Retention of a recombinant GFP protein expressed by the yellow fever 17D virus in the E/NS1 intergenic region in the endoplasmic reticulum

The flaviviral envelope proteins, E protein and precursor membrane protein, are mainly associated with the endoplasmic reticulum (ER) through two transmembrane (TM) domains that are exposed to the luminal face of this compartment. Their retention is associated with the viral assembly process. ER-retrieval motifs were mapped at the carboxy terminus of these envelope proteins. A recombinant yellow fever (YF) 17D virus expressing the reporter green fluorescent protein (GFP) with the stem-anchor (SA) region of E protein fused to its carboxy terminus was subjected to distinct genetic mutations in the SA sequence to investigate their effect on ER retention. Initially, we introduced progressive deletions of the stem elements (H1, CS and H2). In a second set of mutants, the effect of a length increase for the first TM anchor region was evaluated either by replacing it with the longer TM of human LAMP-1 or by the insertion of the VALLLVA sequence into its carboxy terminus. We did not detect any effect on the GFP localisation in the cell, which remained associated with the ER. Further studies should be undertaken to elucidate the causes of the ER retention of recombinant proteins expressed at the intergenic E/NS1 region of the YF 17D virus polyprotein.

The Expression of GFP Gene in Transformed and Tumor Cells / 上海第二医科大学学报

ObjectiveTo search the appropriate experimental conditions for using green fluores-cent protein (GFP) as a reporter gene in tumor gene therapy. Methods The plasmids carrying mu-tated GFP gene were transfected into the eukaryotic cells to observe transient gene expression. Thesestudies were conducted to 1. directly determine GFP expression and express stability in the COS- 7cells, 2. compare the transfection efficiency of two plasmids pcDNA3 - EGFP, pSVKa - S65T with dif-ferent promoters in different tumor cell lines, 3. determine two genes expression in a single cell usingLacZ cotransfected with GFP by FACS. ResultsFluorescence could be detected in intact viable cellsunder different sets of conditions. The expression of GFP might last two weeks or more and the ex-pressed fluorescence was stable. The transfection rate of pcDNA3 - EGFP expressed was different inthree tumor cell lines examined. But pSVK3 - GFP expressed similarly in four tumor cell lines exam-ined. FACS showed the probability of two genes entering a single cell is above >85% at the ratio 1:4.ConclusionThe above data indicate that the GFP can be visualized continuously and directly for geneexpression in living cells. GFP may also be used for quicklly selecting cells carrying a target gene.