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KMID : 0980720030220010104
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Keimyung Medical Journal
2003 Volume.22 No. 1 p.104 ~ p.113
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Development of Permanent Cell Lines for HIV-1 Tat Inhibitor Screening
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Park Jong-Gu
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Abstract
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Among potential genetic targets for intervention of HIV-1 replication, the tat gene product is a key target. In this study, two permanent cell lines, HeLa-LT2 and CCRF-CEM-LT1, have been developed for screening of Tat inhibitors. pcDNA3-LT1 and pcDNA3-LT2 plasmids that contain the firefly luciferase and HIV-1 tat genes were constructed to this end. The luciferase reporter gene is under the control of the HIV-1 long terminal repeat (LTR) and the HIV-1 tat gene is expressed constitutively from the cytomegalovirus (CMV) promoter. These plasmids were stably transfected into HeLa and CCRF-CEM cell lines and maintained under stable selection. G418-selected cell lines were assayed for luciferase activity, identified by polymerase chain reaction (PCR), and further analyzed for expression of the HIV-1 tat gene by RT-PCR. The inhibitory activity of Tat inhibitors is assessed by measuring suppressed expression of the luciferase gene. This in vitro assay system can be a useful tool for screening of new Tat inhibitors such as tat antisense and TAR decoy.
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KEYWORD
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HIV-1, Permanent cell line, Tat inhibitor
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