Berlivet Soizik, Houlard Martin, Gérard Matthieu
Epigenetic Regulation and Cancer Group, CEA, iBiTecS, Gif-sur-Yvette Cedex, France.
Methods Mol Biol. 2010;650:85-100. doi: 10.1007/978-1-60761-769-3_7.
RNA interference is widely used for loss-of-function studies in mammalian cells. As an alternative to the transfection of small RNAs, plasmid vectors have been developed to express short hairpin RNAs (shRNAs). We engineered the pHYPER shRNA vector, which is based on a 2.5-kb mouse genomic fragment encompassing the H1 gene. We have previously shown that this shRNA vector is highly efficient for both transient transfection studies in embryonic stem (ES) cells and generation of stable ES cell lines. Following ES cell transfection, the H1 promoter of pHYPER is recognized by the RNA polymerase III machinery, which directs the transcription of the shRNA. We provide here detailed protocols that explain how to optimize the use of pHYPER in ES cells.
RNA干扰在哺乳动物细胞的功能缺失研究中被广泛应用。作为小RNA转染的替代方法，已开发出质粒载体来表达短发夹RNA（shRNA）。我们构建了基于包含H1基因的2.5kb小鼠基因组片段的pHYPER shRNA载体。我们之前已表明，这种shRNA载体在胚胎干细胞（ES细胞）的瞬时转染研究以及稳定ES细胞系的生成中都非常高效。ES细胞转染后，pHYPER的H1启动子被RNA聚合酶III机制识别，从而指导shRNA的转录。我们在此提供详细方案，解释如何在ES细胞中优化使用pHYPER。
