Targeting repair proteins to the mitochondria of mammalian cells through stable transfection, transient transfection, viral transduction, and TAT-mediated protein transduction.
The mitochondrial genome represents a target for exogenous and endogenous damage. Its necessity for successful electron transport makes its repair valuable to the cell. Previous work from our lab has shown that mitochondrial DNA (mtDNA) can be repaired in mammalian cells, and the use of mitochondrial-targeted repair proteins can augment repair to enhance viability following genotoxic stress. In addition, it has also been shown that other repair enzymes that are targeted to the mitochondria can sensitize the cell to DNA damaging agents, thereby aiding the effectiveness of certain chemotherapeutic agents. The methods herein describe the development of mitochondrial-targeted proteins using plasmids or protein transduction domains. It includes the utilization of these constructs to create stably transfected cell lines, transiently transfected cell lines, viral-mediated transduction, and protein transduction domain-mediated mitochondrial protein localization. The end result will be a mammalian cell that expresses the mitochondrial-targeted protein of interest.
Koczor
Christopher A
CA
Department of Cell Biology and Neuroscience, College of Medicine, University of South Alabama, Mobile, AL, USA.
Snyder
Janet W
JW
Shokolenko
Inna N
IN
Dobson
Allison W
AW
Wilson
Glenn L
GL
Ledoux
Susan P
SP
D000818:Animals; D015139:Blotting, Southern; D002470:Cell Survival; D004249:DNA Damage; D004260:DNA Repair; D004272:DNA, Mitochondrial / Q000235:genetics* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D015696:Gene Products, tat / Q000378:metabolism*; D018014:Gene Transfer Techniques; D006801:Humans; D008928:Mitochondria / Q000235:genetics* / Q000378:metabolism*; D010957:Plasmids; D021381:Protein Transport; D014161:Transduction, Genetic; D014162:Transfection
D004272:DNA, Mitochondrial; D004268:DNA-Binding Proteins; D015696:Gene Products, tat