ISOLATION OF VIABLE RECONSTITUTED CELLS FROM HUMAN KARYOPLASTS FUSED TO MOUSE CYTOPLASTS

Long-term survivors of reconstituted human-mouse cells were isolated and characterized by utilizing nuclear and cytoplasmic genetic markers. Karyoplasts were derived from the human SV40-transformed fetal lung fibroblast strain WI38-VA13, while cytoplasts were obtained from the mouse fibroblast A9 cell line which was both hypoxanthine-aminopterin-thymidine-sensitive (HATs; nuclear marker) and chloramphenicol-resistant (CAPr; cytoplasmic marker). The fusion products were isolated in medium containing HAT and CAP. Clones initially showed a growth pattern different from either human or mouse parental cell, but after repeated subculturing, morphologically resembled the nuclear donor cell. The human and mouse components in these cells were identified from other possible fusion combinations by karyotypic, enzymatic and mitochondrial DNA analyses. The karyotype, using both Q-banding and C-banding revealed only human chromosomes. Electrophoretic mobility of the enzyme malate dehydrogenase, a nuclear controlled enzyme, confirmed the human nucleus. Buoyant density centrifugation of radioactive-labeled isolated mitochondrial DNA from the reconstituted cells provided evidence that the cytoplasm was of marine origin.