FVB/N-Tg(ACTA1-TPM3*M9R)4Hrd/MbngJ

Stock number: 018304
Research areas: Developmental Biology Research, Mouse/Human Gene Homologs, Neurobiology Research

Description

HSA-αTm_slow(Met9Arg) transgenic mice exhibit an overexpression of a dominant-negative alpha-tropomyosin slow Met9Arg mutation associated with human nemaline myopathy (NM) directed to fast-twitch glycolytic skeletal muscle (type 2B) fibers. Transgenic mice display features of NM in skeletal muscle tissue, including electron-dense accumulations (nemaline rods), increased slow/oxidative fiber content, and late-onset skeletal muscle weakness. These HSA-αTm_slow(Met9Arg) transgenic mice may be useful in studying nemaline myopathy, skeletal muscle repair, and congenital muscular dystrophy.

Detailed description

Human nemaline myopathy (NM) is a hereditary disease of skeletal muscle defined by a distinct pathology of electron-dense accumulations (rods) within the sarcomeric units. HSA-αTm_slow(Met9Arg) transgenic mice express a human αTm_slow cDNA sequence modified to have the dominant-negative Met9Arg mutation associated with NM, all under the direction of the 2.2 kbp human α-skeletal actin (ACTA1 or HSA) promoter. As expected for this HSA promoter, αTm_slow(Met9Arg) expression is directed primarily to fast-twitch glycolytic skeletal muscle (type 2B) fibers in the transgenic mice.

The specific phenotype of hemizygous HSA-αTm_slow(Met9Arg) transgenic mice from founder line 4 is described below.
HSA-αTm_slow(Met9Arg) transgenic mice are viable and fertile, with αTm_slow(Met9Arg) mRNA expression levels ~160% that of the endogenous αTm_slow gene in slow fibers. Such overexpression of the dominant-negative Met9Arg mutant isoform in type 2B fibers results in clinical and pathological features of NM in multiple different skeletal muscles, including the presence of nemaline rods, increased slow/oxidative fiber content, and late-onset skeletal muscle weakness. As determined by immunofluorescent α-actinin 2 staining, nemaline rods are observed at the center and periphery of all fast glycolytic skeletal muscle (type 2B) fibers, and are commonly clustered around the Z-line of the sarcomere. The percentage of rods containing fibers increases between 2-12 months of age, and the percentage varies considerably between different muscles (but does not correlate with amount of mutant protein present). Transgenic mice exhibit a non-progressive increase in slow/fast oxidative fiber types 1 and 2A by ~2 months of age. Hypertrophy of type 2B fibers is apparent by ~2 months of age, but declines over the next 3-5 months. This age-related decline in hypertrophy correlates with late-onset muscle weakness beginning ~5-6 months of age, with muscle weakness progressing in severity out to 10-13 months of age. Like human nemaline disease, the muscle weakness in transgenic mice does not correlate with the numbers of rod-containing fibers or with the degree of type 1 fiber predominance. In contrast to human muscles, cytoplasmic bodies and tubular aggregates were observed in superficial gastrocnemius of transgenic mice (and not in any other mouse muscles examined). The hypertrophy of type 2B fibers observed in these mice may be sufficient to prevent overt muscle weakness in an unchallenged environment.

Regarding any differences between hemizygous and homozygous mice: The donating investigator (Dr. Edna C. Hardeman) maintained her transgenic colony by backcrossing with FVB/NJ wildtype mice (total of six generations by 2009). In their colony, while the level of transgene expression was increased in homozygous mice, they observed no significant differences in clinical pathology, breeding, or lifespan between hemizygous and homozygous mice.

Development

The HSA-αTm_slow(Met9Arg) transgene was created by Dr. Edna C. Hardeman (University of New South Wales, Sydney, Australia). The human tropomyosin 3 gene was used to isolate a full-length αTm_slow cDNA sequence. The isolated sequence was modified by site-directed mutagenesis to have the ATG->AGG base pair substitution encoding a methionine-to-arginine substitution at codon 9 (M9R); resulting in the dominant-negative αTm_slow(Met9Arg) mutation associated with human nemaline myopathy. The mutant cDNA sequence was placed downstream of a 2.2 kbp human α-skeletal actin (ACTA1) promoter sequence, and upstream of a 1022 bp cassette containing the SV40 small t antigen intron and 3' untranslated region. The transgene was injected into fertilized FVB/NJ eggs, which were then transferred to pseudopregnant foster females (ARC/S). Founder mice were identified and bred to FVB/NJ mice to generate each founder line. Three founder lines developed nemaline rod pathology with αTm_slow(Met9Arg) transcript expression levels in slow fibers of ~160% (line 4), ~120% (line 14) and ~70% (line 9) that of the endogenous αTm_slow gene. HSA-αTm_slow(Met9Arg) transgenic mice from founder line 4 were then backcrossed to FVB/NJ inbred mice for six generations. In 2009, frozen embryos were sent to Dr. Marie-Louise Bang (Dulbecco Telethon Institute IRGB-CNR, Milan, Italy). There, transgenic mice were additionally backcrossed four generations with FVB/NCrl mice, and then sent to The Jackson Laboratory Repository in 2012. Upon arrival, mutant mice were bred to FVB/NJ inbred mice (Stock No. 001800) for at least one generation to establish The Jackson Laboratory Repository colony.

Allele

Symbol: Tg(ACTA1-TPM3*M9R)4Hrd
Name: transgene insertion 4, Edna Hardeman
MGI accession ID: MGI:5444208
Generation method: Transgenic
Attributes: Dominant negative; Inserted expressed sequence; Humanized sequence
Marker symbol: Tg(ACTA1-TPM3*M9R)4Hrd
Marker name: transgene insertion 4, Edna Hardeman
Chromosome: UN
Strain of origin: FVB/NJ
Expressed genes: TPM3,tropomyosin 3,human
Molecular note: First, the human tropomyosin 3 gene was used to isolate a full-length alphaTm^slow cDNA sequence. Next, the sequence was modified by site-directed mutagenesis to have the ATG-to-AGG base pair substitution encoding a methionine-to-arginine substitution at codon 9 (M9R); resulting in the dominant-negative alphaTm^slow(Met9Arg) mutation associated with human nemaline myopathy. The mutant cDNA sequence was placed downstream of a 2.2 kbp human alpha-skeletal actin (ACTA1) promoter sequence, and upstream of a 1022 bp cassette containing the SV40 small t antigen intron and 3' untranslated region. Lines 4, 9 and 14 were generated with high, low and moderate expression levels, respectively.