Delivery of mtZFNs into early mouse embryos

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McCann, Beverly 
Cox, Andy 
Gammage, Payam 
Stewart, James 
Zernicka-Goetz, Magdalena  ORCID logo

Mitochondrial diseases often result from mutations in the mitochondrial genome (mtDNA). In most cases, mutant mtDNA coexists with wild-type mtDNA, resulting in heteroplasmy. One potential future approach to treat heteroplasmic mtDNA diseases is the specific elimination of pathogenic mtDNA mutations, lowering the level of mutant mtDNA below pathogenic thresholds. Mitochondrially-targeted zinc finger nucleases (mtZFNs) have been demonstrated to specifically target and introduce double-strand breaks in mutant mtDNA, facilitating substantial shifts in heteroplasmy. One application of mtZFN technology, in the context of heteroplasmic mtDNA disease, is delivery into the heteroplasmic oocyte or early embryo to eliminate mutant mtDNA, preventing transmission of mitochondrial diseases through the germline. Here we describe a protocol for efficient production of mtZFN mRNA in vitro, and delivery of these into 0.5 dpc mouse embryos to elicit shifts of mtDNA heteroplasmy.

Germline, In vitro transcription, Micromanipulation, Mitochondrial disease, mtZFN, Animals, Cells, Cultured, DNA Breaks, Double-Stranded, DNA, Mitochondrial, Embryo, Mammalian, Female, Gene Transfer Techniques, Genome, Mitochondrial, Male, Mice, Mice, Inbred C57BL, Mitochondria, Mutation, Zinc Finger Nucleases
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Zinc Finger Proteins
Champ Foundation (unknown)
Medical Research Council (MC_U105697135)
Medical Research Council (MC_UU_00015/4)