Transferring isolated mitochondria into tissue culture cells

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.

Figure 1.

Overview of the mitocytoplast fusion method. This is a two-step procedure: Step 1—mitochondria injection and cytoplast generation and Step 2—mitocytoplast-cell fusion and culture of the cybrids. Specific details are illustrated in Supplementary Figure S1.

Figure 2.

Demonstration of the mitochondrial injection, mitocytoplast generation and mitocytoplast-cell fusion. The injection shown in these photographs was performed using one-cell mouse embryos and fluorescent beads as the injection marker. (a) Images of mitochondrial injection and pulling a mitocytoplast; (b) examples of mitocytoplast-cell fusion; and (c) examples of mitocytoplast-fused cells. The presence of the fluorescent beads in the cells demonstrates successful transfer of exogenous material into these cells.

Figure 3.

Mitocytoplast-introduced, GFP-labeled mouse mitochondria fused to endogenous DsRed-labeled mitochondrial networks in a mouse cell. Mitocytoplast-fused cells were cultured in a chambered cover glass and imaged 3-4 h after mitocytoplast fusion using Olympus Fluoview® FV1000 confocal laser scanning microscope. A large quantity of isolated green mouse mitochondria was introduced into this mouse recipient cell using the mitocytoplast fusion method as shown in the left panel. The co-localization of the green and red signals indicates the active fusion of the introduced green mitochondria to the endogenous red mouse mitochondria, and the preservation of the biological activity of the isolated mitochondria during the procedure.

Figure 4.

The mouse cell lines generated from this procedure are homoplasmic for the injected mouse mtDNA genomes. (a) Species-specific mtDNA PCR results from cybrid mouse cells made using gerbil mitocytoplasts, assayed after 10 days in culture. Top: gerbil mtDNA PCR assay; 642 bp product (ND3-ND4 junction) indicated by arrow. Bottom: mouse mtDNA PCR assay; 1 175 bp product (15 200 nt - 75 nt) indicated by arrow. Mouse but not gerbil mtDNA genomes were detected in these two clones analyzed. M: 1 kb plus DNA ladder; 1: LL/2-STOmt-gerbil-G1; 2: LL/2-STOmt-gerbil-A2; rho0: mouse LL/2 rho0 recipient cell; C+: genomic DNA from gerbil liver. (b) Species-specific mtDNA PCR results from cybrid mouse cells made using rat mitocytoplasts, assayed after 30 days in culture. Top: rat mtDNA PCR assay; 558 bp product (15 417–15 974 nt) indicated by arrow. Bottom: mouse mtDNA PCR assay (as above); product indicated by arrow. Mouse but not rat mtDNA genomes were detected in all of the 18 clones assayed. M: 1 kb plus DNA ladder; C−: water control; C+: genomic DNA from rat YB2/O cells.