Skowyra Michael L, Doering Tamara L
Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, MO, USA.
Methods Mol Biol. 2012;845:165-86. doi: 10.1007/978-1-61779-539-8_11.
RNA interference (RNAi) is an experimental technique used to suppress individual gene expression in eukaryotic cells in a sequence-dependent manner. The process relies on double-stranded RNA (dsRNA) to target complementary messenger RNA for degradation. Here, we describe two plasmid-based strategies we have developed for RNAi in Cryptococcus neoformans. The pFrame vector utilizes the ACT1 promoter to enable the constitutive synthesis of hairpin RNA (hpRNA), the stem of which constitutes the dsRNA trigger. The pIBB103 vector relies on convergent, inducible GAL7 promoters to independently drive the synthesis of the sense and antisense strands of the interfering sequence; these strands anneal to form the initiating dsRNA molecule. Both vectors are designed to co-silence a "sentinel" gene with an easily scored phenotype to help identify clones in which RNAi is most effective. We provide guidelines for selecting a suitable interfering sequence to trigger RNAi in C. neoformans and describe the steps for subcloning into either vector, transforming C. neoformans by electroporation, screening clones for RNAi-related phenotypes, and evaluating the efficacy and specificity of gene silencing by RNAi.
RNA干扰（RNAi）是一种实验技术，用于以序列依赖的方式抑制真核细胞中的单个基因表达。该过程依赖于双链RNA（dsRNA）靶向互补信使RNA进行降解。在这里，我们描述了我们开发的两种用于新型隐球菌RNAi的基于质粒的策略。pFrame载体利用ACT1启动子实现发夹RNA（hpRNA）的组成型合成，其茎构成dsRNA触发物。pIBB103载体依赖于收敛的、可诱导的GAL7启动子来独立驱动干扰序列的正义链和反义链的合成；这些链退火形成起始dsRNA分子。两种载体都设计用于与具有易于评分表型的“哨兵”基因共同沉默，以帮助鉴定RNAi最有效的克隆。我们提供了选择合适的干扰序列以触发新型隐球菌RNAi的指南，并描述了亚克隆到任一载体、通过电穿孔转化新型隐球菌、筛选具有RNAi相关表型的克隆以及评估RNAi基因沉默的效力和特异性的步骤。
