A quantitative assay for trans-activation 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, 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.
Watson
M E
ME
Xenova Limited, Slough, Berkshire, England.
Moore
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D004274:DNA, Recombinant; D004279:DNA, Viral / Q000235:genetics*; D004797:Enzyme-Linked Immunosorbent Assay; D015967:Gene Expression Regulation, Viral; D015696:Gene Products, tat / Q000235:genetics / Q000502:physiology*; D016325:HIV Long Terminal Repeat; D015497:HIV-1 / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D008081:Liposomes; D011993:Recombinant Fusion Proteins / Q000032:analysis* / Q000096:biosynthesis; D015533:Transcriptional Activation; D014162:Transfection; D054322:tat Gene Products, Human Immunodeficiency Virus
D004274:DNA, Recombinant; D004279:DNA, Viral; D015696:Gene Products, tat; D008081:Liposomes; D011993:Recombinant Fusion Proteins; D054322:tat Gene Products, Human Immunodeficiency Virus