129-Acta1^tm1Hrd/J

Stock number: 018284
Product name: Acta1(H40Y)
Research areas: Cardiovascular Research, Developmental Biology Research, Mouse/Human Gene Homologs, Neurobiology Research, Reproductive Biology Research, Research Tools

Description

The Acta1(H40Y) knock-in allele has the endogenous α-skeletal actin sequences replaced by a mutant α-skeletal actin containing the single amino acid change (H40Y) associated with dominantly-inherited severe nemaline myopathy in humans. Acta1(H40Y) heterozygotes display clinical and pathological features of patients with nemaline myopathy, and may be useful in studying nemaline myopathy and congenital muscular dystrophy.

Detailed description

The Acta1(H40Y) knock-in allele has the endogenous α-skeletal actin sequences replaced by a mutant α-skeletal actin containing the single amino acid change (H40Y) associated with dominantly-inherited severe nemaline myopathy in humans. Acta1(H40Y) heterozygotes display clinical and pathological features of patients with nemaline myopathy, including severe, early onset limb and eyelid muscle weakness, decreased mobility, presence of cytoplasmic and nuclear nemaline rods, and increased mortality. Specifically, heterozygous mice of both sexes display severe muscle weakness (beginning ~15 days of age), and decreased ambulation, reduced body weight (loss of body and facial muscle mass/emaciated appearance), ruffled hair, kyphosis (hunchbacked appearance), watery eyes and ptosis (drooping eyelids) all by 21 days of age. Heterozygous mice have a shortened lifespan; ~61% of males and ~3% of females die by 13 weeks of age. Nemaline rods are detected in the cytoplasm and nucleus of muscle fibers of all muscles tested from heterozygous animals. Heart function may also be impaired in heterozygous mice as cytoplasmic rods are detected in cardiomyocytes. Heterozygous males have reduced fertility. Heterozygous females may appear sick but most survive and breed by 12 weeks of age. The donating investigator reports breeding wildtype males with heterozygous females is irregular with one litter born every 1-2 months, but females can breed up to 14 months of age. Older aged breeders (more than 4 months old) are recommended as breeding improves with age. The donating investigator suggests providing crushed food beginning at pre-weaning age in case mice will not be able to reach the food hopper. In addition, one may employ antibiotic eye ointment (e.g., chloromycetin ophthalmic ointment or Bactrim) to treat eye infections that can accompany the eye phenotype. The donating investigator has not attempted to generate homozygous mice to date (June 2012), and reports they observe white, agouti, and ginger (chinchilla) coat color.

In an attempt to offer the Acta1(H40Y) knock-in allele on the same genetic background on which it was first characterized ("R1-129" genetic background (mixed 129X1/SvJ and 129S1/Sv)), The Jackson Laboratory imported and maintained the Acta1(H40Y) knock-in mouse line by breeding heterozygous females with wildtype males for several years. However in May 2015, to prevent its extinction, we had to begin breeding the colony to 129S1/SvImJ inbred mice (Stock No. 002448). Therefore, the Acta1(H40Y) knock-in mouse line distributed after this (and later cryopreserved) was on a genetic background that was similar, but not identical, to the "R1-129" mix upon which we received it.

Development

To create the targeting vector, Dr. Edna C. Hardeman (University of New South Wales, Sydney, Australia) isolated a mouse α-skeletal actin (Acta1) genomic DNA sequence (spanning ~1.8 kbp upstream of exon 1 through the 3' UTR ~2.3 kbp downstream of exon 7) and introduced a C->T base pair substitution (CAC->TAC) in exon 2 by site-directed mutagenesis. The mutation results in a single amino acid substitution of histidine to tyrosine at codon 40 (H40Y), a mutation associated with dominantly-inherited severe nemaline myopathy in humans (see note below). A loxP-flanked PGKneo cassette was also placed downstream of the 3' UTR. This construct replaced the endogenous α-skeletal actin locus via electroporation into (129X1/SvJ x 129S1/Sv)F1-Kitl⁺ derived R1 embryonic stem (ES) cells. Correctly targeted ES cells (clone #65) were injected into recipient (ARC or ARC/Balb/c) blastocysts, and the resulting chimeric males were mated with "R1-129" females to generate the Acta1(H40Y) knock-in mouse line. Acta1(H40Y) heterozygous females were then bred to "R1-129" males for 11 generations. In 2012, Dr. Hardeman sent heterozygous females and wildtype males from the colony (on "R1-129" genetic background (mixed 129X1/SvJ and 129S1/Sv)) to The Jackson Laboratory Repository. Upon arrival, these mice were maintained several years by breeding heterozygous females with wildtype males. However in May 2015, to prevent its extinction, we had to begin breeding the colony to 129S1/SvImJ inbred mice (Stock No. 002448). Therefore, the Acta1(H40Y) knock-in mouse line distributed after this (and later cryopreserved) was on a genetic background that was similar, but not identical, to the "R1-129" mix upon which we received it.

Additional note: Both the human and mouse α-skeletal actin genes encode two N-terminal amino acids (Met and Cys) which are removed from the protein coincident with translation. Therefore, conventional numbering of the α-skeletal actin amino acid sequence begins with the third encoded amino acid (Asp). Hence, the H40Y mutation refers to the 40th amino acid in the protein (although this is amino acid 42 in the gene sequence).

Allele

Symbol: Acta1^tm1Hrd
Name: targeted mutation 1, Edna C Hardeman
MGI accession ID: MGI:5424775
Generation method: Targeted
Attributes: Dominant negative; Humanized sequence
Synonyms: Acta1(H40Y); Acta1^{tm1(H40Y;neo)Hrd}
Marker symbol: Acta1
Marker name: actin, alpha 1, skeletal muscle
Marker synonyms: ACTA; Acts; Actsk-1; ASMA; CFTD; CFTD1; CFTDM; CMYO2A; CMYO2B; CMYO2C; CMYP2A; CMYP2B; CMYP2C; MPFD; NEM1; NEM2; NEM3; SHPM
Chromosome: 8
Strain of origin: (129X1/SvJ x 129S1/Sv)F1-Kitl⁺
Molecular note: A C to T base pair substitution in exon 2 was introduced into the mouse alpha-skeletal actin (Acta1) genomic DNA sequence (construct spanning ~1.8 kbp upstream of exon 1 through the 3' UTR about 2.3 kbp downstream of exon 7) by site-directed mutagenesis. The CAC to TAC mutation of codon 40 results in a single amino acid substitution of histidine to tyrosine in the encoded protein. A loxP-flanked PGKneo cassette was also placed downstream of the 3' UTR. Both the human and mouse alpha-skeletal actin genes encode two N-terminal amino acids (Met and Cys) which are removed from the protein coincident with translation. Therefore, conventional numbering of the alpha-skeletal actin amino acid sequence begins with the third encoded amino acid (Asp). Hence, the H40Y mutation refers to the 40th amino acid in the protein (although this is amino acid 42 in the gene sequence).