shRNA Off-Target Effects <i>In Vivo</i>: Impaired Endogenous siRNA Expression and Spermatogenic Defects

<div><p>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 <u>r</u>eproductive <u>h</u>omeob<u>ox</u> 3 (<i>Rhox3</i>) gene paralogs. Using a <i>Rhox3</i> short hairpin (sh) RNA with 100% complementarity to all 8 <i>Rhox3</i> paralogs, expressed from a CRE-regulated transgene, we successfully knocked down <i>Rhox3</i> expression in male germ cells <i>in vivo</i>. These <i>Rhox3</i>-shRNA transgenic mice had dramatic defects in spermatogenesis, primarily in spermatocytes and round spermatids. To determine whether this phenotype was caused by reduced <i>Rhox3</i> expression, we generated mice expressing the <i>Rhox3</i>-shRNA but lacking the intended target of the shRNA—<i>Rhox3</i>. These double-mutant mice had a phenotype indistinguishable from <i>Rhox3</i>-shRNA-expressing mice that was different from mice lacking the <i>Rhox3</i> paralogs, indicating that the <i>Rhox3</i> shRNA disrupts spermatogenesis independently of <i>Rhox3</i>. <i>Rhox3</i>-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 <i>Rhox3</i> 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 <i>in vivo</i>.</p>