title: 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 function, a testis-determining gene was transfected into gonads and its ability to sex-reverse was examined. When Sry was transfected into female (XX) gonads, upregulation of its target gene Sox9 was observed, as well as a downregulation of the ovarian gene Foxl2. Conversely, when shSox9 was introduced into male (XY) gonads, reduction of Sox9 and its target gene, Amh was observed, with a concomitant upregulation of Foxl2. Nucleofection-based gene delivery can recapitulate in vivo events of gonadal development that demonstrates 'proof-of-principle' of the method as a screening tool to evaluate the cellular function of potential sex-determining and gonadal differentiation genes.
authors.affiliation: Prince Henry's Institute of Medical Research, Melbourne, Vic., Australia.
authors.forename: J
authors.initials: J
authors.lastname: Ryan
authors.forename: L
authors.initials: L
authors.lastname: Ludbrook
authors.forename: D
authors.initials: D
authors.lastname: Wilhelm
authors.forename: A
authors.initials: A
authors.lastname: Sinclair
authors.forename: P
authors.initials: P
authors.lastname: Koopman
authors.forename: P
authors.initials: P
authors.lastname: Bernard
authors.forename: V R
authors.initials: VR
authors.lastname: Harley
mesh_terms: D000818:Animals; D046149:Embryo Culture Techniques; D005260:Female; D000074842:Forkhead Box Protein L2; D051858:Forkhead Transcription Factors / Q000235:genetics; D018507:Gene Expression Regulation, Developmental; D055785:Gene Knockdown Techniques; D024282:Genes, sry; D049452:Green Fluorescent Proteins / Q000235:genetics; D008297:Male; D051379:Mice; D010053:Ovary / Q000196:embryology* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics; D055758:SOX9 Transcription Factor / Q000037:antagonists & inhibitors / Q000235:genetics; D019849:Sex Determination Processes / Q000235:genetics*; D012733:Sex Differentiation / Q000235:genetics*; D051876:Sex-Determining Region Y Protein / Q000235:genetics; D013737:Testis / Q000196:embryology* / Q000378:metabolism*; D014162:Transfection
chemical_list: D000074842:Forkhead Box Protein L2; D051858:Forkhead Transcription Factors; C498893:Foxl2 protein, mouse; D011993:Recombinant Fusion Proteins; D055758:SOX9 Transcription Factor; D051876:Sex-Determining Region Y Protein; C527933:Sox9 protein, mouse; C497746:Sry protein, mouse; D049452:Green Fluorescent Proteins