Off-Target Effect of doublecortin Family shRNA on Neuronal Migration Associated with Endogenous MicroRNA Dysregulation
Copyright policy )Off-Target Effect of doublecortin Family shRNA on Neuronal Migration Associated with Endogenous MicroRNA Dysregulation
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.
- University of California, San Francisco United States
- University of California System United States
- Howard Hughes Medical Institute United States
- University of California, San Diego United States
- UNIVERSITY OF CALIFORNIA SAN DIEGO
Doublecortin Domain Proteins, Doublecortin Protein, Mice, 129 Strain, Neuroscience(all), Mice, Transgenic, DOUBLE-STRANDED-RNA, SEQUENCE, NEUROGENESIS, Gene Knockout Techniques, Mice, Cell Movement, KINASE, Animals, Humans, RNA, Small Interfering, INTERFERENCE, Neurons, CENTRAL-NERVOUS-SYSTEM, Neuropeptides, PROLIFERATION, NEOCORTEX, Mice, Inbred C57BL, MICE, MicroRNAs, Phenotype, Gene Knockdown Techniques, Microtubule-Associated Proteins, RADIAL MIGRATION
Doublecortin Domain Proteins, Doublecortin Protein, Mice, 129 Strain, Neuroscience(all), Mice, Transgenic, DOUBLE-STRANDED-RNA, SEQUENCE, NEUROGENESIS, Gene Knockout Techniques, Mice, Cell Movement, KINASE, Animals, Humans, RNA, Small Interfering, INTERFERENCE, Neurons, CENTRAL-NERVOUS-SYSTEM, Neuropeptides, PROLIFERATION, NEOCORTEX, Mice, Inbred C57BL, MICE, MicroRNAs, Phenotype, Gene Knockdown Techniques, Microtubule-Associated Proteins, RADIAL MIGRATION
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