A chemical enucleation method for the transfer of mitochondrial DNA to rho(o) cells.
The study of pathogenic mitochondrial DNA mutations has, in most cases, relied on the production of transmitochondrial cybrids. Although the procedure to produce such cybrids is well established, it is laborious and cumbersome. Moreover, the mechanical enucleation procedure is inefficient and different techniques have to be used depending on the adherence properties of the cell. To circumvent these difficulties, we developed a chemical enucleation method that can have wide applicability for the production of transmitochondrial cybrids. The method is based on the use of actinomycin D to render the nuclear genome transcription/replication inactive and unable to recover after treatment. Such treated cells are fused to cells devoid of mitochondrial DNA and selected for the presence of a functional oxidative phosphorylation system. Our results showed that 95% of the clones recovered by this procedure are true transmitochondrial cybrids. This method greatly facilitates the production of transmitochondrial cybrids, thereby increasing the number of mtDNA mutations and the recipient cell types that can be studied by this system.
Bayona-Bafaluy
Maria Pilar
MP
Department of Neurology, University of Miami School of Medicine, Miami, FL 33136, USA.
Manfredi
Giovanni
G
Moraes
Carlos T
CT
D000818:Animals; D002459:Cell Fusion / Q000379:methods; D002460:Cell Line; D002467:Cell Nucleus / Q000187:drug effects* / Q000502:physiology; D003593:Cytoplasm / Q000235:genetics; D004272:DNA, Mitochondrial / Q000235:genetics*; D003609:Dactinomycin / Q000494:pharmacology*; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D018014:Gene Transfer Techniques; D006071:Gorilla gorilla; D006801:Humans; D006822:Hybrid Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology*; D002679:Pan troglodytes; D011092:Polyethylene Glycols / Q000494:pharmacology; D015203:Reproducibility of Results; D014407:Tumor Cells, Cultured
D004272:DNA, Mitochondrial; D019384:Nucleic Acid Synthesis Inhibitors; D003609:Dactinomycin; D011092:Polyethylene Glycols