The 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 (342369, 352500, 352517)

3 homozygous mutant females (342362, 352521, 352522)

Gene 1521
Expression Summary

Taqman Summary:
RNA transcripts are detectable in all tissues analyzed.

The highest levels of RNA transcripts are detectable in lung.

Moderate levels of RNA transcripts are detectable in: liver, kidney, lymph nodes, skin, gallbladder, urinary bladder, pituitary gland, adrenal gland, tongue, coagulating gland, prostate gland, uterus and white fat.

Lower levels of RNA transcripts are also detectable in: whole brain, cortex, subcortical region, cerebellum, brainstem, olfactory bulb, spinal cord, eye, Harderian glands, heart, pancreas, spleen, thymus, bone marrow, salivary gland, skeletal muscle, stomach, small intestine, cecum, colon, testis, epididymis, seminal vesicle and ovary.

LacZ Summary:
In the heart, strong lacZ expression was detected in blood vessels with moderate expression in valves.  Other organs with expression showed weak to moderate staining in some blood vessels.  
 
LacZ expression was detected in:  brain, spinal cord, spleen, aorta and heart.

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

Gene 1521
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:
4 homozygous mutant females (300629, 315134, 328205, 328210)
3 homozygous mutant males (300637, 339132, 341033)
2 wild-type control females (300630, 328203)
2 wild-type control males (300639, 300640)

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 1521
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:
4 homozygous mutant females (300629, 315134, 328205, 328210)
3 homozygous mutant males (300637, 339132, 341033)
2 wild-type control females (300630, 328203)
2 wild-type control males (300639, 300640)

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, adrenal glands, 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 are 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 are not considered to be genotype related.

Gene 1521
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:
4 homozygous mutant females (300629, 315134, 328205, 328210)
3 homozygous mutant males (300637, 339132, 341033)
2 wild-type control females (300630, 328203)
2 wild-type control males (300639, 300640)

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 1521
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:
4 homozygous mutant females (300629, 315134, 328210, 339140)
3 homozygous mutant males (300637, 339132, 341033)
2 wild-type control females (300630, 328203)
2 wild-type control males (300639, 300640)

When compared to age- and gender-matched wild-type control mice, two of three homozygous mutant males (300637, 339132)  had increased levels of cholesterol and high density lipoprotein. 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 1521
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:
4 homozygous mutant females (300629, 328205, 328206, 328210)
3 homozygous mutant males (300637, 339132, 341033)
2 wild-type control females (300630, 328203)
2 wild-type control males (300639, 300640)

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 1521
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:
4 homozygous mutant females (300629, 315134, 328205, 328210)
3 homozygous mutant males (300637, 339132, 341033)
2 wild-type control females (300630, 328203)
2 wild-type control males (300639, 300640)

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 are 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 was regarded as biologically significant or genotype related.