title: Engineered mtZFNs for Manipulation of Human Mitochondrial DNA Heteroplasmy.
abstract: 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.
authors.affiliation: MRC Mitochondrial Biology Unit, Wellcome Trust/MRC Building, Hills Road, Cambridge, CB2 0XY, UK. pag44@mrc-mbu.cam.ac.uk.
authors.forename: Payam A
authors.initials: PA
authors.lastname: Gammage
authors.affiliation: MRC Mitochondrial Biology Unit, Wellcome Trust/MRC Building, Hills Road, Cambridge, CB2 0XY, UK.
authors.forename: Lindsey
authors.initials: L
authors.lastname: Van Haute
authors.affiliation: MRC Mitochondrial Biology Unit, Wellcome Trust/MRC Building, Hills Road, Cambridge, CB2 0XY, UK. mam@mrc-mbu.cam.ac.uk.
authors.forename: Michal
authors.initials: M
authors.lastname: Minczuk
mesh_terms: D002478:Cells, Cultured; D053903:DNA Breaks, Double-Stranded; D004272:DNA, Mitochondrial / Q000235:genetics*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics / Q000378:metabolism*; D018628:Gene Dosage / Q000235:genetics*; D014644:Genetic Variation / Q000235:genetics; D006239:Haplotypes / Q000235:genetics; D006801:Humans; D008928:Mitochondria / Q000235:genetics*; D028361:Mitochondrial Diseases / Q000235:genetics; D016335:Zinc Fingers / Q000235:genetics*
chemical_list: D004272:DNA, Mitochondrial; C035042:endodeoxyribonuclease FokI; D015252:Deoxyribonucleases, Type II Site-Specific
keywords: Gene therapy; Genetic engineering; Heteroplasmy; Mitochondrial disease; Zinc finger; Zinc finger nuclease