Rinne Andreas, Littwitz Christoph, Bender Kirsten, Kienitz Marie-Cécile, Pott Lutz
Department of Physiology, Stritch School of Medicine, Loyola University Chicago, Maywood, IL 60153, USA.
Methods Mol Biol. 2009;515:107-23. doi: 10.1007/978-1-59745-559-6_7.
RNA interference (RNAi) represents the most frequently utilized technique to analyze proteins by loss of function assays. Protein synthesis is impaired by sequence-specific degradation of mRNA, which is triggered by short (19-28 nt) silencing RNAs (siRNA). Efficient gene silencing using RNAi has been demonstrated in numerous cell lines and primary cultured cells. Incorporation of siRNA into terminally differentiated mammalian cells, such as adult cardiac myocytes is limited by their resistance to standard transfection protocols. Viral delivery of short-hairpin RNA (shRNA) overcomes these limitations and allows efficient gene silencing in these cells. This chapter describes the generation and characterization of recombinant siRNA-encoding adenoviruses and their application to adult cardiac myocytes, which represent a standard experimental model in research related to cardiac physiology and pathophysiology. Feasibility of this approach is demonstrated by effective ablation (>80%) of both, a transgene encoding for eGFP and the endogenous muscarinic M(2) acetylcholine receptor.
RNA干扰（RNAi）是功能缺失分析中最常用的蛋白质分析技术。短（19 - 28个核苷酸）的沉默RNA（siRNA）引发mRNA的序列特异性降解，从而损害蛋白质合成。RNAi介导的高效基因沉默已在众多细胞系和原代培养细胞中得到证实。将siRNA导入终末分化的哺乳动物细胞（如成年心肌细胞）受到限制，因为它们对标准转染方案具有抗性。短发夹RNA（shRNA）的病毒递送克服了这些限制，并能在这些细胞中实现高效基因沉默。本章描述了编码重组siRNA的腺病毒的产生、特性及其在成年心肌细胞中的应用，成年心肌细胞是心脏生理学和病理生理学相关研究中的标准实验模型。通过有效敲除（>80%）编码绿色荧光蛋白（eGFP）的转基因和内源性毒蕈碱M(2)型乙酰胆碱受体，证明了该方法的可行性。
