Modification of the mouse mitochondrial genome by insertion of an exogenous gene

Abstract

Using homologous recombination in yeast we have inserted a synthetic gene encoding human ornithine transcarbamylase (sOTC), designed to allow mitochondrial (mt) translation, into the mouse mt genome. Modification of the mt genome was facilitated by its cloning into a yeast centromeric plasmid. The sOTC gene was initially flanked by 25 bp of the mt tRNA(His) gene at its 5' end and by 23 bp of the mt tRNA(Ser (AGY)) gene at its 3' end (Wheeler et al., 1996). In order to achieve homologous recombination the flanking homology was subsequently extended to 525 and 362 bp by the polymerase chain reaction (PCR). The sOTC gene was thus inserted into the cloned mt genome at a unique location between the tRNA(His) and tRNA(Ser (AGY)) genes. Positioning of the sOTC gene between these normally contiguous tRNA genes should allow its processing from the mt polycistronic transcript. The ability to modify the mammalian mt genome in this way is a valuable step towards a functional analysis of mt genetic mechanisms and possibly also towards a gene therapy approach for mt disorders.