Pharmaceutical Research

, 28:2863 | Cite as

Trial and Error: How the Unclonable Human Mitochondrial Genome was Cloned in Yeast

  • Brian W. Bigger
  • Ai-Yin Liao
  • Ana Sergijenko
  • Charles Coutelle
Research Paper



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 tRNAThr gene.


We tested multi- and single-copy vector systems for cloning human mitochondrial DNA in E. coli and Saccharomyces cerevisiae, including transformation-associated recombination.


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 tRNAThr were both stable and unmutated.


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.


gene therapy human mitochondrial DNA mitochondrial disease unclonable yeast 



autonomous replicating sequence


bacterial artificial chromosome


displacement loop


mitochondrial DNA


P1 phage artificial chromosome


transformation-associated recombination


terrific broth

Supplementary material

11095_2011_527_MOESM1_ESM.docx (21 kb)
Supplementary Table 1PCR and sequencing primers (DOCX 21 kb)
11095_2011_527_MOESM2_ESM.docx (13 kb)
Supplementary Table 2PCR conditions (DOCX 12.8 kb)


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Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Brian W. Bigger
    • 1
  • Ai-Yin Liao
    • 1
  • Ana Sergijenko
    • 1
  • Charles Coutelle
    • 2
  1. 1.Stem Cell & Neurotherapies, Faculty of Medical and Human SciencesUniversity of ManchesterManchesterUK
  2. 2.Gene Therapy Research Group, Sir Alexander Fleming BuildingImperial College LondonLondonUK

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