MODIFICATION OF THE MOUSE MITOCHONDRIAL GENOME BY INSERTION OF AN EXOGENOUS GENE

Using homologous recombination in yeast we have inserted a synthetic g ene encoding human ornithine transcarbamylase (sOTC), designed to allo w mitochondrial (mt) translation, into the mouse mt genome. Modificati on of the mt genome was facilitated by its cloning into a yeast centro meric 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)) ge ne at its 3' end (Wheeler et al., 1996). In order to achieve homologou s 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 t he tRNA(His) and tRNA(Ser (AGY)) genes. Positioning of the sOTC gene b etween these normally contiguous tRNA genes should allow its processin g from the mt polycistronic transcript. The ability to modify the mamm alian mt genome in this way is a valuable step towards a functional an alysis of mt genetic mechanisms and possibly also towards a gene thera py approach for mt disorders. (C) 1997 Elsevier Science B.V.