Analysis of Gene Function in Cultured Embryonic Mouse Gonads Using Nucleofection
J Ryan, L Ludbrook, D Wilhelm, A Sinclair, P Koopman, P Bernard, VR Harley
Sexual Development | KARGER | Published : 2011
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..View full abstract
Awarded by National Health and Medical Research Council
We thank Dr. Laura Parry for her helpful advice. We acknowledge funding from the National Health and Medical Research Council Program Grant #334314 (V.R.H., P.K., A.S.). L.L. was supported by an Australian Postgraduate Award.