4.8 Article

Off-Target Effect of doublecortin Family shRNA on Neuronal Migration Associated with Endogenous MicroRNA Dysregulation

Journal

NEURON
Volume 82, Issue 6, Pages 1255-1262

Publisher

CELL PRESS
DOI: 10.1016/j.neuron.2014.04.036

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Funding

  1. National Institutes of Health [R01NS41537]
  2. EMBO Long Term Fellowship
  3. Giannini Fellowship
  4. Brain Behavior Research Foundation

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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.

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