We have developed a method for transferring exogenous DNA molecules into isolated mammalian mitochondria using bacterial conjugation. In general, we accomplish this by (i) inserting an origin of DNA transfer (oriT) sequence into a DNA construct, (ii) transforming the construct into an appropriate Escherichia coli strain and then (iii) introducing the mobilizable DNA into mitochondria through conjugation. We tested this approach by transferring plasmid DNA containing a T7 promoter sequence into mitochondria that we had engineered to contain T7 RNA polymerase. After conjugation between E.coli and mitochondria, we detected robust levels of T7 transcription from the DNA constructs that had been transferred into the mitochondria. This approach for engineering DNA constructs in vitro and subsequent transfer into mitochondria by conjugation offers an attractive experimental system for studying many aspects of vertebrate mitochondrial gene expression and is a potential route for transforming mitochondrial networks within mammalian cells.
Bibliographical noteFunding Information:
The authors would like to thank Michael Sadowsky for the gifts of plasmids and E.coli strains, and the generous financial support from the Minnesota Medical Foundation, the Academic Health Center and the Institute of Human Genetics of the University of Minnesota. Funding to pay the Open Access publication charges for this article was provided by a grant from NIH (NINDS grant number NS052612).
PubMed: MeSH publication types
- Evaluation Study
- Journal Article
- Research Support, N.I.H., Extramural
- Research Support, Non-U.S. Gov't
- Research Support, U.S. Gov't, P.H.S.