Trial and Error: How the Unclonable Human Mitochondrial Genome was Cloned in Yeast
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.
KEY WORDSgene therapy human mitochondrial DNA mitochondrial disease unclonable yeast
autonomous replicating sequence
bacterial artificial chromosome
P1 phage artificial chromosome
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