Analysis of gene function in cultured embryonic mouse gonads using nucleofection

Abstract

Mammalian sex determination is a dynamic process involving balanced gene expression leading to the development of either a testis or an ovary. Candidate sex-determining genes have been identified through microarray-based studies of gonadal gene expression; however, few methods exist for validation. This study describes a new technique for transfecting gonads using nucleofection. Fifteen micrograms of expression plasmid DNA was transfected into E11.5 gonads, cultured for 3 days and gene expression analyzed. Following optimization, we consistently achieved cell transfection efficiencies of 11% of cells using pMax-GFP plasmid. To test the applicability of nucleofection to studies of gene functi..