title: Ex vivo magnetofection: a novel strategy for the study of gene function in mouse organogenesis.
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.
authors.affiliation: Division of Molecular Genetics and Development, Institute for Molecular Bioscience, The University of Queensland, Brisbane, Australia.
authors.forename: Terje
authors.initials: T
authors.lastname: Svingen
authors.forename: Dagmar
authors.initials: D
authors.lastname: Wilhelm
authors.forename: Alexander N
authors.initials: AN
authors.lastname: Combes
authors.forename: Brett
authors.initials: B
authors.lastname: Hosking
authors.forename: Vincent R
authors.initials: VR
authors.lastname: Harley
authors.forename: Andrew H
authors.initials: AH
authors.lastname: Sinclair
authors.forename: Peter
authors.initials: P
authors.lastname: Koopman
mesh_terms: 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*
chemical_list: D055758:SOX9 Transcription Factor; D051876:Sex-Determining Region Y Protein; C527933:Sox9 protein, mouse; C497746:Sry protein, mouse