Joseph J. Lucas

The construction of viable nuclear-cytoplasmic hybrid cells by nuclear transplantation

Using the mouse L-cell line as a model system, a generalized approach is presented for nuclear transplantation in cultured cells resulting in the construction of cytoplasmic-nuclear hybrid cells. Techniques were developed for the preparation of cytoplast and karyoplasts having minimum contamination by parent whole cells. Sendai virusmediated fusion was performed in a manner which maximized the formation of the desired fusion products-cells having one cell equivalent of cytoplasm from one parent and a nucleus from a second parent. The viability of the fusion products was established by examination of photographic records of the developing cultures. Using these techniques, we found that nuclei could be introduced routinely into 10-30% of a cytoplast culture. From determinations of the increase in cell number with time, it was estimated that at least 30% of the reconstructed cells were capable of division. The approach was next applied to the formation of hybrid cells from L-cell cytoplasts and A9 cell karyoplasts. The A9 cell line is an azaguanine-resistant derivative of L cells. Thus any whole cells remaining in the culture of fused cells were readily eliminated by treatment with the purine analogue. The culture of remaining cytoplasmic-nuclear hybrid cells grew to confluence in the presence of azaguanine. The applicability of the approach to the construction of hybrid cells using parent lines from different organisms is briefly discussed.

Purification and characterization of regenerating mouse L929 karyoplasts

Within 72-96 hr after preparation, about 10% of the karyoplasts made from mouse L929 cells regenerated to reform whole viable cells. As soon as 30 hr after preparation, however, nearly all of the remaining 90% of karyoplasts were dead. By separating living and dead karyoplasts at 30 hr, therefore, that fraction destined to complete regeneration was effectively purified. Complete separation was accomplished by sedimentation through Ficoll-paque (Pharmacia), a patented preparation originally developed for the separation of monocytes from whole blood. With the addition of this technique to the previously reported purification scheme for karyoplasts, various biochemical and morphological studies were attempted. Of particular importance are results indicating that karyoplasts that regenerate do not initially contain any more cytoplasm than the average karyoplasts in a preparation--that is, about 10% of the cytoplasm within a whole cell. Electron microscopy of karyoplasts immediately after preparation indicated an unequal partitioning of cytoplasmic organelles at the time of enucleation. For example, karyoplasts initially contain about 11.4% of the mitochondrial volume of whole cells, but only 2.9% of the Golgi apparatus. The size of the karyoplasts and the volume occupied by a variety of organelles was followed throughout the process of regeneration. Although there was an approximately linear increase in the diameter of regenerating karyoplasts, there appeared not to be a simple concordant increase in the volume occupied by all cellular organelles. An extensive investigation was performed to determine whether or not karyoplasts contained centrioles. Immediately after enucleation, 15,000 random thin sections through karyoplasts, which represented about 100 complete bodies, were examined for the presence or absence of centrioles. No centrioles were observed. Examination of the cytoplasts revealed that they contained a sufficient number of centrioles to account for all of the centrioles that were present in the whole cells before enucleation. Centrioles were first detected in karyoplasts in 24 hr after preparation, about the same time that karyoplasts regained the ability to adhere to the surface of tissue culture dishes. At this time, however, the average karyoplast had less than one centriole. By 72 hr, the regenerated karyoplasts had approximately the same number of centrioles as whole cells.

The regeneration and division of mouse L-cell karyoplasts

A technique for efficient cytochalasin-induced enucleation was used to prepare karyoplasts--nuclei surrounded by a thin shell of cytoplasm and an outer cell membrane. Methods for estimating the quantity of cytoplasm remaining in karyoplasts indicated that they contained less than 10% of the amount found in whole cells. Procedures for separating karyoplasts from contaminating cytoplasmic fragments and whole cells are also described. Freshly prepared L-cell karyoplasts were unable to adhere to and spread upon a surface. However, after incubation for several days, about 30% of the karyoplasts regained these abilities to some degree. A portion of the regenerating karyoplasts were then observed to divide. These events were confirmed and recorded by time-lapse cinematography. In addition, by culturing karyoplasts under appropriate conditions, clones were isolated.

A staining procedure for identifying viable cell hybrids constructed by somatic cell fusion, cybridization, or nuclear transplantation

A general procedure for identifying viable hybrid cells was developed. One cell type was labeled by a brief incubation in the Kodak laser dye rhodamine 123, which accumulates in the mitochondria; a second cell type was labeled by a brief incubation in the Hoechst fluorochrome 33258, which binds to chromatin. The substances, which are eventually lost from the organelles, appeared to be nontoxic; the plating efficiencies of numerous cell lines tested was unaffected. Either whole cells or cytoplasts labeled with rhodomine 123 were fused, using inactivated Sendai virus, to whole cells or karyoplasts labeled with Hoechst 33258. When living cells were illuminated with ultraviolet light, individual whole cell hybrids, cybrids, or cytoplasmicnuclear hybrid cells could be rapidly identified by the appropriate staining pattern.

