Eliceiri B, Labella T, Hagino Y, Srivastava A, Schlessinger D, Pilia G, Palmieri G, D'Urso M
Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, MO 63110.
Proc Natl Acad Sci U S A. 1991 Mar 15;88(6):2179-83. doi: 10.1073/pnas.88.6.2179.
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.
我们已经开发出一种方法，可为酵母人工染色体（YAC）配备能用于筛选稳定转化的哺乳动物细胞的标记物，并确定了此类包含人类核糖体RNA（rDNA）或葡萄糖-6-磷酸脱氢酶（G6PD）基因的YAC的命运和表达情况。通过与一个载体臂的URA3基因进行同源重组，酵母细胞中的YAC被“改造”上可选择标记物。引入的DNA片段包含一个在酵母中具有选择性的LYS2标记物和一个在TK缺陷细胞中具有选择性的胸苷激酶（TK）标记物，两侧是URA3序列的部分片段，这些片段在重组事件中会破坏内源基因。对转化的L-M TK-小鼠细胞的分析表明，包含rDNA或G6PD的YAC以基本完整的形式整合到哺乳动物细胞DNA中。对于G6PD，在分析的两个转化体中，每个转化体都发现了一个转染YAC的单拷贝，并且该YAC完全表达，产生了预期的人类同工酶以及由人类基因产物和内源小鼠基因产物组成的异二聚体。
