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

Brian W Bigger, Ai-Yin Liao, Ana Sergijenko, Charles Coutelle

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)2863-2870
Number of pages8
JournalPharmaceutical Research
Volume28
Issue number11
DOIs
Publication statusPublished - Nov 2011

Keywords

  • base sequence
  • clone cells
  • DNA, mitochondrial
  • drug compounding
  • drug delivery systems
  • Escherichia coli
  • genetic therapy
  • genetic vectors
  • genome, human
  • genome, mitochondrial
  • humans
  • mitochondria
  • molecular sequence data
  • molecular targeted therapy
  • plasmids
  • recombination, genetic
  • Saccharomyces cerevisiae
  • sequence analysis, DNA
  • yeasts

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