Nuclear transplantation with mammalian cells.

Early microsurgical nuclear transplantation experiments with nonmammalian systems suggested that cytoplasmic elements participate in the regulation of nuclear gene expression and replication. With cells from Rana pipiens (Briggs and King, 1960), Xenopus leavis (Gurdon, 1962), and Drosophila melanogaster (Okada et al., 1974), it was shown that egg cell cytoplasm could redirect the differentiative pathway of nuclei from cells at much later stages of development. Moreover, in Stentor coeruleus, for example, nuclear DNA synthesis likewise appeared to be regulated, at least in part, by cytoplasmic factors (deTerra, 1967). With mammalian cells, numerous somatic cell hybridization experiments demonstrated that the patterns of gene expression of two parental cell types could be stably altered when a hybrid cell containing a mixed genome was constructed [reviewed by Ringertz and Savage (1976); Lucas (1982)]. For example, rat hepatoma cells that secreted albumin were fused to mouse fibroblasts that did not. Some hybrid clones secreted only mouse or rat albumin, while others secreted both rat and mouse albumin (Peterson and Weiss, 1972). Results of this and other similar experiments showed that a complex array of interactions is possible when two very different cell types are fused. They also suggested the existence, in animal cells, of elements which can interact functionally with a foreign nucleus and either positively or negatively regulate the expression of certain genes.

Polypeptide synthesis in enucleated mouse fibroblasts

The polypeptides synthesized in cytoplasts prepared from mouse L929 cells by cytochalasin-induced enucleation were analyzed by two-dimensional gel electrophoresis. Nearly all detectable polypeptides made in parental whole cells were likewise made in cytoplasts, though in decreasing amounts, for up to at least 12 hours after enucleation. A similar analysis of cells treated with actinomycin D, an inhibitor of transcription, showed that such cells initially synthesized all of the polypeptides made by untreated cells. However, by 12 hours after treatment, the electrophoretic patterns produced by preparations of radiolabeled cells were highly aberrant, with some polypeptides apparently greatly overproduced relative to others. The results are consistent with the notion that physical removal of the nucleus disrupts the mechanisms regulating mRNA degradation in eukaryotic cells.

Chapter 20 Nuclear Transplantation with Mammalian Cells

Publisher Summary This chapter describes the techniques required to construct large, viable, homogeneous populations of true cytoplasmic-nuclear hybrids. These techniques are being applied to the construction of novel systems for investigating gene expression and its control in mammalian cells. Thus, the cytoplasts and karyoplasts prepared from various differentiated and transformed cell lines that exhibit distinct morphological and/or biochemical traits are fused to form true cytoplasmic-nuclear hybrid cells. The hybrids and their progeny are analyzed for the maintenance, loss, or acquisition of the parental cell traits. The chapter outlines the methods for the Preparation and Characterization of Karyoplasts and of Cytoplasts. The procedure for nuclear transplantation involves the preparation of Sendai virus, virus-mediated fusion, characterization of hybrids, and genetic selection techniques. The selection procedure should be applicable for the selection of cytoplasmic-nuclear hybrids. Thus, before fusing cytoplasts and karyoplasts derived from cell lines of interest, the mutants resistant to appropriate drugs, and toxins can first be selected.

NUCLEAR TRANSPLANTATION WITH MAMMALIAN CELLS

Using the technique of cytochalasin-induced enucleation in a centrifugal field, cultures of cytoplasts and karyoplasts were prepared from many mammalian cell lines, including mouse L929, A9 and neuroblastoma cells and Chinese hamster ovary cells. Detailed characterization of karyoplasts from the L929 and A9 lines indicated that they contained no more than 10-20% of the cytoplasm found within whole cells. After separation of karyoplasts from contaminating cytoplasmic fragments and whole cells, they were fused to cytoplasts by Sendai virus-mediated fusion. Up to 30% of an L-cell cytoplast culture could be re-nucleated using karyoplasts from either L929 or A9 cells. The reconstituted and hybrid cells were viable, that is, capable of division. Consideration of these results and of observations made using other cell lines demonstrated that the techniques should be of general applicability in the investigation of cytoplasmic-nuclear interactions.