Stable integration and expression in mouse cells of yeast artificial chromosomes harboring human genes.
Stable integration and expression in mouse cells of yeast artificial chromosomes harboring human genes.
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含有人类基因的酵母人工染色体在小鼠细胞中稳定整合和表达。
DOI：
10.1073/pnas.88.6.2179
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发表时间：
1991
影响因子：
11.1
通讯作者：
D'Urso,M
中科院分区：
文献类型：
--
作者：
Eliceiri,B;Labella,T;Hagino,Y;Srivastava,A;Schlessinger,D;Pilia,G;Palmieri,G;D'Urso,M
我们已经开发了一种方法，以适应酵母人工染色体（YAC）的标记，允许选择稳定转化的哺乳动物细胞，并确定了这样的YAC的命运和表达含有人类核糖体RNA（rDNA）或葡萄糖-6-磷酸脱氢酶（G6 PD）的基因。酵母细胞中的YAC通过与一个载体臂的URA 3基因同源重组而用选择性标记物“改造”。引入的DNA片段含有在酵母中选择性的LYS 2标记物和在TK缺陷细胞中选择性的胸苷激酶（TK）标记物，由在重组事件期间破坏内源基因的URA 3序列的部分包围。转化的L-M TK-小鼠细胞的分析显示，含有rDNA或G6 PD的YAC以基本上完整的形式掺入哺乳动物细胞DNA中。对于G6 PD，在分析的两个转化体中的每一个中发现了转染的YAC的单拷贝，并且其完全表达，产生预期的人同工酶以及由人基因产物和内源性小鼠基因产物组成的异源二聚体。
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.
影响因子：
4.3
作者：
H. Sambrook
通讯作者：
H. Sambrook