Browsing School, Graduate by Subject "Green Fluorescent Proteins"
Now showing items 1-1 of 1
Study of the human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus LTR directed gene expression using the green fluorescent protein (GFP) as the reporterUsing enhanced green fluorescence protein (EGFP-1), a transient transfection reporter system was established to monitor the transcriptional activity of the long terminal repeats (LTR) of several primary strains of human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus SIVmac239. After transient transfection of HeLa cells with variant HIV-1 LTR-EGFP-1 constructs, we scanned the cell culture using fluorescent activated cell sorter (FACS). Using FACS it was possible to simultaneously estimate for transfection efficiency and to quantitatively determine the fluorescence intensity of the transfected population. Data showed that expression of EGFP-1 was DNA dose dependent. FACS enabled the visualization of heterogeneity in the level of reporter gene expression in a transiently transfected population. The distribution of the fluorescent intensity of transfected cells was treated as a frequency distribution, and different statistical estimators were used to quantitate the amount of EGFP-1 expression. The sensitivity of the system was compared to that of chloramphenicol acetyl transferase (CAT) assay. In HeLa cells the EGFP-1 reporter assay was more sensitive than CAT assay in the absence of Tat transactivation. In HeLa cells using the EGFP-1 reporter system it was possible to demonstrate that the two coding exons of SIVmac Tat was required for optimal transactivation of the SIVmac LTR, while the first coding exon of HIV-1 Tat was sufficient to transactivate both the HIV-1 and the SIVmac LTR. However, in T cells lines while we observed low levels of transactivation from the HIV-1 and SIVmac LTR by HIV-1 Tat, we were unable to detect enhanced expression of EGFP-1 in the presence of the two coding exons of SIV mac Tat. Since the EGFP-1 reporter assay is not an enzyme based assay, in the absence of signal amplification the sensitivity of the assay in T cell lines was poor. Hence, it was difficult to detect small differences in Tat activated gene expression in the presence of either the single coding exon or both the coding exons of SIVmac Tat in T cell lines.