30] Use of ethidium bromide to manipulate ratio of mutated and wild-type mitochondrial DNA in cultured cells

Abstract

Publisher Summary This chapter discusses the use of ethidium bromide to manipulate the ratio of mutated and wild-type mitochondrial DNA in cultured cells. In many cases, they exist in heteroplasmic form—that is, both mutated and wild-type genomes are present within cells. To investigate the effects of these mutations on mitochondrial function, it is desirable to isolate clonal cell lines that contain exclusively mutated or exclusively wildtype mitochondrial DNA (mtDNA). The chapter describes a system is for the characterization of mutations of the mtDNA. This system is based on the isolation of human cell lines that completely lack mtDNA (ρ˚0 cells). This system is used to investigate the pathogenetic mechanisms of mutations of mtDNA. In addition, one usually selects for mitochondrial transformants of the ρ0 cells by selecting for mitochondrial respiratory chain function. Therefore, to compare the phenotype of cells possessing 100% wild-type and 100% mutated mtDNA, or to investigate the threshold level of mutation resulting in phenotypic changes, it may be necessary to manipulate the mtDNA complement of cells for isolating cell lines that possess varying levels of mutation.