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 (CAPr) and sensitive to hypoxanthine/aminopterin/thymidine (HATS). CAPr 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 CAPr 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.Cytochalasin B can be used to separate animal cells growing in monolayer culture into nuclear [karyoplasts (1, 2) and minicells (3)] and cytoplasmic [cytoplasts (1, 2)] components. As previously reported (2), these cell fragments die within 48-72 hr after separation, but in the past 2 years, workers in several laboratories have reported reconstitution and subsequent viability of mammalian cells by fusing karyoplasts from a specific cell line to cytoplasts from the same cell line (4-6) (homospecific) or to other cell lines (7, 8) (heterospecific). These studies indicate that such reconstitution experiments are feasible, but convincing evidence that such reconstituted cells after several mitotic divisions are not whole cell contaminants that detached during enucleation or that failed to enucleate during the centrifugation is still lacking. The main reason such reconstituted cells have not been satisfactorily identified, as opposed to whole cell contaminants, is due primarily to the lack of both nuclear and cytoplasmic markers that would permit the identification of reconstituted cells, at a time when the properties of such cells may have been modified by the influence of a new combination of nucleus and cytoplasm.

Analysis of muscle protein expression in polyethylene glycol-induced chicken: rat myoblast heterokaryons.

“…Aliquots of unmixed and of mixed parental cells were plated at a density, of l0 ~ cells/60-mm dish to serve as non-PEG-treated controls. The:tnixed cell sus-pension, containing 2-3 x 106 cells of each parental type, was then centrifuged at 250 g for 5 rain, the medium was removed, and the pellet was resuspended in 0.5 ml of 50% PEG 1500 in DME lacking HS (25). After 90 s, the mixture was diluted gently with l0 ml DME and pelleted, and the cells were resuspended in 5 ml DME and 10% HS.…”

Analysis of myogenesis by somatic cell hybridization. II. Retention of myogenic competence and suppression of transformed properties in hybrids between differentiation competent and incompetent rat L6 myoblasts

“…LGJl-Nl cells. Following the protocol of Shay (1977) cells were suspended for 75 seconds in 1 ml of 50% polyethylene glycol (PEG, M.W. 6,000) in MEM containing 10% dimethylsulfoxide.…”

Assignment of the gene for cytosolic alanine aminotransferase (AAT1) to human chromosome 8

“…Hybrids were produced by the fusion of rat hepatoma FU5AH cells and the foetal human (liver) fibroblasts. For fusion, the cells were mixed together in serum free DMEM containing 47 yo polyethylene glycol (molecular weight 6000) and 15 Yo dimethylsulphoxide for 2 niin (Shay, 1977). Additional serum free medium was added, and the cells were washed twice before plating in DMEM containing 10 yo foetal calf serum (GIBCO), 10 mM hypoxanthine, 40 ,UM aminopterin, 16 ,UM thymidine and 5 PM ouabain.…”