Ma Hoi Tang, Poon Randy Y C
Division of Life Science, Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong.
Curr Protoc Cell Biol. 2010 Dec;Chapter 27:Unit 27.2. doi: 10.1002/0471143030.cb2702s49.
The principal problem with RNA interference (RNAi) experiments is off-target effects. The most vigorous demonstration of the specificity is the rescue of the RNAi effects with an RNAi-resistant target gene. By combining the expression of short hairpin RNA (shRNA) and rescue cDNA in the same vector, both the validation of shRNA specificity and the generation of shRNA-expressing cell lines can easily be accomplished. If the compensatory cDNA is under the control of an inducible promoter, stable shRNA-expressing cells can be generated before the knockdown phenotypes are studied, by conditionally turning off the rescue protein. The use of model systems is detailed in these protocols.
RNA干扰（RNAi）实验的主要问题是脱靶效应。特异性的最有力证明是用RNAi抗性靶基因挽救RNAi效应。通过在同一载体中组合短发夹RNA（shRNA）和挽救cDNA的表达，可以轻松完成shRNA特异性的验证以及产生表达shRNA的细胞系。如果补偿性cDNA受诱导型启动子控制，则可以在研究敲低表型之前，通过有条件地关闭挽救蛋白来产生稳定表达shRNA的细胞。这些方案中详细介绍了模型系统的使用。
