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Neuron. 2014 Jun 18;82(6):1255-1262. doi: 10.1016/j.neuron.2014.04.036.

Off-target effect of doublecortin family shRNA on neuronal migration associated with endogenous microRNA dysregulation.

Author information

1
Neurogenetics Laboratory Howard Hughes Medical Institute, Department of Neurosciences, University of California, San Diego La Jolla, CA 92093.
#
Contributed equally

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.

PMID:
24945770
PMCID:
PMC4086250
DOI:
10.1016/j.neuron.2014.04.036
[Indexed for MEDLINE]
Free PMC Article

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