A simple assay system for examination of the inhibitory potential in vivo of decoy RNAs, ribozymes and other drugs by measuring the Tat-mediated transcription of a fusion gene composed of the long terminal repeat of HIV-1 and a gene for luciferase.
Nucleic acid-based drugs, including antisense RNA and DNA, ribozymes and decoys appear to have potential for the suppression of the expression of specific genes. To allow the examination of the potential of such agents in vivo as anti-HIV drugs in standard laboratories, where facilities for handling live virions are not available, we constructed a simple assay system (HIV-1 model) that allows measurement of the extent of inhibition of Tat-mediated transcription of HIV-1 by nucleic acid-based drugs and other agents. In cells that harbor a stable chimeric long terminal repeat (LTR)-Luc construct (a fusion gene consisting of the LTR of HIV-1 and the gene for luciferase), total luciferase activity in an aliquot of cell lysate is dose- and promoter-dependent on transfection with a Tat expression plasmid, reflecting the character of the LTR promoter of HIV. When HeLa cells were co-transfected with the Tat expression plasmid and another plasmid that encoded the U6 promoter or the promoter of the gene for tRNA(Val) linked to the trans-activating response (TAR) sequence, total luciferase activity was inhibited by 60 or 40%, respectively. The inhibition was also dependent on the dose of the TAR expression plasmid. These results demonstrate the usefulness of this simple assay system for detection of the efficacy of a decoy RNA or a ribozyme in vivo, without a requirement for HIV-infected cells, by measurement of luciferase activity in vitro.
Koseki
S
S
National Institute of Bioscience and Human Technology, MITI, Tsukuba Science City, Japan.
Ohkawa
J
J
Yamamoto
R
R
Takebe
Y
Y
Taira
K
K
D019677:Artificial Gene Fusion; D001483:Base Sequence; D015696:Gene Products, tat / Q000502:physiology*; D016325:HIV Long Terminal Repeat / Q000235:genetics*; D015497:HIV-1 / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D008156:Luciferases / Q000235:genetics*; D009690:Nucleic Acid Conformation; D010957:Plasmids; D012313:RNA / Q000737:chemistry / Q000494:pharmacology*; D016337:RNA, Catalytic / Q000494:pharmacology*; D014158:Transcription, Genetic / Q000187:drug effects* / Q000502:physiology; D054322:tat Gene Products, Human Immunodeficiency Virus
D015696:Gene Products, tat; D016337:RNA, Catalytic; D054322:tat Gene Products, Human Immunodeficiency Virus; D012313:RNA; D008156:Luciferases