Ex vivo magnetofection: a novel strategy for the study of gene function in mouse organogenesis.
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.
Svingen
Terje
T
Division of Molecular Genetics and Development, Institute for Molecular Bioscience, The University of Queensland, Brisbane, Australia.
Wilhelm
Dagmar
D
Combes
Alexander N
AN
Hosking
Brett
B
Harley
Vincent R
VR
Sinclair
Andrew H
AH
Koopman
Peter
P
D000818:Animals; D002460:Cell Line; D004622:Embryo, Mammalian / Q000196:embryology / Q000378:metabolism; D005260:Female; D018507:Gene Expression Regulation, Developmental / Q000235:genetics*; D006801:Humans; D008297:Male; D051379:Mice; D038081:Organogenesis; D010053:Ovary / Q000196:embryology / Q000378:metabolism; D034622:RNA Interference; D055758:SOX9 Transcription Factor; D012730:Sex Chromosomes / Q000235:genetics; D051876:Sex-Determining Region Y Protein / Q000235:genetics / Q000378:metabolism; D013737:Testis / Q000196:embryology / Q000378:metabolism; D046509:Tissue Culture Techniques / Q000379:methods*
D055758:SOX9 Transcription Factor; D051876:Sex-Determining Region Y Protein; C527933:Sox9 protein, mouse; C497746:Sry protein, mouse