Bosch Justin A, Sumabat Taryn M, Hariharan Iswar K
Department of Molecular and Cell Biology, University of California, Berkeley, California 94720.
Department of Molecular and Cell Biology, University of California, Berkeley, California 94720
Genetics. 2016 May;203(1):109-18. doi: 10.1534/genetics.116.187062. Epub 2016 Mar 16.
RNA interference (RNAi) has emerged as a powerful way of reducing gene function in Drosophila melanogaster tissues. By expressing synthetic short hairpin RNAs (shRNAs) using the Gal4/UAS system, knockdown is efficiently achieved in specific tissues or in clones of marked cells. Here we show that knockdown by shRNAs is so potent and persistent that even transient exposure of cells to shRNAs can reduce gene function in their descendants. When using the FLP-out Gal4 method, in some instances we observed unmarked "shadow RNAi" clones adjacent to Gal4-expressing clones, which may have resulted from brief Gal4 expression following recombination but prior to cell division. Similarly, Gal4 driver lines with dynamic expression patterns can generate shadow RNAi cells after their activity has ceased in those cells. Importantly, these effects can lead to erroneous conclusions regarding the cell autonomy of knockdown phenotypes. We have investigated the basis of this phenomenon and suggested experimental designs for eliminating ambiguities in interpretation. We have also exploited the persistence of shRNA-mediated knockdown to design a sensitive lineage-tracing method, i-TRACE, which is capable of detecting even low levels of past reporter expression. Using i-TRACE, we demonstrate transient infidelities in the expression of some cell-identity markers near compartment boundaries in the wing imaginal disc.
RNA干扰（RNAi）已成为一种在黑腹果蝇组织中降低基因功能的强大方法。通过使用Gal4/UAS系统表达合成短发夹RNA（shRNA），可以在特定组织或标记细胞的克隆中有效地实现基因敲低。在这里，我们表明shRNA介导的基因敲低非常有效且持久，以至于即使细胞短暂暴露于shRNA也能降低其后代的基因功能。当使用FLP-out Gal4方法时，在某些情况下，我们观察到与表达Gal4的克隆相邻的未标记“影子RNAi”克隆，这可能是由于重组后但细胞分裂前短暂的Gal4表达所致。同样，具有动态表达模式的Gal4驱动系在其活性在这些细胞中停止后也可以产生影子RNAi细胞。重要的是，这些效应可能导致关于基因敲低表型细胞自主性的错误结论。我们研究了这种现象的基础，并提出了消除解释歧义的实验设计。我们还利用了shRNA介导的基因敲低的持久性来设计一种敏感的谱系追踪方法，即i-TRACE，它甚至能够检测到过去低水平的报告基因表达。使用i-TRACE，我们证明了在翅成虫盘中隔界附近一些细胞身份标记表达的短暂不忠实性。
