Please use this identifier to cite or link to this item:
|Scopus||Web of Science®||Altmetric|
|Title:||shRNA off-target effects in vivo: impaired endogenous siRNA expression and spermatogenic defects|
De Rooij, D.
|Citation:||PLoS One, 2015; 10(3):e0118549-1-e0118549-23|
|Publisher:||Public Library of Science|
|Hye-Won Song, Anilkumar Bettegowda, Daniel Oliver, Wei Yan, Mimi H. Phan, Dirk G. de Rooij, Mark A. Corbett, Miles F. Wilkinson|
|Abstract:||RNA interference (RNAi) is widely used to determine the function of genes. We chose this approach to assess the collective function of the highly related reproductive homeobox 3 (Rhox3) gene paralogs. Using a Rhox3 short hairpin (sh) RNA with 100% complementarity to all 8 Rhox3 paralogs, expressed from a CRE-regulated transgene, we successfully knocked down Rhox3 expression in male germ cells in vivo. These Rhox3-shRNA transgenic mice had dramatic defects in spermatogenesis, primarily in spermatocytes and round spermatids. To determine whether this phenotype was caused by reduced Rhox3 expression, we generated mice expressing the Rhox3-shRNA but lacking the intended target of the shRNA-Rhox3. These double-mutant mice had a phenotype indistinguishable from Rhox3-shRNA-expressing mice that was different from mice lacking the Rhox3 paralogs, indicating that the Rhox3 shRNA disrupts spermatogenesis independently of Rhox3. Rhox3-shRNA transgenic mice displayed few alterations in the expression of protein-coding genes, but instead exhibited reduced levels of all endogenous siRNAs we tested. This supported a model in which the Rhox3 shRNA causes spermatogenic defects by sequestering one or more components of the endogenous small RNA biogenesis machinery. Our study serves as a warning for those using shRNA approaches to investigate gene functions in vivo.|
|Keywords:||Hela Cells; Animals; Mice, Transgenic; Humans; Mice; Homeodomain Proteins; RNA, Small Interfering; DNA Primers; Microarray Analysis; In Situ Nick-End Labeling; Spermatogenesis; RNA Interference; Models, Biological; Male; Gene Knockdown Techniques|
|Rights:||© 2015 Song et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited|
|Appears in Collections:||Paediatrics publications|
Files in This Item:
|hdl_94227.pdf||Published version||3.1 MB||Adobe PDF||View/Open|
Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.