RESEARCH ARTICLE shRNA Off-Target Effects In Vivo: Impaired Endogenous siRNA Expression and Spermatogenic Defects Hye-Won Song 1 , Anilkumar Bettegowda 1 , Daniel Oliver 2 , Wei Yan 2 , Mimi H. Phan 1 , Dirk G. de Rooij 3 , Mark A. Corbett 4 , Miles F. Wilkinson 1 * 1 Department of Reproductive Medicine, School of Medicine, University of California San Diego, La Jolla, California, United States of America, 2 Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada, United States of America, 3 Center for Reproductive Medicine, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands, 4 School of Pediatrics and Reproductive Health, The University of Adelaide, Adelaide, Australia * mfwilkinson@ucsd.edu 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 transgen- ic mice had dramatic defects in spermatogenesis, primarily in spermatocytes and round spermatids. To determine whether this phenotype was caused by reduced Rhox3 expres- sion, 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, in- dicating 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 sup- ported 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. Introduction RNA interference (RNAi) is a widely used approach to silence genes and thereby deduce their function. While primarily used for in vitro studies, RNAi has also been instrumental in deci- phering the functions of many genes in vivo. A particularly useful application is to elucidate the functions of gene paralog families. As long as the family members are sufficiently related, a PLOS ONE | DOI:10.1371/journal.pone.0118549 March 19, 2015 1 / 23 OPEN ACCESS Citation: Song H-W, Bettegowda A, Oliver D, Yan W, Phan MH, de Rooij DG, et al. (2015) shRNA Off- Target Effects In Vivo: Impaired Endogenous siRNA Expression and Spermatogenic Defects. PLoS ONE 10(3): e0118549. doi:10.1371/journal.pone.0118549 Academic Editor: Helen White-Cooper, Cardiff University, UNITED KINGDOM Received: October 4, 2014 Accepted: January 20, 2015 Published: March 19, 2015 Copyright: © 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. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Microarray data are available from the GEO database (accession number GSE64324). Funding: This work was supported by the National Institutes of Health (www.nih.gov/) R01-HD053808 and—HD45595 (to MFW). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist.