Gammage Payam A, Van Haute Lindsey, Minczuk Michal
MRC Mitochondrial Biology Unit, Wellcome Trust/MRC Building, Hills Road, Cambridge, CB2 0XY, UK.
Methods Mol Biol. 2016;1351:145-62. doi: 10.1007/978-1-4939-3040-1_11.
Enrichment of desired mitochondrial DNA (mtDNA) haplotypes, in both experimental systems and the clinic, is an end sought by many. Through use of a designer nuclease platform optimized for delivery to mitochondria-the mitochondrially targeted zinc finger-nuclease (mtZFN)-it is possible to discriminate between mtDNA haplotypes with specificity to the order of a single nucleotide substitution. Site-specific cleavage of DNA produces a shift in the heteroplasmic ratio in favor of the untargeted haplotype. Here, we describe protocols for assembly of paired, conventional tail-tail mtZFN constructs and experimental approaches to assess mtZFN activity in mammalian cell cultures.
在实验系统和临床中，富集所需的线粒体DNA（mtDNA）单倍型是许多人追求的目标。通过使用针对线粒体递送进行优化的设计核酸酶平台——线粒体靶向锌指核酸酶（mtZFN），可以区分mtDNA单倍型，其特异性可达单核苷酸取代水平。DNA的位点特异性切割会导致异质性比例发生变化，有利于未靶向的单倍型。在这里，我们描述了配对的传统尾对尾mtZFN构建体的组装方案以及评估哺乳动物细胞培养物中mtZFN活性的实验方法。
