Selection of reconstituted cells from karyoplasts fused to chloramphenicol-resistant cytoplasts

Abstract

Murine Balb/3T3 and murine A-MT-BU-A1 mammary tumor cells were separated in the presence of cytochalasin B into enucleated cytoplasmic components (cytoplasts) and nucleated subcellular components (karyoplasts). Karyoplasts were derived from 3T3 cells, while cytoplasts were derived from A-MT-BU-A1 cells that were both chloramphenicol-resistant (CAP(r)) and sensitive to hypoxanthine/aminopterin/thymidine (HAT(s)). CAP(r) has been shown to be cytoplasmically transmitted (possibly a mitochondrial gene mutation), while sensitivity to medium containing HAT has been shown to be transmitted by the nucleus (i.e., nuclear gene mutation). Such CAP(r) cytoplasts derived from A-MT-BU-A1 cells were then fused, using polyethylene glycol, to HAT-resistant 3T3 karyoplasts. The mononucleated reconstituted cells produced by such procedures were cloned in medium containing both HAT and CAP. Some of the reconstituted cells survived, because they were resistant to both drugs, while the nuclear and cytoplasmic whole cell contaminants were killed by one or the other of the two drugs. The results of these experiments indicate that reconstituted cells that are derived from two different cell lines are viable, as indicated by their ability for long-term proliferation in culture. Most of the clones derived resembled morphologically the 3T3 nuclear donor parent cells, but some of the clones did not resemble either parental cell line. It is anticipated that such selection techniques will permit more complete analysis of interrelationships between nucleus and cytoplasm.