A microtransfection method using the luciferase-encoding reporter gene for the assay of human immunodeficiency virus LTR promoter activity.
A microtransfection method, using either the DEAE-dextran or the Ca.phosphate procedure has been developed. A plasmid expressing the luciferase-encoding gene under the control of the human immunodeficiency virus (HIV) LTR promoter was constructed. Transfections were performed in 96-well plates, allowing statistical evaluation of the results. This microtransfection method requires the use of 100- to 1000-fold less plasmid and cells than in a conventional chloramphenicol acetyl transferase (CAT) assay. A Luciferase index which takes into account cell viability after transfection has been defined using a semi-automated absorbance assay. A 20-h incubation period post-transfection is sufficient for optimal results. Basal long terminal repeat activity and autologous Tat transactivation were studied in various lymphoid, monocytic and adherent human cell lines. Infection of microtransfected cells by HIV activated luc expression. This assay can thus also be used for rapid detection and quantitation of HIV. Antiviral activities of drugs can be assessed in a two-day test.
Unité d'Oncologie Virale, Institut Pasteur, Paris, France.
O
O
Schwartz
J L
JL
Virelizier
L
L
Montagnier
U
U
Hazan
D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D004274:DNA, Recombinant; D015967:Gene Expression Regulation, Viral; D015696:Gene Products, tat / Q000235:genetics*; D006678:HIV / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D008156:Luciferases / Q000235:genetics*; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics*; D015203:Reproducibility of Results; D015534:Trans-Activators / Q000235:genetics*; D015533:Transcriptional Activation; D014162:Transfection; D014779:Virus Replication; D054322:tat Gene Products, Human Immunodeficiency Virus
D004274:DNA, Recombinant; D015696:Gene Products, tat; D015534:Trans-Activators; D054322:tat Gene Products, Human Immunodeficiency Virus; D008156:Luciferases; D015500:Chloramphenicol O-Acetyltransferase