CELLS DERIVED FROM TWO hIUMAN DIPLOID STRAINS EACH CARRYING AN X-LINKED MUTATION*

Two male diploid fibroblast strains, each carrying deficiency mu- tations at different X-linked loci (glucose-6-phosphate dehydrogenase and hy- poxanthine-guanine-phosphoribosyltransferase) have been successfully hybrid- ized. The resulting mononucleated hybrid cells have been shown to synthesize both normal gene products, indicating that both X chromosomes are function- ally active in the hybrid cells. We believe this is the first reported example of intergenic complementation in fused human diploid cells. Introduction.-Although the possibility of applying the techniques of cell hybridization1' 2 to human diploid cells for studies of somatic cell genetics has been discussed,3 the difficulty of finding suitable cells has been a major deterrent. Ideally the cells to be used should carry several markers, each the primary gene product of a single chromosomal gene. IMIoreover, genic polymorphism should exist at each locus, the allelic products should be identifiable at the level of the single cultured cell, and the cells should be amenable to in vitro selection pro- cedures. Of the several thousand examples of simnple Mendelian traits in man,4 only a dozen or so can be identified at the cell level.5 Three involve mutations of genes located on the X chromosome: glucose-6-phosphate dehydrogenase de- ficiency (g-6-pd),6 hypoxanthine-guanine phosphoribosyltransferase deficiency (HGPRT),7 and the Hurler-Hunter syn(drome or gargoylism I1.8 Cells from patients with one of the first two deficiencies have been used in the experiments here described. Diploid fibroblasts derived from a male with the g-6-pd de- ficiency were fused with those from a male with the HGPRT deficiency in an attempt to obtain a hybrid tetraploid str-ain of cells which, in respect to their two X chromosomes, would be equivalent to cells doubly heterozygous in re- pulsion. Hybrid cells were obtained in which gene products of the X chromo- somes of both parental strains could be identified. Materials and Methods.-Skin biopsies were obtained from male patients known to be affected either by the Lesch-Nyhan syndrome9 or by a severe form of congenital non- spherocytic anemia associated with g-6-pd deficiency and from females heterozygous at these loci. Bits of tissue approximately 1-2 mm3 were placed in a 60 mm Petri dish with 1-1.5 ml of minimum growth medium10 containing 5%,0 calf, 5%,0 fetal calf, and 5%0 human AB sera; they were then incubated in a 5% C02 atmosphere. A normal amount of medium (5-10 ml) was added after 1-2 days. After 5 to 10 days, when there had been extensive growth of a fibroblast layer, the culture was trypsinized and grown in stoppered bottles with the same growth medium, omitting human serum. All experiments were conducted before the strains had undergone 20 culture passages (30-40 cell divisions).