Nonreplicating Intracellular Bacterial Vector for Conjugative DNA Transfer into Mitochondria
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We have previously shown that DNA constructs can be introduced into isolated mitochondria through the process of conjugative transfer from an E. coli host. We set out to generate a conjugative E. coli strain that would be able to introduce itself into the cytoplasm of a mammalian cell for the purpose of transferring DNA into the mitochondria in the cell.
We have now developed a method for making E. coli strains from which nonreplicating populations of daughter cells can be generated. We used this approach to modify a facultative intracellular enteroinvasive E. coli (EIEC) and introduced conjugative functions to this new strain.
We demonstrate that this new strain can generate large populations of nonreplicating cells that are capable of conjugative transfer to other cells and can readily invade mammalian tissue culture cells, live in the cytoplasm of the cell for several days, and that do not kill the invaded mammalian cell.
We successfully constructed an E. coli host suitable for intracellular conjugative transfer but, due to the lack of suitable mitochondrial screening or selectable markers, we have not yet been able to determine if these bacterial vectors can in fact transfer DNA into intracelluar mitochondria.
KEY WORDSconjugative DNA transfer facultative intracellular enteroinvasive Escherichia coli mitochondria mitochondrial DNA nonreplicating bacterial cell
enhanced green fluorescent protein
enteroinvasive Escherichia coli
Polymerase Chain Reaction
Acknowledgments & Disclosures
We thank Michael Sadowsky for the gifts of plasmids and E.coli strains. This work was funded by the National Institutes of Health (NS052612) and by the Minnesota Partnership for Biotechnology and Medical Genomics.