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Ex vivo magnetofection: A novel strategy for the study of gene function in mouse organogenesis
Author(s) -
Svingen Terje,
Wilhelm Dagmar,
Combes Alexander N.,
Hosking Brett,
Harley Vincent R.,
Sinclair Andrew H.,
Koopman Peter
Publication year - 2009
Publication title -
developmental dynamics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.634
H-Index - 141
eISSN - 1097-0177
pISSN - 1058-8388
DOI - 10.1002/dvdy.21919
Subject(s) - biology , testis determining factor , gene knockdown , ectopic expression , transgene , gonadal ridge , knockout mouse , gene , function (biology) , gene expression , microbiology and biotechnology , gene knockout , microinjection , genetics , embryogenesis , y chromosome
Abstract Gene function during mouse development is often studied through the production and analysis of transgenic and knockout models. However, these techniques are time‐ and resource‐consuming, and require specialized equipment and expertise. We have established a new protocol for functional studies that combines organ culture of explanted fetal tissues with microinjection and magnetically induced transfection (“magnetofection”) of gene expression constructs. As proof‐of‐principle, we magnetofected cDNA constructs into genital ridge tissue as a means of gain‐of‐function analysis, and shRNA constructs for loss‐of‐function analysis. Ectopic expression of Sry induced female‐to‐male sex‐reversal, whereas knockdown of Sox9 expression caused male‐to‐female sex‐reversal, consistent with the known functions of these genes. Furthermore, ectopic expression of Tmem184a , a gene of unknown function, in female genital ridges, resulted in failure of gonocytes to enter meiosis. This technique will likely be applicable to the study of gene function in a broader range of developing organs and tissues. Developmental Dynamics 238:956–964, 2009. © 2009 Wiley‐Liss, Inc.