Establishment Of Mouse Leukemia Cell Lines Expressing Human HCD4/CCR5/cyclin T1 Using Lentiviral Vectors

Objective: We established a mouse leukemia cell line that can highly express hCD4/CCR5/cyclinT1,and this cell line will be used in HIV-1 infection research.Human Immunodeficiency Virus(HIV)infects the cells of the immune system thereby destroying or impairing their functions,resulting in acquired immunodeficiency syndrome(AIDS).Since the first AIDS case was reported in America in 1981,AIDS has become a public health problem and a social issue that severely harms the existence and development of human beings.Since HIV is highly species-specific,establishing a cell/animal model by using method of chimeric simian-human immunodeficiency virus(SHIV)in non-human primates.And creating a mouse animal model by implanting the severe combined immunodeficiency(SCID)mice with human tissues necessary to develop HIV-1 infection.Therefore,the aim of this research is to develop a cell model of supporting normal entry and efficient replication.Methods: Firstly,the plasmid expressing hCD4,hCCR5 and hcyclinT1 vector of pLV[Exp]-EGFP/Neo-CMV>hCD4:T2A:hCCNT1:IRES:hCCR5 was established using Gateway technology,and the plasmid was detected by sequencing identification.Next lentiviral was packaged in the 293 T cells,and the expression of hCD4 and the hCCR5 genes in 293 T cells was detected by qRT-PCR method.Subsequently,the mouse leukemia cell line L1210 and L615 cells were transfected with the lentiviral vectors at an optimum MOI,and the expression of hCD4,hCCR5 and hcyclinT1 genes were analyzed by PCR,Western blot analysis and immunofluorescrne staining compared with the cells transfected with empty lentiviral vectors.In the next experiment,the transfected L615 and L1210 cells infected with HIV-1,HIV-1RNA in the cell culture supernatants and cell body was detected by RT-PCR,the sensitivity of human CD4/CCR5/cyclinT1 transgenic mouse cells to HIV infection was confirmed.Results: The results of sequencing identification and fluorescence expression show that the plasmid expressing hCD4,hCCR5 and hcyclinT1 vector of pLV[Exp]-EGFP/Neo-CMV>hCD4:T2A:hCCNT1:IRES:hCCR5 was successfully constructed and wrapped as the lentiviral vector,which mediated hCD4,hCCR5 and hcyclinT1 genes to express specially and efficiently in 293 T cells.And the L615 and L1210 cells were transfected with lentiviral vector.qRT-PCR results revealed that the hCD4 and CCR5 proteins were highly expressed in mouse leukemia cell L615 and L1210.Western blot analysis illustrated that the L1210 and L615 cells expressed CD4,CCR5 and hcyclinT1 proteins compared with the cells transfected with empty lentiviral vectors.The results of immunofluorescence staining revealed that CD4,CCR5 and hcyclinT1 were expressed on the cell surface,which demonstrating that the L615 and L1210 cells were humanized and that they possess the characteristics necessary for HIV infection of human host cells.In our research,viral entry and replication was further indicated by detection of HIV-1RNA in culture supernatants and cell body.The transgenic mice cells L615 and L1210 expressed significantly higher levels of HIV-1RNA than the non-transfection group.The transgenic mice L615 and L1210 cells were possibly infected by HIV-1 and could support efficient HIV-1 invasion and replication as the host cell.Conclusion: Mouse leukemia cell lines that could express hCD4,hCCR5 and hcyclinT1 were established to facilitate normal entry and efficient replication of HIV-1,and such cell model can further promote research of HIV-1 infected mouse animal model.The cross-species transgenic mice cell and animal model can be used to investigate the pathogenesis of HIV/AIDS,candidate vaccines and potential antiviral drugs against this disease.