shRNA off-target effects in vivo: impaired endogenous siRNA expression and spermatogenic defects

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dc.contributor.authorSong, H.
dc.contributor.authorBettegowda, A.
dc.contributor.authorOliver, D.
dc.contributor.authorYan, W.
dc.contributor.authorPhan, M.
dc.contributor.authorDe Rooij, D.
dc.contributor.authorCorbett, M.
dc.contributor.authorWilkinson, M.
dc.contributor.editorWhite-Cooper, H.
dc.date.issued2015
dc.description.abstractRNA 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.
dc.description.statementofresponsibilityHye-Won Song, Anilkumar Bettegowda, Daniel Oliver, Wei Yan, Mimi H. Phan, Dirk G. de Rooij, Mark A. Corbett, Miles F. Wilkinson
dc.identifier.citationPLoS ONE, 2015; 10(3):e0118549-1-e0118549-23
dc.identifier.doi10.1371/journal.pone.0118549
dc.identifier.issn1932-6203
dc.identifier.issn1932-6203
dc.identifier.orcidCorbett, M. [0000-0001-9298-3072]
dc.identifier.urihttp://hdl.handle.net/2440/94227
dc.language.isoen
dc.publisherPublic Library of Science
dc.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
dc.source.urihttps://doi.org/10.1371/journal.pone.0118549
dc.subjectHela Cells
dc.subjectAnimals
dc.subjectMice, Transgenic
dc.subjectHumans
dc.subjectMice
dc.subjectHomeodomain Proteins
dc.subjectRNA, Small Interfering
dc.subjectDNA Primers
dc.subjectMicroarray Analysis
dc.subjectIn Situ Nick-End Labeling
dc.subjectSpermatogenesis
dc.subjectRNA Interference
dc.subjectModels, Biological
dc.subjectMale
dc.subjectGene Knockdown Techniques
dc.titleshRNA off-target effects in vivo: impaired endogenous siRNA expression and spermatogenic defects
dc.typeJournal article
pubs.publication-statusPublished

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