Azacytidine Enhances Exogenous Gene Expression in Skeletal Muscle

DNA methylation is one of the mechanism for regulating gene expression. Transfected cells in tissue culture are a mosaic of expression of the exogenous gene. 5’ azacytidine (Aza C), inhibiting nuclear DNA methyltransferase, causes an increase of expression of E.coli a-galactosidase (LacZ) in transfected CHO cells. Murine muscle cell lines G8 and C2C12 were transfected by biolistic technique with plasmids carrying a reporter gene pCMVlacZ and pY3 that confers resistance to Hygromycin B. Resistant clones have been treated with different concentrations of 5’ azacytidine and analyzed by X-gal stain and Southern Blotting of DNA, after various time intervals up to one month. Hygromycin resistant cells were transplanted in the tibialis anterior muscle of living mice. Aza C was injected subcutaneously above the treated muscle. Histochemistry with X-gal was performed after different treatment periods, in order to evaluate the reporter gene expression timings. The myogenic cell lines, after treatment with 5’ azacytidine in vitro , express the exogenous gene at different levels, with four fold enhancement in G8 cells, after 15 days from transfection. The enhancement of expression is directly derived from the inhibition of methylation as shown by DNA restriction analysis. Mouse muscular samples treated subcutaneously with 5’ azacytidine showed an enhancement of lacZ expression in vivo up to 20 days after transfection. 5’ azacytidine enhances the level and the time span of exogenous gene expression in mu scle cells facilitating gene therapy studies.