title: Trial and error: how the unclonable human mitochondrial genome was cloned in yeast.
abstract: OBJECTIVE
  Development of a human mitochondrial gene delivery vector is a critical step in the ability to treat diseases arising from mutations in mitochondrial DNA. Although we have previously cloned the mouse mitochondrial genome in its entirety and developed it as a mitochondrial gene therapy vector, the human mitochondrial genome has been dubbed unclonable in E. coli, due to regions of instability in the D-loop and tRNA(Thr) gene.
  
  
  METHODS
  We tested multi- and single-copy vector systems for cloning human mitochondrial DNA in E. coli and Saccharomyces cerevisiae, including transformation-associated recombination.
  
  
  RESULTS
  Human mitochondrial DNA is unclonable in E. coli and cannot be retained in multi- or single-copy vectors under any conditions. It was, however, possible to clone and stably maintain the entire human mitochondrial genome in yeast as long as a single-copy centromeric plasmid was used. D-loop and tRNA(Thr) were both stable and unmutated.
  
  
  CONCLUSIONS
  This is the first report of cloning the entire human mitochondrial genome and the first step in developing a gene delivery vehicle for human mitochondrial gene therapy.
authors: Stem Cell & Neurotherapies, Faculty of Medical and Human Sciences, University of Manchester, 3.721 Stopford Building, Manchester M13 9PT, UK. brian.bigger@manchester.ac.uk
authors: Brian W
authors: BW
authors: Bigger
authors: Ai-Yin
authors: AY
authors: Liao
authors: Ana
authors: A
authors: Sergijenko
authors: Charles
authors: C
authors: Coutelle
mesh_terms: D001483:Base Sequence; D002999:Clone Cells; D004272:DNA, Mitochondrial / Q000235:genetics*; D004339:Drug Compounding; D016503:Drug Delivery Systems; D004926:Escherichia coli / Q000235:genetics; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D015894:Genome, Human; D054629:Genome, Mitochondrial; D006801:Humans; D008928:Mitochondria / Q000235:genetics; D008969:Molecular Sequence Data; D058990:Molecular Targeted Therapy; D010957:Plasmids; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics; D017422:Sequence Analysis, DNA; D015003:Yeasts / Q000235:genetics
chemical_list: D004272:DNA, Mitochondrial