Green fluorescence protein as a transcriptional reporter for the long terminal repeats of the human immunodeficiency virus type 1.
Using the enhanced green fluorescence protein (EGFP), a transient reporter expression system was established to assess the transcriptional activity of the long terminal repeats (LTR) of primary isolates of the human immunodeficiency virus type 1 (HIV-1). Consistent with the conventional chloramphenicol acetyl transferase (CAT) reporter, EGFP expression, under the direction of HIV-1 LTR, was readily detected in the transient transfection and was elevated by co-transfection of HIV-1 tat-expression vector. Comparing to CAT, however, EGFP expression system has two advantages: (i) Using a fluorescence activated cell sorter (FACS), it was possible to simultaneously measure transfection efficiency and fluorescence intensity of the transfected live cells without the necessity of co-transfection of a reference plasmid for comparing the transcriptional activity of two promoters; and (ii) EGFP expression was readily detected at a DNA concentration where CAT activity was not detectable possibly because the transfectants could be 'gated'. On the other hand, at a higher concentration of DNA, CAT signal became more prominent than that of EGFP, possibly because the enzymatic activity of CAT 'amplified' the signal. EGFP fluorescence detected by FACS was a direct measurement of the expressed chromophore. It is concluded that the system is rapid, reproducible, convenient and useful for quantitative analysis of transcription.
Kar-Roy
A
A
Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore 21201, USA.
Dong
W
W
Michael
N
N
Li
Y
Y
D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D004279:DNA, Viral / Q000032:analysis; D005434:Flow Cytometry; D015696:Gene Products, tat / Q000235:genetics / Q000378:metabolism; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D016325:HIV Long Terminal Repeat / Q000235:genetics*; D015497:HIV-1 / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D012680:Sensitivity and Specificity; D014158:Transcription, Genetic; D014162:Transfection; D054322:tat Gene Products, Human Immunodeficiency Virus
D004279:DNA, Viral; D015696:Gene Products, tat; D008164:Luminescent Proteins; D054322:tat Gene Products, Human Immunodeficiency Virus; D049452:Green Fluorescent Proteins; D015500:Chloramphenicol O-Acetyltransferase