1. Gene silencing of myostatin in differentiation of chicken embryonic myoblasts by small interfering RNA
- Author
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Sato, Fuminori, Kurokawa, Masatoshi, Yamauchi, Nobuhiko, and Hattori, Masa-aki
- Subjects
Chick embryo -- Research ,Chick embryo -- Genetic aspects ,Gene silencing -- Research ,Biological sciences - Abstract
Myostatin (GDF-8) is known to negatively regulate skeletal muscle mass in myogenesis, but few studies have been conducted on the function of endogenous GDF-8 in primary myoblasts. The present study was performed to assess the function of GDF-8 by RNA interference using primary culture of chicken embryonic myoblasts in which myoblasts were differentiated into myotubes. An active form of small interfering RNA (siRNA-1) targeting GDF-8 mRNA was introduced into myoblasts, and an inactive form of siRNA (siRNA-2) was used as a negative control. GDF-8 transcript level was significantly reduced 24 h after the introduction of siRNA-1 to 25% of the control, whereas a 52-kDa GDF-8 precursor was reduced to 45% of the control at 48 h. However, siRNA-2 did not decrease GDF-8 transcript level. When GDF-8-mediated promoter activity was measured chronologically by means of a pGL(CAGA)w-constructed luciferase reporter assay, a concomitant change in activity was initiated after 24 h. The activity rapidly decreased 30 h after siRNA-1 introduction, whereas high activity was maintained at 30-42 h in the control and siRNA-2-treated myoblasts. Myogenic factors such as MyoD and p21, but not myogenin, were altered after 72 h. Cell fusion of the multinucleated myotubes was delayed by the siRNA-1 introduction, and myotubes with aggregated nuclei were shorter and wider. These results strongly suggest that deficiency of GDF-8 delays cell differentiation and causes great alterations in the cellular morphology of chicken embryonic myotubes. GDF-8; GDF-8-mediated promoter activity; multinucleated myotubes doi:10.1152/ajpcell.00543.2005
- Published
- 2006