PARENT SESSION

(T491) GROWTH DIFFERENTIATION FACTOR-8 FUNCTION IN CHICKEN EMBRYONIC MYOBLASTS ANALYZED BY RNA INTERFERENCE.

Sato, Fuminori ¹, Soh, Tomoki ¹, Yamauchi, Nobuhiko¹, Hattori, Masa-aki¹, ¹ Kyushu University, Fukuoka, Japan

ABSTRACT- Growth differentiation factor-8 (GDF-8) negatively regulates skeletal muscle mass in myogenesis from the early embryonic stage. The function of GDF-8 has been investigated in a C2C12 myoblast cell line using its overexpression and recombinant, but there are little studies on the function of endogenous GDF-8 in primary myoblasts. According to the recent progress in chicken genome researches, chicken embryo has been useful as a vertebrate model system in embryogenesis. More recently, we have developed the application of RNA interference targeting unidentified genes to chicken embryo, and it will be a powerful tool for the functional analysis of endogenous GDF-8 during embryogenesis. In this study, the GDF-8 function was analyzed using a primary culture of chicken embryonic myoblasts by RNA interference. The myoblasts, purified from pectoralis major muscles of 12-day chicken embryos by Percoll density gradient, were cultured in MEM containing chicken serum and sodium butyrate. GDF-8-siRNA targeting GDF-8 cDNA at nucleotide positions 880-898 was introduced into cultured myoblasts by lipofection. The cells were then cultured in Opti-MEM containing KSR. GDF-8 expression was estimated by RT-PCR and western blotting, and the activity of Smad3, binding to the regulatory consensus sequence (CAGA) upon GDF-8 stimulation, was measured chronologically by means of pGL(CAGA) ₁₀ -constructed luciferase reporter assay. RT-PCR and western blotting analyses revealed that GDF-8 expression was decreased by introduction of siRNA. Smad3 activity monitored was clearly decreased 12 h after introduction of GDF-8-siRNA, indicating a decreased action of endogenous GDF-8. In the control cells, myotubes that were differentiated from myoblasts increased within 30 h after culture in Opti-MEM containing KSR. In contrast, myoblasts transfected with GDF-8-siRNA significantly proliferated for the first 48 h, but many myotubes were consequently formed after introduction. These results suggest that the deficiency of GDF-8 is effective in the proliferation of myoblasts and delays the formation of myotubes. Thus, endogenous GDF-8 is well related with myogenesis in chicken embryonic myoblasts as a negative regulator.

KEY WORDS: GDF-8, chicken embryonic myoblasts, RNA interference, Smad3