title: Introduction of chloramphenicol resistance into the modified mouse mitochondrial genome: cloning of unstable sequences by passage through yeast.
abstract: Despite increasing awareness of the importance of the mitochondrial genome in human pathology, very few attempts have been made so far toward genetic engineering of mitochondrial DNA (mtDNA). One of the reasons for this slow progress is the difficulty of cloning mtDNA in Escherichia coli, a trait in common with repetitive or palindromic sequences, and some viral sequences. We have previously made a construct containing the entire mouse mitochondrial genome and a cDNA sequence coding for human ornithine transcarbamylase in a yeast/bacterial shuttle vector, which can be stably maintained in E. coli. We wished to modify this vector for mitochondrial gene therapy by the addition of mitochondrial chloramphenicol resistance, conferred by a point mutation in the 16S rRNA gene. Attempts to modify this construct by a straightforward cloning approach in E. coli proved unsuccessful. Two successful strategies for modification of large unstable constructs in both E. coli and the yeast Saccharomyces cerevisiae are compared here.
authors.affiliation: Cystic Fibrosis Gene Therapy Research Group, Section of Molecular Genetics, Division of Biomedical Sciences, Imperial College School of Medicine, Sir Alexander Fleming Building, South Kensington, London, SW7 2AZ, United Kingdom. b.bigger@ic.ac.uk
authors.forename: B
authors.initials: B
authors.lastname: Bigger
authors.forename: O
authors.initials: O
authors.lastname: Tolmachov
authors.forename: J M
authors.initials: JM
authors.lastname: Collombet
authors.forename: C
authors.initials: C
authors.lastname: Coutelle
mesh_terms: D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D002701:Chloramphenicol / Q000494:pharmacology*; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004272:DNA, Mitochondrial; D004352:Drug Resistance, Microbial / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D015316:Genetic Therapy; D005822:Genetic Vectors; D006801:Humans; D051379:Mice; D009154:Mutation; D009954:Ornithine Carbamoyltransferase / Q000235:genetics; D012441:Saccharomyces cerevisiae
chemical_list: D000900:Anti-Bacterial Agents; D018076:DNA, Complementary; D004272:DNA, Mitochondrial; D002701:Chloramphenicol; D009954:Ornithine Carbamoyltransferase