Construction of a cell-cell fusion system and its primary application

Objective: To construct a cell-cell fusion system and to examine the effect of thrombin on fusion mediated by Human immunodeficiency virus type I(HIV-1) envelope glycoproteins(Env). Methods: 293T cells wer cotransfected with HIV-1 Env expression plasmid pSV-III-JRFL-dCT and Tat expression plasmid pcTat. The transfected cells were harvested at different time and HIV-1 Env proteins expressed on the surface of 293T cells were detected by flow cytometry. Then the 293T cells expressing Env proteins were cocultured with Magic5A cells for 20 h. Fused cells were stained by X-gal as blue cells and were examined by microscopy. When a stable fusion system was constructed, the 293T cells were treated by thrombin and the effect of thrombin was exainined by counting the fused cells. Results: The cotransfection with pSV- III -JRFL-dCT and pcTat at 1 : 1 and 2 : I could render a stable fusion system. After the treatment on 293T cells expressing Env, Th promoted the fusion effect in a time-dependent manner. Conclusions: Via coculturing 293T cells cotransfected with pSV-III-JRFL-dCT and pcTat at 1 : 1 or 2 : 1 with Magic5A cells, the HlV-1 envelope-mediated cell-cell fusion system has been constructed. The enhancing effect of Th on the fusion has been proven.