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 immunodef iciency virus type 1 (HIV-1). Consistent with the conventional chlorampheni col acetyl transferase (CAT) reporter, EGFP expression, under the direction of HIV-1 LTR, was readily detected in the transient transfection and was e levated by co-transfection of HIV-1 tat-expression vector. Comparing to CAT , however, EGFP expression system has two advantages: (i) Using a fluoresce nce 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 expr ession was readily detected at a DNA concentration where CAT activity was n ot detectable possibly because the transfectants could be 'gated'. On the o ther hand, at a higher concentration of DNA, CAT signal became more promine nt than that of EGFP, possibly because the enzymatic activity of CAT 'ampli fied' the signal. EGFP fluorescence detected by FAGS was a direct measureme nt of the expressed chromophore. It is concluded that the system is rapid, reproducible, convenient and useful for quantitative analysis of transcript ion. (C) 2000 Elsevier Science B.V. All rights reserved.