A QUANTITATIVE ASSAY FOR TRANSACTIVATION BY HIV-1 TAT, USING LIPOSOME-MEDIATED DNA UPTAKE AND A PARALLEL ELISA SYSTEM

A cellular assay is described in which transient high-level expression of a heterologous reporter gene (chloramphenicol acetyltransferase, C AT) driven by the HIV LTR is used to determine trans-activation in a c ell line constitutively expressing Tat. The use of a parallel ELISA sy stem to determine effects on expression of CAT and of the neomycin pho sphotransferase (NPT) marker gene effectively eliminated sample variab ility caused by cumulative processing errors or cell culture condition s. In addition the use of cationic liposome-mediated transfection mini mized delay between DNA treatment that initiates trans-activation and addition of inhibitors, thereby eliminating background expression leve ls in treated samples. The assay has the potential to discriminate bet ween inhibition of trans-activation and nonspecific effects such as in hibition of transfection and cytotoxicity. It has been adapted to a 96 -well format suitable for high-throughput screening of natural product s and synthetic chemicals.