Modin C, Pedersen F S, Duch M
Department of Molecular and Structural Biology, University of Aarhus, DK-8000 Aarhus C, Denmark.
J Virol. 2000 Dec;74(24):11697-707. doi: 10.1128/jvi.74.24.11697-11707.2000.
A major determinant for transcriptional incompetence of murine leukemia virus (MLV) and MLV-derived vectors in embryonal cells is located at the proline primer binding site (PBS). The mechanism of silencing is unknown, yet the effect is capable of spreading to adjacent promoters. Based on a retroviral vector containing an internal promoter and the escape mutant B2 PBS with expressional capacity in embryonal cells, we have developed an assay to test the ability of putative insulators to shield the silencer at the PBS. Since the B2 PBS reverts to the wild-type PBS at high frequency, a shielding ability of a putative insulator can be assessed from the ratio of expressing B2 PBS to proline PBS proviruses in the target embryonal carcinoma cell population as measured by primer extension. Our results show that none of the possible insulators, scs, BEAD-1, or HS4, is able to shield an internal promoter from the repressive effect of the silencer at the PBS region when inserted between the silencer and the promoter.
小鼠白血病病毒（MLV）及MLV衍生载体在胚胎细胞中转录无活性的一个主要决定因素位于脯氨酸引物结合位点（PBS）。沉默机制尚不清楚，但这种效应能够扩散至相邻启动子。基于一种含有内部启动子以及在胚胎细胞中具有表达能力的逃逸突变体B2 PBS的逆转录病毒载体，我们开发了一种检测方法，以测试假定绝缘子屏蔽PBS处沉默子的能力。由于B2 PBS会高频回复为野生型PBS，通过引物延伸法在靶胚胎癌细胞群体中测量表达B2 PBS与脯氨酸PBS前病毒的比例，即可评估假定绝缘子的屏蔽能力。我们的结果表明，当将scs、BEAD-1或HS4这几种可能的绝缘子插入沉默子与启动子之间时，没有一种能够使内部启动子免受PBS区域沉默子的抑制作用。
