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Keimyung Medical Journal 2003;22(1):104-113.
Development of Permanent Cell Lines for HIV-1 Tat Inhibitor Screening
HIV-I 성장억제제 발굴을 위한 영구 세포주 개발
박종구
Abstract
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.
Key Words: HIV-1, Permanent cell line, Tat inhibitor
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