Alternative method for identifying reconstituted cells

Abstract

Enucleation techniques combining mild centrifugation in the presence of cytochalasin B permit cells to be separated into nuclear fragments (karyoplasts) and cytoplasmic fragments (cytoplasts). These fragments, though stable for a short time, will ultimately degenerate by the procedures described in this report. One can, however, fuse cytoplasts to karyoplasts by using polyethylene glycol and obtain viable reconstituted cells whose properties may be useful for understanding some aspects of the nuclear-cytoplasmic interactions associated with tumorigenicity and steroidogenesis. However, the presence of cybrids, hybrids, and parental whole cell contaminants along with the reconstituted cell population make it necessary to have genetic markers that reside in both the nucleus and cytoplasm in order to preferentially identify reconstituted cells derived from a karyoplast fused to a cytoplast. By utilizing the Y-1 cell line, which is tumorigenic and responds to corticotropin b secreting steroids, and the AMT-BU-A1 (AMT) cell line, which is nontumorigenic and does not respond to corticotropin but has a nuclear marker, BrdUrdr, anda cytoplasmic marker, CAPrF we have reconstituted cells containing Y-1 karyoplasts and AMT cytoplasts. In this report we extend our previous techniques by describing an identification procedure that allowed us to isolate cells reconstituted from AMT karyoplasts fused to Y-1 cytoplasts. The results of these experiments support the concept that with these cell lines the nucleus (karyoplast) is ultimately sufficient to control the phenotypic expression or suppression of tumorigenicity and steroidogenesis.Recent technological advances have allowed investigators to remove the nucleus (karyoplast) from one cell and place that karyoplast into another enucleated cell (cytoplast) (1). The properties of such reconstituted cells may be a potentially valuable tool useful in extending our understanding of nuclear-cytoplasmic interactions. This reconstitution procedure begins by centrifuging monolayers of cells in the presence of cytochalasin B, which results in the separation of the cells into two fragments. One of the fragments contains the nucleus surrounded by variable amounts of cytoplasm and an intact plasma envelope and has been termed karyoplast (2, 3); the other fragment is composed of t90-95% of the cell's original cytoplasm and has been termed cytoplast (2, 3). These fragments may be fused together by using Sendai virus or polyethylene glycol; this process is called cell reconstitution. Because neither of these procedures (enucleation or reconstitution) is 100% efficient, it is necessary to have some way to identify or select reconstituted cells from contaminating parental whole cells, cybrids (the result of the fusion of a cytoplast to a whole cell) (4), and hybrids (one whole cell fused to another whole cell). Previous techniques have made use of physical markers such as latex spheres or genetic markers, such as resistance to bromodeoxyuridine (BrdUrd) and chloramphenicol (CAP), to ide...