Intense Expression of GFP Gene in Gonads of Chicken Embryos by Transfecting Circulating Primordial Germ Cells <I>in vitro</I> and <I>in vivo</I>

Abstract

The present study was carried out to develop techniques for introducing exogenous DNA into primordial germ cells (PGCs) and expressing the introduced DNA efficiently in the gonads of developing chicken embryos. PGCs circulating in the bloodstream (stages 14-15) were transfected with GFP gene in vitro or in vivo by lipofection or nucleofection. The manipulated PGCs successfully migrated to the germinal ridges and expressed GFP gene efficiently in the gonads of developing embryos. Intense GFP gene expression was observed in the gonads during the first 8 days following the transfection, during which period the sexual differentiation of gonads and germ cells (GCs) takes place. The GFP gene expression then gradually declined during subsequent embryonic development until hatching. When PGCs were transfected in vivo by lipofection with linearised plasmid DNA, GFP gene was detected in the gonads of 4.3% (19/442) of embryos examined at 20.5 days of culture, whereas less than 1% of embryos detected GFP gene in the gonads of embryos in which PGCs were transfected in vitro or with circular form plasmid DNA. In two of the embryos in which PGCs were transfected in vivo by lipofection with linearised plasmid DNA, GFP gene was expressed clearly in limited areas of the gonads of 20.5-day cultured embryos. The results obtained in this study suggest that the present in vitro and in vivo techniques for PGC manipulation provide a useful experimental system for studying gene functions in the sexual differentiation of gonads and GCs in early chicken embryos.