Development of
Permanent Cell Lines for
HIV-1 Tat Inhibitor Screening |
HIV-I 성장억제제 발굴을 위한 영구 세포주 개발 |
박종구 |
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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 |