Stable integration and expression in mouse cells of yeast artificial chromosomes harboring human genes

Abstract

We have developed a way to fit yeast artificial chromosomes (YACs) with markers that permit the selection of stably transformed mammalian cells, and have determined the fate and expression of such YACs containing the genes for human ribosomal RNA (rDNA) or glucose-6-phosphate dehydrogenase (G6PD). The YACs in the yeast cell are "retrofitted" with selectable markers by homologous recombination with the URA3 gene of one vector arm. The DNA fragment introduced contains a LYS2 marker selective in yeast and a thymidine kinase (TK) marker selective in TK-deficient cells, bracketed by portions of the URA3 sequence that disrupt the endogenous gene during the recombination event. Analyses of transformed L-M TK- mouse cells showed that YACs containing rDNA or G6PD were incorporated in essentially intact form into the mammalian cell DNA. For G6PD, a single copy of the transfected YAC was found in each of two transformants analyzed and was fully expressed, producing the expected human isozyme as well as the heterodimer composed of the human gene product and the endogenous mouse gene product.