Reid L H, Gregg R G, Smithies O, Koller B H
Department of Pathology, University of North Carolina, Chapel Hill 27599.
Proc Natl Acad Sci U S A. 1990 Jun;87(11):4299-303. doi: 10.1073/pnas.87.11.4299.
We have examined the expression of transfected human hypoxanthine phosphoribosyltransferase minigenes (HPRT) in mouse embryonic stem (ES) cells. cDNA constructs of this gene that have been successfully used in somatic cell lines failed to confer hypoxanthine/aminopterin/thymidine (HAT) resistance in ES cells. In contrast, constructs containing introns 1 and 2 from the HPRT gene produced a high frequency of HAT-resistant colonies. This observation allowed us to identify two sequences in these introns that influence expression of the HPRT gene in ES cells. One element, located in intron 2, is required for effective HPRT expression in these cells; the other element, located in intron 1, acts as an enhancer of HPRT expression. Using this information, we have constructed an HPRT minigene that can be used for either positive or negative selection in ES cell experiments. This dual capability allows the design of "in-out" procedures to create subtle changes in target genes by homologous recombination with the aid of this selectable minigene.
我们检测了转染的人次黄嘌呤磷酸核糖转移酶小基因（HPRT）在小鼠胚胎干细胞（ES细胞）中的表达情况。该基因的cDNA构建体已成功应用于体细胞系，但在ES细胞中未能赋予次黄嘌呤/氨基蝶呤/胸腺嘧啶核苷（HAT）抗性。相比之下，含有HPRT基因第1和第2内含子的构建体产生了高频的HAT抗性菌落。这一观察结果使我们能够在这些内含子中鉴定出两个影响HPRT基因在ES细胞中表达的序列。一个元件位于第2内含子中，是这些细胞中有效表达HPRT所必需的；另一个元件位于第1内含子中，作为HPRT表达的增强子。利用这些信息，我们构建了一个可用于ES细胞实验中正向或负向选择的HPRT小基因。这种双重功能使得能够设计“进出”程序，借助这个可选择的小基因通过同源重组在靶基因中产生细微变化。
