Construction and testing of engineered zinc-finger proteins for sequence-specific modification of mtDNA.
Nat Protoc
; 5(2): 342-56, 2010 Feb.
Article
em En
| MEDLINE
| ID: mdl-20134433
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.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
DNA Mitocondrial
/
Engenharia Genética
/
Dedos de Zinco
Idioma:
En
Revista:
Nat Protoc
Ano de publicação:
2010
Tipo de documento:
Article
País de afiliação:
Reino Unido