Teratocarcinoma cells as vehicles for introducing specific mutant mitochondrial genes into mice

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Abstract
A means of introducing specific mitochondrially encoded mutant genes into mice was established. Mouse teratocarcinoma stem cells were used as vehicles for the cytoplasmic markers because of their known capacity for normal somatic and germinal differentiation after injection into blastocysts. The mutation of choice, chloramphenicol resistance (CAPR), was produced in a melanoma cell line by mutagenesis and selection. The CAPR trait was transferred from a resistant melanoma cell to a sensitive (CAPS) teratocarcinoma cell by fusing to the latter only the cytoplasmic portion of the CAPR donor. This indirect route demonstrated the cytoplasmic provenance of the mutation. Protein synthesis in mitochondria isolated from the cybrid cells was barely affected by chloramphenicol, in contrast to the inhibitory influence of the drug on mitochondria of the parent teratocarcinoma line. Cells of the cybrid clone resembled teratocarcinoma cells and retained their ability to form diverse tissues in solid tumors produced from subcutaneous grafts. Cells from the tumors were retransplanted and tested periodically by culture in chloramphenicol; they were stably CAPR even after 16 wk in vivo in the absence of the selective agent. The CAPR cybrids were microinjected into blastocysts of another inbred strain and, after transfer to foster mothers, mosaic mice were obtained. They comprised both cybrid- and blastocyst-derived cells in various tissues, as indicated by strain-specific nuclear markers. Successful normal differentiation of the CAPR lineage in vivo is demonstrated. Teratocarcinoma cybrids offer a practical portal of entry of preselected mitochondrial genes into mice. This permits in vivo investigation of maternally transmitted traits, of mitochondrial genetic influences in specialized cells and of possible roles of cytoplasmic genes in clinical and disease states.