Watson M E, Moore M
Xenova Limited, Slough, Berkshire, England.
AIDS Res Hum Retroviruses. 1993 Sep;9(9):861-7. doi: 10.1089/aid.1993.9.861.
A cellular assay is described in which transient high-level expression of a heterologous reporter gene (chloramphenicol acetyltransferase, CAT) driven by the HIV LTR is used to determine trans-activation in a cell line constitutively expressing Tat. The use of a parallel ELISA system to determine effects on expression of CAT and of the neomycin phosphotransferase (NPT) marker gene effectively eliminated sample variability caused by cumulative processing errors or cell culture conditions. In addition the use of cationic liposome-mediated transfection minimized delay between DNA treatment that initiates trans-activation and addition of inhibitors, thereby eliminating background expression levels in treated samples. The assay has the potential to discriminate between inhibition of trans-activation and nonspecific effects such as inhibition of transfection and cytotoxicity. It has been adapted to a 96-well format suitable for high-throughput screening of natural products and synthetic chemicals.
本文描述了一种细胞分析方法，其中由HIV长末端重复序列（LTR）驱动的异源报告基因（氯霉素乙酰转移酶，CAT）的瞬时高水平表达用于确定在组成性表达Tat的细胞系中的反式激活。使用平行ELISA系统来确定对CAT表达和新霉素磷酸转移酶（NPT）标记基因的影响，有效地消除了由累积处理误差或细胞培养条件引起的样品变异性。此外，使用阳离子脂质体介导的转染使启动反式激活的DNA处理与添加抑制剂之间的延迟最小化，从而消除了处理样品中的背景表达水平。该分析方法有潜力区分反式激活抑制与非特异性效应，如转染抑制和细胞毒性。它已被调整为适用于天然产物和合成化学品高通量筛选的96孔形式。
