xxx

Gene: 1406	Name: Cln2	Family: Protease	Subfamily: Aspartyl	Accession: AJ011912	GI: 3766470

Gene 1406
Summary of Phenotypic Analysis

Changes related to genotype at 49 days:

Behavior: Homozygous mutant mice exhibited a significant increase in thermal response latency during tail flick testing when compared with age- and gender-matched wild-type control mice.

Changes related to genotype at 100-144 days:

Lethality: Death at 100 to 130 days, mainly homozygous females.
Home Cage Observation : Seizure like behavior, hunched posture, lack of coordination, abnormal gait, tremors, and/or tachypnea.
Physical examination: Hunched posture, dehydrated, ataxia, weak, tremors, abnormal gait, abnormal activity level and/or gasping respirations .

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:

Home cage observation
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
Behavioral tests
Aging studies
Fertility

Behavior:
When compared to age- and gender-matched wild-type control mice, homozygous mutant mice exhibited a significant increase in thermal response latency during tail flick testing, indicating a possible increased pain threshold phenotype.

Home Cage Observation:
When compared to age- and gender-matched wild-type control mice, the homozygous mice, mainly females, had a combination of one or more of the following findings: seizure like behavior, hunched posture, lack of coordination, abnormal gait, tremors, and/or tachypnea.

Physical Examination:
When compared to age- and gender-matched wild-type control mice at 100-130 days, the homozygous mice had a combination of one or more of the following findings: hunched posture, dehydrated, ataxia, weak, tremors, abnormal gait, abnormal activity level and/or gasping respirations.

Gene 1406
Behavior

Homozygous mutant mice exhibited a significant increase in thermal response latency during tail flick testing 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 (240302, 240327, 250262, 250264, 258170, 267422, 267427, 273521, 277156, 277159)
12 wild-type control males (240298, 240299, 240300, 240325, 250259, 250261, 258165, 267423, 267426, 273519, 273522, 277158)

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 exhibited a significant increase in thermal response latency during tail flick testing, indicating a possible increased pain threshold 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 1406
Fertility

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

Two 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. Both matings pairs survived to have had only two litters because the homozygous mutant mice for this gene mutation die prematurely. Three weeks later, the live pups were counted and weaned.

Mouse ID numbers are as follows:

2 homozygous mutant males (250242, 312268)

2 homozygous mutant females (250256, 312265)

Gene 1406
Expression Summary

RT-PCR Summary:
RNA transcripts are detectable in all tissues analyzed: brain, cortex, subcortical region, cerebellum, brainstem, olfactory bulb, spinal cord, eye, Harderian glands, heart, lung, liver, pancreas, kidney, spleen, thymus, lymph nodes, bone marrow, skin, gallbladder, urinary bladder, pituitary gland, adrenal gland, salivary gland, skeletal muscle, tongue, stomach, small intestine, large intestine, cecum, testis, epididymis, seminal vesicle, coagulating gland, prostate gland, ovaries, uterus and white fat.

LacZ Summary:
LacZ (beta-galactosidase) expression is detectable in brain, spinal cord, eyes, pancreas, pituitary gland and testis.

Expression:
Brain
In wholemount staining, strong lacZ expression is detectable in practically all areas of the brain: olfactory bulb, cerebrum, cerebellum and brainstem. On coronal sections of the cerebrum strong lacZ expression is detectable in cortex and hippocampus. Moderate lacZ expression is detectable in amygdala, habenular nuclei, hypothalamus and ventricles. Weak lacZ expression is detectable in thalamus and caudate putamen. On coronal sections of the cerebellum strong lacZ expression is detectable in a few cells in white matter. Moderate lacZ expression is detectable in Purkinje cell, granular and molecular layers and fourth ventricle. Many nuclei in brainstem express lacZ.

Spinal cord
Moderate to strong lacZ expression is detectable in gray matter including in motor neurons. Further a few epithelial cells of the central canal express lacZ.

Eyes
LacZ expression is detectable in the inner nuclear and ganglion cell layers of the retina.

Pancreas
Moderate lacZ expression is detectable in a few acinar cells.

Pituitary Gland
Weak lacZ expression is detectable in pars distalis.

Male Reproductive Systems
Testis
LacZ expression is detectable in spermatogenic cells of distinct tubule cross sections of the seminiferous tubules.

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

Gene 1406
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. The data are compiled from the N0F2 and N1F2 generations.

49 Day Cohort Mouse ID numbers are as follows:
3 homozygous mutant females (175170, 181409, 186764)
3 homozygous mutant males (192605, 208204, 208215)
2 wild-type control females (181407, 182666)
2 wild-type control males (181405, 181410)

100-130 Day Cohort Mouse ID numbers are as follows:
4 homozygous mutant females (218984, 219246, 219250, 277149)
2 wild-type control females (221735, 221736)
1 wild-type control male (291156)

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 1406
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. The data are compiled from the N0F2 and N1F2 generations.

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 1406
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. The data are compiled from the N0F2 and N1F2 generations.

