Levine F, Yee J K, Friedmann T
Department of Pediatrics, UCSD School of Medicine, La Jolla 92093-0634.
Gene. 1991 Dec 15;108(2):167-74. doi: 10.1016/0378-1119(91)90431-a.
We have studied the properties of dicistronic transcriptional units in retroviral vectors. In these vectors, the promoter in the 5' retroviral long terminal repeat (LTR) controls expression of both an upstream cistron (luc) encoding firefly luciferase and a downstream cistron (neo), a selectable marker encoding neomycin phosphotransferase (NPTII). By assaying for simultaneous expression of luc and neo after transfection or infection of hamster BHK, rat 208F, and mouse retroviral packaging cell lines, we have identified important factors that affect expression from the downstream cistron, including the presence of intercistronic ATG sequences, the length of the intercistronic sequence and conformity of the sequence surrounding the downstream start codon to the eukaryotic consensus sequence. Optimized dicistronic vectors produced amounts of NPTII comparable to a vector in which neo was driven by a strong internal promoter consisting of a modified Rous sarcoma virus LTR. Additionally, they produced higher virus titers and demonstrated improved stability of gene expression in the absence of selection. By virtue of their physical compactness and elimination of the need for a separate promoter for every gene, dicistronic transcriptional units allow the introduction of larger genes into retroviral vectors and may allow for more than two genes to be placed in a single vector.
我们研究了逆转录病毒载体中双顺反子转录单元的特性。在这些载体中，5' 逆转录病毒长末端重复序列（LTR）中的启动子控制着上游编码萤火虫荧光素酶的顺反子（luc）和下游编码新霉素磷酸转移酶（NPTII）的可选择标记顺反子（neo）的表达。通过检测转染或感染仓鼠 BHK、大鼠 208F 和小鼠逆转录病毒包装细胞系后 luc 和 neo 的同时表达，我们确定了影响下游顺反子表达的重要因素，包括顺反子间 ATG 序列的存在、顺反子间序列的长度以及下游起始密码子周围序列与真核生物共有序列的一致性。优化后的双顺反子载体产生的 NPTII 量与由修饰的劳氏肉瘤病毒 LTR 组成的强内部启动子驱动 neo 的载体相当。此外，它们产生了更高的病毒滴度，并在无选择条件下表现出改善的基因表达稳定性。由于其物理紧凑性以及无需为每个基因单独设置启动子，双顺反子转录单元允许将更大的基因引入逆转录病毒载体，并且可能允许在单个载体中放置两个以上的基因。
