Nayak Arabinda, Andino Raul
Department of Microbiology and Immunology, University of California, San Francisco, CA, USA.
Methods Mol Biol. 2011;721:231-44. doi: 10.1007/978-1-61779-037-9_14.
Reconstitution of RNA-inducing silencing complex (RISC) in vitro is a powerful biochemical technique to analyze crucial steps in RNA interference (RNAi) pathways. RISC contains an RNase enzyme, Argonaute, which is guided by small interfering RNA (siRNA) to recognize and silence its targets. Drosophila S2 cell extract is a good source of enzymes and factors that faithfully recapitulate essential steps in RISC assembly and function. In this chapter, we will describe how to prepare enzymatically active cell-free S2 extract to analyze the Slicer activity of RISC as well as the effects of viral RNAi suppressors which block this process.
在体外重建RNA诱导沉默复合体（RISC）是一种强大的生化技术，用于分析RNA干扰（RNAi）途径中的关键步骤。RISC包含一种核糖核酸酶——AGO蛋白，它由小干扰RNA（siRNA）引导，以识别并沉默其靶标。果蝇S2细胞提取物是酶和因子的良好来源，能忠实地重现RISC组装和功能的基本步骤。在本章中，我们将描述如何制备具有酶活性的无细胞S2提取物，以分析RISC的切割活性以及阻断该过程的病毒RNAi抑制因子的作用。
