Ex vivo magnetofection: A novel strategy for the study of gene function in mouse organogenesis

Svingen, T, Wilhelm, D, Combes, A. N., Hosking, B, Harley, V. R., Sinclair, A. H. and Koopman, Peter (2009) Ex vivo magnetofection: A novel strategy for the study of gene function in mouse organogenesis. Developmental Dynamics, 238 4: 956-964. doi:10.1002/dvdy.21919


Author Svingen, T
Wilhelm, D
Combes, A. N.
Hosking, B
Harley, V. R.
Sinclair, A. H.
Koopman, Peter
Title Ex vivo magnetofection: A novel strategy for the study of gene function in mouse organogenesis
Journal name Developmental Dynamics   Check publisher's open access policy
ISSN 1058-8388
1097-0177
Publication date 2009-04-01
Year available 2009
Sub-type Article (original research)
DOI 10.1002/dvdy.21919
Open Access Status Not Open Access
Volume 238
Issue 4
Start page 956
End page 964
Total pages 9
Editor Gary C Schoenwolf
Place of publication Hoboken, NJ, U. S. A.
Publisher John Wiley & Sons
Language eng
Subject 1116 Medical Physiology
C1
970106 Expanding Knowledge in the Biological Sciences
060403 Developmental Genetics (incl. Sex Determination)
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.
Formatted 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.
© 2009 Wiley-Liss, Inc.
Keyword Gene delivery
Sex determination
Germ cells
Meiosis
Tmem184a
Sry
Sox9
Q-Index Code C1
Q-Index Status Confirmed Code
Grant ID HD049431
Institutional Status UQ

 
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Created: Thu, 03 Sep 2009, 18:22:13 EST by Mr Andrew Martlew on behalf of Institute for Molecular Bioscience