A MICROTRANSFECTION METHOD USING THE LUCIFERASE-ENCODING REPORTER GENE FOR THE ASSAY OF HUMAN-IMMUNODEFICIENCY-VIRUS LTR PROMOTER ACTIVITY

被引：105

作者：

SCHWARTZ, O ^{[1
]}

VIRELIZIER, JL ^{[1
]}

MONTAGNIER, L ^{[1
]}

HAZAN, U ^{[1
]}

机构：

[1] INST PASTEUR,IMMUNOL VIRALE LAB,F-75724 PARIS 15,FRANCE

来源：

GENE | 1990年 / 88卷 / 02期

关键词：

long terminal repeat; Recombinant DNA; reporter gene; Tat transactivation; transient expression; virus titration;

D O I：

10.1016/0378-1119(90)90032-M

中图分类号：

Q3 [遗传学];

学科分类号：

071007 ; 090102 ;

摘要：

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 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 huc 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. © 1990.

引用

页码：197 / 205

页数：9

共 26 条

[1] TRANS-ACTIVATOR GENE OF HUMAN T-LYMPHOTROPIC VIRUS TYPE-III (HTLV-III) [J].