caa a22 4500 445314311 CHVBK 20180317142641.0 cr unu---uuuuu 170323e20111101xx s 000 0 eng 10.1007/s11095-011-0527-1 doi (NATIONALLICENCE)springer-10.1007/s11095-011-0527-1 Trial and Error: How the Unclonable Human Mitochondrial Genome was Cloned in Yeast [Elektronische Daten] [Brian Bigger, Ai-Yin Liao, Ana Sergijenko, Charles Coutelle] 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 tRNAThr 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 tRNAThr 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. Springer Science+Business Media, LLC, 2011 gene therapy nationallicence human mitochondrial DNA nationallicence mitochondrial disease nationallicence unclonable nationallicence yeast nationallicence ARS : autonomous replicating sequence nationallicence BAC : bacterial artificial chromosome nationallicence D-loop : displacement loop nationallicence mtDNA : mitochondrial DNA nationallicence PAC : P1 phage artificial chromosome nationallicence TAR : transformation-associated recombination nationallicence TB : terrific broth nationallicence Bigger Brian Stem Cell & Neurotherapies, Faculty of Medical and Human Sciences, University of Manchester, 3.721 Stopford Building, M13 9PT, Manchester, UK aut Liao Ai-Yin Stem Cell & Neurotherapies, Faculty of Medical and Human Sciences, University of Manchester, 3.721 Stopford Building, M13 9PT, Manchester, UK aut Sergijenko Ana Stem Cell & Neurotherapies, Faculty of Medical and Human Sciences, University of Manchester, 3.721 Stopford Building, M13 9PT, Manchester, UK aut Coutelle Charles Gene Therapy Research Group, Sir Alexander Fleming Building, Imperial College London, SW7 5AZ, London, UK aut Pharmaceutical Research Springer US; http://www.springer-ny.com 28/11(2011-11-01), 2863-2870 0724-8741 28:11<2863 2011 28 11095 https://doi.org/10.1007/s11095-011-0527-1 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/s11095-011-0527-1 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Bigger Brian Stem Cell & Neurotherapies, Faculty of Medical and Human Sciences, University of Manchester, 3.721 Stopford Building, M13 9PT, Manchester, UK aut NATIONALLICENCE 700 1- Liao Ai-Yin Stem Cell & Neurotherapies, Faculty of Medical and Human Sciences, University of Manchester, 3.721 Stopford Building, M13 9PT, Manchester, UK aut NATIONALLICENCE 700 1- Sergijenko Ana Stem Cell & Neurotherapies, Faculty of Medical and Human Sciences, University of Manchester, 3.721 Stopford Building, M13 9PT, Manchester, UK aut NATIONALLICENCE 700 1- Coutelle Charles Gene Therapy Research Group, Sir Alexander Fleming Building, Imperial College London, SW7 5AZ, London, UK aut NATIONALLICENCE 773 0- Pharmaceutical Research Springer US; http://www.springer-ny.com 28/11(2011-11-01), 2863-2870 0724-8741 28:11<2863 2011 28 11095 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer