Establishment Of A Mouse Leukemia Cell Line Expressing Human CD4 And Study Of Its Cell Cycle

Recently, molecular biology has achieved great progress. Gene transfection has been widely used in many fields of experiments and therapies. To obtain stable and efficient expression of a specific molecule in leukemia cells for further study, a pLXIN-hCD4 vector encoding human CD4 gene was constructed. This paper was divided into two parts.Experimentâ… Establishment of retroviral vector System expressing human CD4 geneObjective To express human CD4 efficiently and stably in leukemia cells, an retroviral vector, pLXIN-hCD4 encoding human CD4 gene was constructed.Methods Human CD4 gene was amplified by PCR, which was subcloned into pLXIN, named as recombinant pLXIN-hCD4. It was subsequently transduced into the E.coli and the positive colony was selected, which was digested and sequenced to be correct. The recombinant pLXIN-hCD4 plasmid was transfected into packing cells PT67 by lipofectamine, the stable clone was then selected and developed by culturing in a medium containing G418. The supernatants containing virus particles were collected.Results Human CD4 was correctly cloned into pLXIN vector, which was demonstrated via PCR and enzyme restriction. The PT67 cells capable of producing virus particulars were obtained.Conclusion The recombinant plasmid, pLXIN-hCD4 was constructed successfully.Experimentâ…¡Establishment of a mouse leukemia cell line expressing human CD4 and study of its cell cycleObjective human CD4 was expressed efficiently and stably in L615 cells, a mouse acute lymphoblastic leukemia cell line. Then the cell cycle was investigated in vitro.Methods L615 cells were infected by the obtained supernatants containing virus particles, the stable clone was selected by G418, which was called L615-hCD4 cells. The expression of human CD4 in L615-hCD4 cells as well as cell cycles of L615 cells and L615-hCD4 cells were analyzed by flow cytometry (FCM).Results The L615-hCD4 cells stably express human CD4 while L615 cells do not. The cell cycle of L615-hCD4 cells has little difference from that of L615 cells.Conclusion L615-hCD4 cells can express human CD4 stably, whose cell cycle was coincident with L615 cells.