49 Day Cohort Mouse ID numbers are as follows:
3 homozygous mutant females (175170, 181409, 186764)
3 homozygous mutant males (192605, 208204, 208215)
2 wild-type control females (181407, 182666)
2 wild-type control males (181405, 181410)

100-130 Day Cohort Mouse ID numbers are as follows:
4 homozygous mutant females (218984, 219246, 219250, 277149)
2 homozygous mutant males (287341, 287342)
2 wild-type control females (221735, 221736)
1 wild-type control male (291156)

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 1406
Clinical Chemistry

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

49 Day Cohort Mouse ID numbers are as follows:
3 homozygous mutant females (175170, 186764, 186766)
3 homozygous mutant males (192605, 208215, 236231)
3 wild-type control females (182666, 182667, 186765)
3 wild-type control males (181410, 182671, 192603)

90 Day Cohort Mouse ID numbers are as follows:
5 homozygous mutant females (208189, 208191, 208198, 239063, 267417)
4 homozygous mutant males (231776, 231777, 250242, 255626)
4 wild-type control females (208169, 208170, 208190, 208192)
4 wild-type control males (208199, 208201, 208202, 219273)

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 1406
Hematology

There were no significant differences in the homozygous 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 are compiled from the N0F2 and N1F2 generations.

49 Day Cohort Mouse ID numbers are as follows:
3 homozygous mutant females (175170, 181409, 192599)
3 homozygous mutant males (192605, 208215, 236231)
3 wild-type control females (181407, 186765, 195971)
3 wild-type control males (181405, 181410, 192603)

90 Day Cohort Mouse ID numbers are as follows:
6 homozygous mutant females (208189, 208198, 239063, 239089, 267417, 273514)
4 homozygous mutant males (231776, 231777, 231779, 255626)
4 wild-type control females (208169, 208170, 208190, 208192)
4 wild-type control males (208199, 208201, 208202, 219273)

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

Gene 1406
Physical Examination

Changes related to genotype at 100-130 days:

General :

Hunched posture, homozygous female and males
Dehydrated, homozygous female

Locomotor:

Ataxia, homozygous females
Weak, homozygous females and males
Tremors, homozygous males
Abnormal gait, homozygous males
Abnormal activity level, homozygous females

Respiration:

Gasping, homozygous female.

The following mice were evaluated by physical examination. The data were compiled from the N0F2 and N1F2 generations.

49 Day Cohort Mouse ID numbers are as follows:
3 homozygous mutant females (175170, 181409, 186764)
3 homozygous mutant males (192605, 208204, 208215)
2 wild-type control females (181407, 182666)
2 wild-type control males (181405, 181410)

100-130 Day Cohort Mouse ID numbers are as follows:
4 homozygous mutant females (218984, 219246, 219250, 277149)
2 homozygous mutant male (287341, 287342)
2 wild-type control females (221735, 221736)
1 wild-type control male (291156)

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.)

When compared to age- and gender-matched wild-type control mice at 100-130 days, the homozygous mice had a combination of one or more of the following findings: hunched posture (female 277149; males 287341, 287342), dehydrated (female 277149), ataxia (females 218984, 219246, 219250), weak (females 218984, 219246, 219250; males 287341, 287342), tremors (males 287341, 287342), abnormal gait (287341, 287342), abnormal activity level (females 218984, 219246, 219250, 277149) and/or gasping respirations (female 277149). The findings correlated with those at home cage observation.

Individual homozygous mutant mice had only other 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.

Gene 1406
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. The data are compiled from the N0F2 and N1F2 generations.

49 Day Cohort Mouse ID numbers are as follows:
6 homozygous mutant females (208189, 208191, 208197, 208198, 267417, 273514)
7 homozygous mutant males (231776, 231777, 231779, 247064, 255626, 287341, 287342)
6 wild-type control females (208169, 208170, 208190, 208192, 273513, 273515)
4 wild-type control males (208199, 208201, 208202, 219273)

90 Day Cohort Mouse ID numbers are as follows:
10 homozygous mutant females (208189, 208191, 208197, 208198, 239063, 239089, 267417, 273514, 277146, 277149)
6 homozygous mutant males (231776, 231777, 231779, 255626, 287341, 287342)
6 wild-type control females (208169, 208170, 208190, 208192, 273513, 273515)
4 wild-type control males (208199, 208201, 208202, 219273)

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.

Gene 1406
Home Cage Observation

Changes related to genotype at 121-149 days:

General appearance: Seizure-like behavior, mainly homozygous females
Body shape: Hunched posture, mainly homozygous females
Movement: Lack of coordination, abnormal gait, and/or tremors, mainly homozygous females
Respiration: Tachypnea, mainly homozygous females

Home cage observations were collected by animal care personnel over the lifetime of the animals. The observables were similar to those found in the physical examination.

The following mice had home cage observations:

124-144 Day Cohort Mouse ID numbers are as follows:
14 homozygous mutant females (208179, 208180, 208181, 208189, 208191, 208197, 208198, 218984, 218985, 219246, 219250, 287346, 287347, 291150)
3 homozygous mutant males (231779, 287341, 287342)

When compared to age- and gender-matched wild-type control mice, the homozygous mutant mice, mainly females, had a combination of one or more of the following findings: seizure like behavior, hunched posture, lack of coordination, abnormal gait, tremors, and/or tachypnea. The findings correlated with those at physical examination.

There were no other genotype-related or biologically significant differences noted between mutant and wild-type control mice for any of the parameters evaluated during home cage observations.