Selection by drug resistance proteins located in the mitochondria of mammalian cells

Abstract

Transformation of mitochondria in mammalian cells is now a technical challenge. In this report, we demonstrate that the standard drug resistant genes encoding neomycin and hygromycin phosphotransferases can potentially be used as selectable markers for mammalian mitochondrial transformation. We re-engineered the drug resistance genes to express proteins targeted to the mitochondrial matrix and confirmed the location of the proteins in the cells by fusing them with GFP and by Western blot and mitochondrial content mixing analyses. We found that the mitochondrially targeted-drug resistance proteins confer resistance to high levels of G418 and hygromycin without affecting the viability of cells.

Figure 1

Subcellular localization of drug-resistance fusion proteins. (A) Mitochondrial targeting of GFP protein using the leader sequence of mouse mitochondrial transcription factor A (TfamL). The GFP was clearly localized only in the mitochondrial networks of HeLa229 cells. (B) Structure of mitochondrial targeting fusion vectors. The GFP and Neo^R genes or the GFP and Hyg^R genes were fused by a linker sequence along with the mitochondrial targeting sequence of mouse Tfam (TfamL). (C and D) Microscopic examination of GFP fusion proteins targeted to the mitochondria. The GFP fusion proteins expressed in the mtGFPNeo (green) (C) and mtGFPHyg (green) (D) cell lines was clearly localized in the mitochondrial networks of HeLa229 cells without visibly detectable protein in the cytosol. The mitochondrial networks stained with a mitochondrial specific dye (M429) (red) were exactly matched with the GFP-labeled mitochondria.

Figure 2

Examination of GFP-fused drug resistance proteins in the mitochondria. (A) Western analysis of GFPNeo fusion proteins. Mitochondria from the mtGFP and mtGFPNeo cells were prepared after mechanical cell lysis followed by differential centrifugation. Proteins from the total cells, mitochondria and supernatant were prepared and then fractionated in a 10% SDS/PAGE. Western blot analysis was performed using the rabbit polyclonal GFP antiserum. The GFP and GFPNeo proteins were clearly accumulated in the mitochondrial matrix since the mouse Tfam mitochondrial leader sequence delivers the proteins to the mitochondria. The weak signal in the supernatant of the mtGFPNeo cells may have originated from broken mitochondria that were disrupted during mechanical mitochondrial preparation. SDS/PAGE gel was used as a loading control for the GFP signal. (B) Fusion of mitochondrial networks between GFP- (green) and DsRed-labelled (red) mitochondria. Rapid mixing and distribution of both fluorescent proteins throughout the fused mitochondria indicated that the GFPNeo fusion proteins were correctly located in the mitochondrial matrix.

Figure 3

Drug resistance conferred by mitochondrially located proteins. (A) Growth curve of the stable mtGFPNeo cell line in G418-containing media. The cell lines mtGFPNeo, GFPNeo and HeLa229 cells stably transfected with pcDNA6 were cultured in MEM-alpha media containing 5% FBS and standard level of G418 (400 μg/ml) for 10 days to compare the cell viability. (B) Growth curve of the stable mtGFPHyg cell line in hygromycin-containing media. The cell lines mtGFPHyg, GFPHyg and HeLa229 cells stably transfected with pcDNA6 were cultured in MEM-alpha media containing 5% FBS and standard level of hygromycin (200 μg/ml) for 10 days. Both mtGFPNeo (A) and mtGFPHyg (B) cells are highly resistant to G418 and hygromycin and continue to grow without difficulty (open circles in A and B) but HeLa229 control cells transfected with pcDNA6 in (A) and (B) quickly disappeared due to the toxicity of the drugs (closed circles in A and B). The patterns of the growth curves of the mtGFPNeo and mtGFPHyg clonal cell lines were very similar with the patterns of growth shown in the GFPNeo and GFPHyg cells (open circles and squares in A and B).