Neurogenetics Laboratory Howard Hughes Medical Institute, Department of Neurosciences, University of California, San Diego La Jolla, CA 92093.
Neuron. 2014 Jun 18;82(6):1255-1262. doi: 10.1016/j.neuron.2014.04.036.
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.
利用短发夹 RNA(shRNA，敲低)在发育中的大脑中进行急性基因失活是研究遗传功能的强大技术；然而，敲低和敲除鼠表型之间的差异仍存在未解决的问题。例如，双皮质素(Dcx)敲低而非敲除显示出新皮层神经元迁移表型。在这里，我们报告说，将 shRNA 而不是 siRNA 或 miRNA 电穿孔到 Dcx 中，在 Dcx 敲除小鼠中表现出类似于野生型小鼠的迁移表型，这表明 shRNA 介导的脱靶毒性。这种效应不仅限于 Dcx，因为它在 Dclk1 敲除小鼠中以及部分乱序 shRNA 中都观察到，表明这是一种序列依赖性但非序列特异性的效应。对电穿孔细胞中的 RNA 进行分析显示，内源性 let7 miRNA 水平存在缺陷，而 let7 或 Dicer 的破坏则重现了迁移缺陷。结果表明，shRNA 介导的敲低可能通过改变内源性 miRNA 途径产生不良的迁移效应。
