Construction and testing of engineered zinc-finger proteins for sequence-specific modification of mtDNA.

TitleConstruction and testing of engineered zinc-finger proteins for sequence-specific modification of mtDNA.
Publication TypeJournal Article
Year of Publication2010
AuthorsMinczuk, MA, Kolasinska-Zwierz, P, Murphy, MP, Papworth, MA
JournalNat Protoc
Volume5
Issue2
Pagination342-56
Date Published2010 Feb
ISSN1750-2799
KeywordsAmino Acid Sequence, Base Sequence, DNA Probes, DNA, Mitochondrial, Genetic Engineering, Genome, Mitochondrial, Mutagenesis, Oligodeoxyribonucleotides, Plasmids, Zinc Fingers
Abstract

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.

DOI10.1038/nprot.2009.245
Alternate JournalNat Protoc
Citation Key10.1038/nprot.2009.245
PubMed ID20134433
Grant ListMC_U105663142 / / Medical Research Council / United Kingdom
MC_U105697135 / / Medical Research Council / United Kingdom
/ / Medical Research Council / United Kingdom