Construction of viable mouse‐human hybrid cells by nuclear transplantation

Hightower, Margaret J.; Lucas, Joseph J.

doi:10.1002/jcp.1041050112

Cited by 9 publications

(10 citation statements)

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“…If the fragments are from two different cell types, cells containing single nuclei within foreign cytoplasm can be constructed (6,7). By judicious choice of the parental cell types and the use of a variety of staining techniques, such reconstituted cells can be produced in large numbers and definitively identified immediately after fusion (8)(9)(10)(11). These cells, having a minimal contribution of cytoplasm from the nuclear donor cell type (12)(13)(14)(15), would appear to be excellent systems for investigating the interaction of cytoplasm and nucleus in determining a variety of cellular properties and events, including cell shape and cytoarchitecture and genome replication and expression.…”

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confidence: 99%

“…As described in detail previously (8)(9)(10), cytoplasts and karyoplasts were prepared and used to construct mouse-human hybrid cells. They could be definitively identified by using a variety of morphological, immunological, and genetic criteria, including nuclear pattern of staining with the fluorochrome Hoechst 33258, appearance of the actin-myosin-containing cytoskeleton, presence of fibronectin, and sensitivity to both azaguanine and diphtheria toxin.…”

mentioning

confidence: 99%

“…They could be definitively identified by using a variety of morphological, immunological, and genetic criteria, including nuclear pattern of staining with the fluorochrome Hoechst 33258, appearance of the actin-myosin-containing cytoskeleton, presence of fibronectin, and sensitivity to both azaguanine and diphtheria toxin. The reconstituted cells gradually became resistant to both agents, as they lost hypoxanthine phosphoribosyltransferase molecules from their cytoplasm and toxin receptors from their surface (8,10). Staining of cell nuclei with the fluorochrome Hoechst 33258 and indirect immunofluorescence using rabbit antiserum to myosin were performed as previously (8).…”

mentioning

confidence: 99%

“…The reconstituted cells gradually became resistant to both agents, as they lost hypoxanthine phosphoribosyltransferase molecules from their cytoplasm and toxin receptors from their surface (8,10). Staining of cell nuclei with the fluorochrome Hoechst 33258 and indirect immunofluorescence using rabbit antiserum to myosin were performed as previously (8). Observation of fluorescent staining, combined with autoradiography, was accomplished by the method devised by Lockwood (24).…”

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confidence: 99%

“…This article must therefore be hereby marked "advertisement" in accordance with 18 (8). One reconstituted cell is shown in Fig.…”

mentioning

confidence: 99%

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Identification of key regulated events early in the life of hybrid animal cells constructed by nuclear transplantation.

Hightower¹,

Bruno²,

Lucas³

1983

Proc. Natl. Acad. Sci. U.S.A.

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Reconstituted cells were constructed by fusion of cytoplasts from the human diploid fibroblast cell strain Detroit 532 and karyoplasts from the mouse fibroblast cell line A9. Several cellular properties were examined during the first 48 hr after nuclear transplantation. (i) The overall morphology of the cells originally resembled that of the cytoplasmic donor, Detroit 532, but rapidly changed to approximate that of the nuclear donor, A9. However, definitive changes in the microfilament structure of the reconstituted cells were not seen until 24-48 hr after fusion. These observations support the idea that the presence or absence of an ordered array of microfilament bundles is not the sole determinant of cell shape. (ii) Although cytoplasts and karyoplasts were prepared from cultures of randomly growing cells, the first division of reconstituted cells occurred in a synchronous manner. However, the initiation of DNA synthesis was not synchronized. It thus appeared that, in their first cell cycle, the cells had a G2 period of variable length. The results further suggest that the cytoplasm of interphase fibroblasts contains the material necessary to initiate or support DNA synthesis in a transplanted nucleus but not entry into mitosis. (iii) A two-dimensional gel electrophoretic analysis of polypeptide synthesis in reconstituted cell cultures showed that synthesis directed by transplanted mouse nuclei could be detected as early as 3-6 hr after fusion. Some of the mouse polypeptides detected at the earliest time points studied were not among the major polypeptides synthesized by the parental A9 cells. By about 48 hr after fusion, the pattern of polypeptides produced by reconstituted cells was almost indistinguishable from that of the nuclear donor parent cells.Cytoplasts and karyoplasts prepared from animal cells by cytochalasin-induced enucleation (1, 2) can be fused together to reconstitute whole viable cells (3)(4)(5). If the fragments are from two different cell types, cells containing single nuclei within foreign cytoplasm can be constructed (6, 7). By judicious choice of the parental cell types and the use of a variety of staining techniques, such reconstituted cells can be produced in large numbers and definitively identified immediately after fusion (8-11). These cells, having a minimal contribution of cytoplasm from the nuclear donor cell type (12-15), would appear to be excellent systems for investigating the interaction of cytoplasm and nucleus in determining a variety of cellular properties and events, including cell shape and cytoarchitecture and genome replication and expression. However, analyses of both reconstructed and cybrid cells performed so far have indicated that, except for a few instances (16-21), exposure to a foreign cytoplasm has little long-term effect on a transplanted nucleus. In this report it is shown that, in most respects, cells constructed by the method of nuclear transplantation are indistinguishable from the nuclear donor parental cell type as early as 48 hr after fusion....

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