Mitochondrial Biology Unit, MRC, Cambridge, UK.
Nat Protoc. 2010 Feb;5(2):342-56. doi: 10.1038/nprot.2009.245. Epub 2010 Feb 4.
Engineered zinc-finger proteins (ZFPs) are hybrid proteins developed to direct various effector domains (EDs) of choice to predetermined DNA sequences. They are used to alter gene expression and to modify DNA in a sequence-specific manner in vivo and in vitro. Until now, ZFPs have mostly been used to target DNA sites in nuclear genomes. This protocol describes how to adapt engineered ZFP technology to specifically modify the mammalian mitochondrial genome. The first step describes how to construct mitochondrially targeted ZFPs (mtZFPs) so that they are efficiently imported into mammalian mitochondria. In the second step, methods to test the basic properties of mtZFPs in vitro are described. Finally, we outline how the mtZFPs can be transiently transfected into mammalian cells and their mitochondrial import tested by both immunofluorescence and biochemical methods. The protocol can be completed within a week, although time-consuming DNA cloning steps may extend this.
基因工程锌指蛋白（ZFPs）是一种杂合蛋白，用于将各种效应结构域（EDs）定向到预定的 DNA 序列。它们被用于在体内和体外以序列特异性的方式改变基因表达和修饰 DNA。到目前为止，ZFPs 主要用于靶向核基因组中的 DNA 位点。本方案描述了如何将基因工程 ZFP 技术用于特异性修饰哺乳动物线粒体基因组。第一步描述了如何构建靶向线粒体的 ZFPs（mtZFPs），使其能够有效地导入哺乳动物线粒体。第二步描述了体外测试 mtZFPs 基本性质的方法。最后，我们概述了如何将 mtZFPs 瞬时转染到哺乳动物细胞中，并通过免疫荧光和生化方法检测其线粒体导入。该方案可以在一周内完成，尽管耗时的 DNA 克隆步骤可能会延长时间。
