A chemical enucleation method for the transfer of mitochondrial DNA to rho(o) cells

Abstract

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.

Figure 1

Chemical enucleation procedure to transfer mtDNA to ρ° cells. The mtDNA donor cell is treated with actinomycin D at conditions that preclude recovery of treated cells. This cell with a degenerating nucleus is a suitable mtDNA donor and can be fused to ρ° cells with PEG as described in Materials and Methods. In our experiment, transmitochondrial cybrid clones were obtained without a nuclear selection (they were isolated in media lacking uridine to inhibit growth of ρ° cells) and later checked for resistance to G418, puromycin and bromodeoxyuridine (BrdU). Most of the cybrids were sensitive to the drug-resistance present in the actinomycin-treated cells and resistant to BrdU.

Figure 2

Genetic make-up of transmitochondrial cybrids produced by chemical enucleation. Twenty individual transmitochondrial cybrid clones were analyzed for the presence of chromosomes from the mtDNA donor cell. Polymorphic tetranucleotide repeat markers located in chromosomes 3, 9, 14, 17 and 21 (see Materials and Methods) were used to distinguish the nuclear background (TEX versus 143B). Nineteen of 20 clones had exclusively 143B-derived chromosomes. Only clone 15, and a pool of clones selected for G418 resistance had TEX-derived chromosomes. The presence of mtDNA was also determined by Southern blot.

Figure 3

Cell respiration in transmitochondrial cybrids. Approximately 10⁶ cells (in 600 µl of no glucose DMEM) were injected in a Hansatech oxygen measurement chamber and oxygen consumption recorded over time. Antimycin A was added to the chamber to inhibit complex I- and II-driven respiration and Ascorbate/TMPD was added to initiate complex IV-driven respiration. The latter was inhibited by KCN. The oxygen consumption rates (endogenous minus antimycin and Asc/TMPD minus KCN) were plotted for representative clones and parental cell lines. Respiration was restored in transmitochondrial cybrids.