Construction And Identification Of Recombinant Lentiviral Vectors For Mouse TREM2 Gene Overexpression And ShRNA Interference

ObjectiveTo construct recombinant lentiviral vector for mouse TREM2(triggering receptor expressed on myeloid cells-2,TREM2)gene overexpression and shRNA interference,and to detect the level of TREM2 gene expression when recombinant lentiviral vector were infected into mouse BV2 microglia.Methods1.Construction and identification of recombinant lentiviral vectors for mouse TREM2 gene overexpressionThe TREM2 gene fragment was amplified by PCR,and the amplified product was ligated to the linearized GV358 vector.Then these recombinant lentiviral vectors were transformed into competent cell E.coli DH5?.After identifying with PCR and DNA sequencing,the PCR product was co-transfected with the packaging plasmid into 293T cells and the virus titer was determined by fluorescence method.The recombinant lentiviral vector for mouse TREM2 gene overexpression was used to infect mouse BV2 microglia,and the TREM2mRNA was detected by Q-PCR.2.Construction and identification of recombinant lentiviral vectors for mouse TREM2 gene shRNA interferenceThree pairs of shRNA of mouse TREM2 gene were designed,synthetized,and ligated separately to the linearized GV248 vector.Then these recombinant lentiviral vectors were transformed into competent cell E.coli DH5?.After Amp screening positive clones,recombinant lentiviral vectors were identified with DNA sequencing.After identifying with DNA sequencing,the recombinant lentiviral vectors was co-transfected with the packaging plasmid into 293T cells and the virus titer was determined by fluorescence method.The three kinds of recombinant lentiviral vectors were used to infect mouse BV2 microglia,and the TREM2 mRNA and protein were detected by Q-PCR and Western blotting,respectively.Results1.Construction and identification of recombinant lentiviral vectors for mouse TREM2 gene overexpressionThe TREM2 gene fragment was successfully obtained by PCR and ligated to the linearized GV358 vector.The recombinant lentiviral vectors was confirmed to carry the correct TREM2 gene by PCR and DNA sequencing.The recombinant lentiviral vectors was packaged in 293T cells and the titer of the concentrated virus was 2.0×10~8 TU/mL.TREM2 expression was significantly increased at mRNA levels compared with those of the non-transfected group and negative control vectors group(p<0.05),when mouse BV2 microglia were infected into recombinant lentiviral vectors for mouse TREM2 gene overexpression.2.Construction and identification of recombinant lentiviral vectors for mouse TREM2 gene shRNA interferenceThe DNA sequencing of positive clones demonstrated that three kinds of recombinant lentiviral vectors for mouse TREM2 gene shRNA interference were constructed successfully.The recombinant lentiviral vectors were packaged in293T cells and the titer of the concentrated virus was 4×10~8 TU/mL,4×10~8TU/mL and 5×10~8 TU/mL respectively.TREM2 expression was significantly decreased at both mRNA and protein levels compared with those of the non-transfected group and negative control vectors group(p<0.05),when mouse BV2 microglia were infected into three kinds of recombinant lentiviral vectors for mouse TREM2 gene shRNA interference.The GV248-shRNA-TREM2-2played the best role and its silence efficiency reached 79.7%.ConclusionsWe constructed the recombinant lentiviral vectors for mouse TREM2 gene overexpression and shRNA interference using lentiviral vector-mediated gene overexpression and RNA interference,and identified them in mouse BV2microglia,which provides an experimental foundation for revealing the mechanism of TREM2 gene involved in the development of late-onset Alzheimer's disease(LOAD).