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Chromosome behaviour in human-mouse somatic-cell hybrids.

The usefulness ofcell fusion in the genetic analysis ofsomatic cells stems largely from two observations. First, Harris & Watkins (1965) demonstrated that not only like cells, as shown by Okada (1962), but also cells of widely different types, even cells from species as different as man and mouse, could be fused together by inactivated Sendai virus. Thus cell fusion can produce recombination of genetic material of different somatic cells. Secondly, Weiss & Green (1967) found that in human-mouse hybrid cells the great majority of the human chromosomes were rapidly eliminated. This preferential loss of chromosomes of one species is a characteristic pattern of behaviour of chromosomes in hybrids between cells of different species. It is the loss of chromosomes from these cells that produces the segregation necessary for genetic analysis. The hybrid cells that have so far been used most often in genetic analysis have been human-mouse hybrids. All of these have been obtained by the use of drug-resistant markers and growth in HAT selective medium (containing hypoxanthine, aminopterin and thymidine), the procedure of selecting hybrid cells that was introduced by Littlefield (1964). There are some reasons for believing that the HAT medium may be a significant factor in the pattern ofchromosome loss: (a) excess of thymidine produces a prolonged S-phase (E. Stubblefield, unpublished work cited by Schmid, 1965), and O'Neill & Miles (1970) have shown that in dikaryons 'pulverized' chromosomes are the last to replicate; (b) excess of thymidine is also reported to cause loss of chromosomes into the cytoplasm (Bootsma, Budke & Vos, 1964); (c) the preferential loss of human chromosomes appears to be more marked in the absence of HAT or in BUDR medium than it does in the HAT medium in which the hybrid was isolated (Weiss & Green, 1967; Matsuya, Green & Basilico, 1968). We have now succeeded in obtaining humanmouse cell hybrids without the use ofHAT medium or any other selective medium by fusing mouse Ehrlich ascites-tumour cells, which are fastgrowing and do not attach to glass, with normal human diploid fibroblasts, which are slow-growing and attach to glass. The parental Ehrlich ascitestumour cells were removed simply by changing the medium, and a hybrid was obtained that attached to glass and grew much faster than the parental human fibroblasts. Chromosome preparations were made and examined. The objectives of this cytogenetic analysis were (a) a consideration of the factors determining chromosome loss and (b) a more complete description of the behaviour of chromosomes in hybrid cells.