Taketo M, Tanaka M
Proc Natl Acad Sci U S A. 1987 Jun;84(11):3748-52. doi: 10.1073/pnas.84.11.3748.
Murine embryonal carcinoma (EC) cells are refractory to infection by retroviruses because retroviral long terminal repeat (LTR) enhancers have little activity in EC cells. A previous report described the isolation of clonal EC cell lines that express the integrated neomycin-resistance gene (neo) linked to the Moloney murine leukemia virus LTR. The expression of the neo gene was explained by a cis-acting mechanism [Taketo, M., Gilboa, E. & Sherman, M. I. (1985) Proc. Natl. Acad. Sci. USA 82, 2422-2426]. From one such EC cell line, we isolated the flanking cellular sequence 5' to the proviral genome, ligated it to various test constructs, and transfected into the parental EC cells. The cellular sequence increased expression of the LTR-linked neo gene significantly, in a manner independent of its orientation and position. The neo mRNA was initiated at the bona fide promoter of the LTR. By deletion analyses, we defined a region of DNA essential for the enhancer activity and determined its sequence. This region contains distinctly characteristic stretches as well as some similarity to various viral and cellular enhancers. Thus the LTR-linked neo gene is expressed because the provirus is integrated in the vicinity of this enhancer that is active in undifferentiated EC cells.
鼠胚胎癌细胞对逆转录病毒感染具有抗性，因为逆转录病毒长末端重复序列（LTR）增强子在胚胎癌细胞中的活性很低。之前的一份报告描述了克隆胚胎癌细胞系的分离，这些细胞系表达与莫洛尼鼠白血病病毒LTR相连的整合型新霉素抗性基因（neo）。neo基因的表达是通过顺式作用机制来解释的[武藤，M.，吉尔博亚，E. & 谢尔曼，M. I.（1985年）美国国家科学院院刊82，2422 - 2426]。从这样一个胚胎癌细胞系中，我们分离出了原病毒基因组5'端的侧翼细胞序列，将其连接到各种测试构建体上，并转染到亲代胚胎癌细胞中。该细胞序列以与其方向和位置无关的方式显著增加了与LTR相连的neo基因的表达。neo mRNA是从LTR的真正启动子开始转录的。通过缺失分析，我们确定了增强子活性所必需的DNA区域并测定了其序列。该区域包含明显的特征性片段以及与各种病毒和细胞增强子的一些相似性。因此，与LTR相连的neo基因得以表达，是因为原病毒整合在这个在未分化的胚胎癌细胞中具有活性的增强子附近。
