title: shRNA off-target effects in vivo: impaired endogenous siRNA expression and spermatogenic defects.
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.
authors: Department of Reproductive Medicine, School of Medicine, University of California San Diego, La Jolla, California, United States of America.
authors: Hye-Won
authors: HW
authors: Song
authors: Department of Reproductive Medicine, School of Medicine, University of California San Diego, La Jolla, California, United States of America.
authors: Anilkumar
authors: A
authors: Bettegowda
authors: Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada, United States of America.
authors: Daniel
authors: D
authors: Oliver
authors: Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada, United States of America.
authors: Wei
authors: W
authors: Yan
authors: Department of Reproductive Medicine, School of Medicine, University of California San Diego, La Jolla, California, United States of America.
authors: Mimi H
authors: MH
authors: Phan
authors: Center for Reproductive Medicine, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.
authors: Dirk G
authors: DG
authors: de Rooij
authors: School of Pediatrics and Reproductive Health, The University of Adelaide, Adelaide, Australia.
authors: Mark A
authors: MA
authors: Corbett
authors: Department of Reproductive Medicine, School of Medicine, University of California San Diego, La Jolla, California, United States of America.
authors: Miles F
authors: MF
authors: Wilkinson
mesh_terms: D000818:Animals; D017931:DNA Primers / Q000235:genetics; D055785:Gene Knockdown Techniques / Q000379:methods; D006367:HeLa Cells; D018398:Homeodomain Proteins / Q000378:metabolism*; D006801:Humans; D020287:In Situ Nick-End Labeling; D008297:Male; D051379:Mice; D008822:Mice, Transgenic; D046228:Microarray Analysis; D008954:Models, Biological; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics* / Q000378:metabolism*; D013091:Spermatogenesis / Q000235:genetics*
chemical_list: D017931:DNA Primers; D018398:Homeodomain Proteins; D034741:RNA, Small Interfering; C000603841:Rhox3 protein, mouse