B6.Cg-Tg(ACTA1-MYOT*T57I)71Mah/J

Stock number: 006615
Product name: TgT57I
Research areas: Mouse/Human Gene Homologs, Neurobiology Research

Description

These transgenic mice express a mutant form of human myotilin (MYOT) directed by the human skeletal muscle alpha 1 actin (ACTA1) promoter in skeletal muscle.

Detailed description

Mice hemizygous for this TgT57I transgene are viable and fertile, with expression of a mutant form of human myotilin (MYOT harboring a T57I point mutation) directed by the human skeletal muscle alpha 1 actin (ACTA1) promoter. RT-PCR reveals transgene expression is specific to skeletal muscle. Mutant mice exhibit progressive muscle pathology. Small myofibrillar aggregates are observed in 2 week old mutant transgenic mice. By age 12 months, aggregates are predominantly found in the quadriceps and triceps (upper forelimb and hindlimb muscles), with the number of affected fibers and pathology increasing with age. Sarcolemmal damage is also observed. Fibrosis, tubular aggregation and adipose infiltration is observed in older transgenic mice. Muscle tissue of the diaphragm, soleus, biceps and ulnar do not form aggregates. Ultrastructural examination of muscle tissue from transgenic mice reveals sarcomeric abnormalities, such as Z-disc streaming. Isolated whole intact extensor digitorum longus muscle exhibits contractile dysfunction with reduced muscle mass and diminished specific maximum force. These mice recapitulate key features of human myotilinopathies and provide a model for studying the underlying mechanism of rela ted diseases such as limb-girdle muscular dystrophy type 1A (LGMD1A), myofibrillar myopathy (MFM), and spheroid body myopathy (SBM). Analysis of transgene inheritance patterns at The Jackson Laboratory colony and the donating investigator's colony suggest that the transgene integrated on the X chromosome. The effects (if any) of X chromosome inactivation in female hemizygotes are not yet characterized (October 2007).

Development

A transgenic construct containing the human myotilin gene, MYOT with a T57I point mutation, a c-myc (Myc) epitope tag, and SV40 polyadenylation site sequence under the control of the human skeletal muscle alpha 1 actin, ACTA1, promoter was injected into fertilized B6SJLF2 mouse eggs. Founder line 71 was established. Founder animals were backcrossed onto the C57BL/6J background for 9 generations by the donating investigator. Analysis of transgene inheritance patterns at The Jackson Laboratory colony and the donating investigator's colony suggest that the transgene integrated on the X chromosome (October 2007).

Allele

Symbol: Tg(ACTA1-MYOT*T57I)71Mah
Name: transgene insertion 71, Michael Hauser
MGI accession ID: MGI:3640478
Generation method: Transgenic
Attributes: Inserted expressed sequence; Humanized sequence
Marker symbol: Tg(ACTA1-MYOT*T57I)71Mah
Marker name: transgene insertion 71, Michael Hauser
Chromosome: UN
Strain of origin: (C57BL/6 x SJL)F2
Expressed genes: MYOT,myotilin,human
Molecular note: The point mutation responsible for limb-girdle muscular dystrophy type 1A (LGMD1A) in a North American family - substitution of threonine for isoleucine at amino acid position 57 (T57I) of the protein - was introduced into the human myotilin cDNA. This mutant cDNA, comprising 1530 bp of coding sequence, 281 bp of 5' UTR and 485 bp of 3' UTR, with sequence encoding a MYC epitope tag inserted at the beginning of the coding region, was cloned into the HAS-VP1 expression vector downstream of nucleotides -2139 through +239 of the human actin, skeletal, alpha 1 gene followed by the splice acceptor from the SV40 P1 intron; two copies of the SV40 polyadenylation signal follow the 3'UTR of the cDNA. RT-PCR analysis of several tissues detected transgene-derived mRNA only in skeletal muscle, and immunoblot analysis with antibody against MYC demonstrated appropriate expression of the transgene product in multiple striated muscles. Immunohistologic examination of muscle sections revealed uniformity of expression in fibers within a muscle group and similarity of expression in slow, type I and in fast, type II fibers. Expression of the mutant human myotilin is 2.6-fold that of the endogenous mouse protein.