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Nucleic Acids Res. 2012 Oct;40(19):e148. doi: 10.1093/nar/gks639. Epub 2012 Jun 29.

Transferring isolated mitochondria into tissue culture cells.

Author information

  • 1Graduate Program in Molecular Cellular Developmental Biology and Genetics, Institutes for Translational Neuroscience & of Human Genetics, University of Minnesota, Medical Bioscience Building, 2101 6th St SE, Minneapolis, Minnesota 55455, USA.

Abstract

We have developed a new method for introducing large numbers of isolated mitochondria into tissue culture cells. Direct microinjection of mitochondria into typical mammalian cells has been found to be impractical due to the large size of mitochondria relative to microinjection needles. To circumvent this problem, we inject isolated mitochondria through appropriately sized microinjection needles into rodent oocytes or single-cell embryos, which are much larger than tissue culture cells, and then withdraw a 'mitocytoplast' cell fragment containing the injected mitochondria using a modified holding needle. These mitocytoplasts are then fused to recipient cells through viral-mediated membrane fusion and the injected mitochondria are transferred into the cytoplasm of the tissue culture cell. Since mouse oocytes contain large numbers of mouse mitochondria that repopulate recipient mouse cells along with the injected mitochondria, we used either gerbil single-cell embryos or rat oocytes to package injected mouse mitochondria. We found that the gerbil mitochondrial DNA (mtDNA) is not maintained in recipient rho0 mouse cells and that rat mtDNA initially replicated but was soon completely replaced by the injected mouse mtDNA, and so with both procedures mouse cells homoplasmic for the mouse mtDNA in the injected mitochondria were obtained.

PMID:
22753025
[PubMed - indexed for MEDLINE]
PMCID:
PMC3479163
Free PMC Article
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