Trial and error: how the unclonable human mitochondrial genome was cloned in yeast.
Pharm Res
; 28(11): 2863-70, 2011 Nov.
Article
em En
| MEDLINE
| ID: mdl-21739320
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.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
DNA Mitocondrial
/
Terapia Genética
/
Genoma Humano
/
Sistemas de Liberação de Medicamentos
/
Vetores Genéticos
Limite:
Humans
Idioma:
En
Ano de publicação:
2011
Tipo de documento:
Article