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

5 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

Fingerprint

Mitochondrial Genome
Human Genome
Yeast
Mitochondrial Genes
RNA, Transfer, Thr
Genes
Yeasts
Mitochondrial DNA
Escherichia coli
Genetic Therapy
Organism Cloning
Gene therapy
Cloning
Genetic Vectors
Aptitude
Human Development
Genetic Recombination
Saccharomyces cerevisiae
Plasmids
Clone Cells

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

Cite this

Trial and error : how the unclonable human mitochondrial genome was cloned in yeast. / Bigger, Brian W; Liao, Ai-Yin; Sergijenko, Ana; Coutelle, Charles.

In: Pharmaceutical Research, Vol. 28, No. 11, 11.2011, p. 2863-2870.

Research output: Contribution to journalArticle

Bigger, Brian W ; Liao, Ai-Yin ; Sergijenko, Ana ; Coutelle, Charles. / Trial and error : how the unclonable human mitochondrial genome was cloned in yeast. In: Pharmaceutical Research. 2011 ; Vol. 28, No. 11. pp. 2863-2870.
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