Analysis of gene function in cultured embryonic mouse gonads using Nucleofection

Ryan, J, Ludbrook, L, Wilhelm, D, Sinlcair, A, Koopman, P, Bernard, P and Harley, V (2011) Analysis of gene function in cultured embryonic mouse gonads using Nucleofection. Sexual Development, 5 1: 7-15. doi:10.1159/000322162


Author Ryan, J
Ludbrook, L
Wilhelm, D
Sinlcair, A
Koopman, P
Bernard, P
Harley, V
Title Analysis of gene function in cultured embryonic mouse gonads using Nucleofection
Journal name Sexual Development   Check publisher's open access policy
ISSN 1661-5425
Publication date 2011-01-01
Year available 2010
Sub-type Article (original research)
DOI 10.1159/000322162
Open Access Status Not yet assessed
Volume 5
Issue 1
Start page 7
End page 15
Total pages 9
Place of publication Basel, Switzerland
Publisher S. Karger
Language eng
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.
Keyword DNA delivery
Nucleofection
Organ culture
Sex determination
SOX9
SRY gonads
Q-Index Code C1
Q-Index Status Confirmed Code
Grant ID 334314
Institutional Status UQ
Additional Notes Published online: 22 November, 2010.

Document type: Journal Article
Sub-type: Article (original research)
Collections: Official 2011 Collection
Institute for Molecular Bioscience - Publications
 
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Citation counts: TR Web of Science Citation Count  Cited 9 times in Thomson Reuters Web of Science Article | Citations
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Created: Mon, 31 Jan 2011, 22:55:29 EST by Susan Allen on behalf of Institute for Molecular Bioscience