Abstract
The avian skeletal alpha-actin gene was used as a template for construction of a myogenic expression vector that was utilized to direct expression of a human IGF-I cDNA in cultured muscle cells and in striated muscle of transgenic mice. The proximal promoter region, together with the first intron and 1.8 kilobases of 3'-noncoding flanking sequence of the avian skeletal alpha-actin gene directed high level expression of human insulin-like growth factor I (IGF-I) in stably transfected C2C12 myoblasts and transgenic mice. Expression of the actin/IGF-I hybrid gene in C2C12 muscle cells increased levels of myogenic basic helix-loop-helix factor and contractile protein mRNAs and enhanced myotube formation. Expression of the actin/IGF-I hybrid gene in mice elevated IGF-I concentrations in skeletal muscle 47-fold resulting in myofiber hypertrophy. IGF-I concentrations in serum and body weight were not increased by transgene expression, suggesting that the effects of transgene expression were localized. These results indicate that sustained overexpression of IGF-I in skeletal muscle elicits myofiber hypertrophy and provides the basis for manipulation of muscle physiology utilizing skeletal alpha-actin-based vectors.
Highlights
The avian skeletal a-actin gene was used as a template for construction of a myogenic expression vector that was utilized to direct expression of a human IGF-1 eDNA in cultured muscle cells and in striated muscle of transgenic mice
Skeletal a-Actin 3'-UTR Enhances the Accumulation ofIGF-1 mRNA through mRNA Stabilization-In order to examine these parameters, we constructed two IGF-I expression vectors based on the avian skeletal a-actin gene which is normally activated during withdrawal from the cell cycle and myoblast fusion [16]
Targeting IGF-1 Expression to Striated Muscle-In most vertebrates, skeletal a -actin is the predominant striated actin isoform expressed in adult skeletal muscle, whereas it is expressed at a much lower level in cardiac muscle in which the a-cardiac isoform predominates [5]
Summary
The avian skeletal a-actin gene was used as a template for construction of a myogenic expression vector that was utilized to direct expression of a human IGF-1 eDNA in cultured muscle cells and in striated muscle of transgenic mice. IGF-1 concentrations in serum and body weight were not increased by transgene expression, suggesting that the effects of transgene expression were localized These results indicate that sustained overexpression of IGF-1 in skeletal muscle elicits myofiber hypertrophy and provides the basis for manipulation of muscle physiology utilizing skeletal a-actinbased vectors. Mouse transgenic studies by Petropulous et al [20] indicated that the conserved sequences in the proximal200 bp of the promoter region were primarily responsible for the avian skeletal a-actin gene's restricted expression pattern in heart and skeletal muscle These studies revealed a high degree of ectopic expression suggesting that additional regulatory elements from the skeletal a-promoter are required for strict striated muscle-specific expression. Recent research implicates the contiguous 3'-flanking region of the human skeletal a-actin gene in directing correct temporal and spatial expression of skeletal actin-based transgenes in
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