Transformation of isolated mammalian mitochondria by bacterial conjugation.
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.
Yoon
Young Geol
YG
Department of Laboratory Medicine and Pathology, University of Minnesota, 420 Delaware Street SE, Minneapolis, MN 55455, USA.
Koob
Michael D
MD
D000818:Animals; D001483:Base Sequence; D002021:Buffers; D002460:Cell Line; D003227:Conjugation, Genetic; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000096:biosynthesis / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D005818:Genetic Engineering / Q000379:methods*; D051379:Mice; D008928:Mitochondria / Q000235:genetics*; D008969:Molecular Sequence Data; D010957:Plasmids; D012313:RNA / Q000096:biosynthesis; D000077278:RNA, Mitochondrial; D014170:Transformation, Genetic; D014764:Viral Proteins / Q000096:biosynthesis / Q000235:genetics
D002021:Buffers; D000077278:RNA, Mitochondrial; D014764:Viral Proteins; D012313:RNA; D004247:DNA; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases