Trial and error: how the unclonable human mitochondrial genome was cloned in yeast

Pharm Res. 2011 Nov;28(11):2863-70. doi: 10.1007/s11095-011-0527-1. Epub 2011 Jul 9.


Purpose: 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • Clone Cells
  • DNA, Mitochondrial / genetics*
  • Drug Compounding
  • Drug Delivery Systems*
  • Escherichia coli / genetics
  • Genetic Therapy / methods*
  • Genetic Vectors*
  • Genome, Human*
  • Genome, Mitochondrial
  • Humans
  • Mitochondria / genetics
  • Molecular Sequence Data
  • Molecular Targeted Therapy
  • Plasmids
  • Recombination, Genetic
  • Saccharomyces cerevisiae / genetics
  • Sequence Analysis, DNA
  • Yeasts / genetics


  • DNA, Mitochondrial

Associated data

  • GENBANK/JN160804