| Objective:to study the possibility of the Red Fluorescent Protein(RFP)gene acts as Reporter Gene in live cells,we specially construct the recombination three-plasmid lentiviral vectors tagged with RFP,and transfect the human bladder carcinommer cell(BCC),T-24 cell line.Observe the result after the transfection accomplished under the Confocal Laser Fluoresecence Microscopy.Method:To transfect the 293T cell with the viral packaging cell by using the RFP-Lentivirus recombination(LV-CMV-RFP)and both pHelper 1.0 and pHelper 2.0 vectors according to the Lipofectamine 2000 method.After incubating them for 3-4 days,harvest and refine the virus solution.Fetch the solution,ingather and concentrate the virus solution.Package some of them at the temperature of -80℃for long-term saving.The left solution was left for the titer test by the Real-Time way or hole dilution way.Culture the T-24 cell line in 96 Well Plates and virus of specific concentration in the air incubator in different MOI proportion for 72-96 hours to grope the best condition for transfecting.Transfect the target cell repeatly according the result of the pre-test above.Observe the cells under the Confocal Laser Fluoresecence Microscopy with the 558nm exciting wavelength and 583 nm emiting wavelength.Identifying the expression of RFP gene and transfection efficiency by Flow Cytometry.After that co-culture the T-24 cell and transfected T-24 cell to observe their growing property.Result:Successfully constructing the recombination of three-plasmid lentiviral vectors tagged with RFP.After transfecting the carcinoma cell line,the cell line strongly emit the red fluorescence under the Confocal Laser Fluoresecence Microscopy.Conclusion:Ds-Red gene can successfully express in T-24 cell in form of three-plasmid lentiviral vectors,so the live carcinoma cells can be diredtly visualized by the Confocal Laser Fluoresecence Microscopy via the red fluoresecent of transfected cells themselves.It is suggested that Ds-Red gene be a favourable reptor gene and screening marker in live cells. |
