title: Off-target effect of doublecortin family shRNA on neuronal migration associated with endogenous microRNA dysregulation.
abstract: Acute gene inactivation using short hairpin RNA (shRNA, knockdown) in developing brain is a powerful technique to study genetic function; however, discrepancies between knockdown and knockout murine phenotypes have left unanswered questions. For example, doublecortin (Dcx) knockdown but not knockout shows a neocortical neuronal migration phenotype. Here we report that in utero electroporation of shRNA, but not siRNA or miRNA, to Dcx demonstrates a migration phenotype in Dcx knockouts akin to the effect in wild-type mice, suggesting shRNA-mediated off-target toxicity. This effect was not limited to Dcx, as it was observed in Dclk1 knockouts, as well as with a fraction of scrambled shRNAs, suggesting a sequence-dependent but not sequence-specific effect. Profiling RNAs from electroporated cells showed a defect in endogenous let7 miRNA levels, and disruption of let7 or Dicer recapitulated the migration defect. The results suggest that shRNA-mediated knockdown can produce untoward migration effects by altering endogenous miRNA pathways.
authors.affiliation: Neurogenetics Laboratory Howard Hughes Medical Institute, Department of Neurosciences, University of California, San Diego La Jolla, CA 92093.
authors.forename: Seung Tae
authors.initials: ST
authors.lastname: Baek
authors.affiliation: Neurogenetics Laboratory Howard Hughes Medical Institute, Department of Neurosciences, University of California, San Diego La Jolla, CA 92093.
authors.forename: Geraldine
authors.initials: G
authors.lastname: Kerjan
authors.affiliation: Neurogenetics Laboratory Howard Hughes Medical Institute, Department of Neurosciences, University of California, San Diego La Jolla, CA 92093.
authors.forename: Stephanie L
authors.initials: SL
authors.lastname: Bielas
authors.affiliation: Neurogenetics Laboratory Howard Hughes Medical Institute, Department of Neurosciences, University of California, San Diego La Jolla, CA 92093.
authors.forename: Ji Eun
authors.initials: JE
authors.lastname: Lee
authors.affiliation: Neurogenetics Laboratory Howard Hughes Medical Institute, Department of Neurosciences, University of California, San Diego La Jolla, CA 92093.
authors.forename: Ali G
authors.initials: AG
authors.lastname: Fenstermaker
authors.affiliation: Neurogenetics Laboratory Howard Hughes Medical Institute, Department of Neurosciences, University of California, San Diego La Jolla, CA 92093.
authors.forename: Gaia
authors.initials: G
authors.lastname: Novarino
authors.affiliation: Neurogenetics Laboratory Howard Hughes Medical Institute, Department of Neurosciences, University of California, San Diego La Jolla, CA 92093.
authors.forename: Joseph G
authors.initials: JG
authors.lastname: Gleeson
mesh_terms: D000818:Animals; D002465:Cell Movement / Q000235:genetics*; D000089622:Doublecortin Domain Proteins; D000089662:Doublecortin Protein; D055785:Gene Knockdown Techniques / Q000379:methods*; D055786:Gene Knockout Techniques / Q000379:methods; D006801:Humans; D051379:Mice; D057507:Mice, 129 Strain; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D035683:MicroRNAs / Q000037:antagonists & inhibitors* / Q000235:genetics*; D008869:Microtubule-Associated Proteins / Q000235:genetics*; D009474:Neurons / Q000166:cytology / Q000502:physiology*; D009479:Neuropeptides / Q000235:genetics*; D010641:Phenotype; D034741:RNA, Small Interfering / Q000235:genetics*
chemical_list: C000714328:DCX protein, human; C000714847:Dcx protein, mouse; D000089622:Doublecortin Domain Proteins; D000089662:Doublecortin Protein; D035683:MicroRNAs; D008869:Microtubule-Associated Proteins; D009479:Neuropeptides; D034741:RNA, Small Interfering