Construction of targeted vector of mouse TLR3 gene double knockout and macrophage RAW264.7TLR3-/-cell line

ObjectiveThe aims of this experiment was through establishing macrophage RAW264.7 cell line lacked Toll-like receptors 3(TLR3)gene to provide theoretical basis for exploring the role of TLR3 in inherent immunity during the process of rabies virus infection.MethodThe Golden Gate Kit was used for constructing transcription activation effect factor nuclease(TALEN)targeting vector pTALEN-TLR3,and correctness was verified by enzyme digestion and se-quencing,RAW264.7 cells by were transiently transfected liposome,and the cells DNA was extracted after transfection, then the shear activity of pTALEN-TLR3 was verified with T7 nucleic acid enzyme.ResultThe connection of left arm and right arm of TALENs was constructed by two steps. In the first step,part A and B were connected separately. Then T1LA and T1LB,T1RA and T1RB,T2LA and T2LB,T2RA and T2RB were connected separately. After the two steps, TALEN module was identified by PCR,the results show that all the four clones of T1L,T1R and T2L were positive, three clones of T2R were positive. The T2L and T2R plasmids were co-transfected to RAW264.7 cells,and then DNA was extracted as template,which was implemented in enzyme digestion by T7 nucleic acid endonuclease after PCR amplifica-tion. The DNA electrophoresis results after enzyme digestion showed that TALEN2 shear activity was strong,and three bands(931,555 and 376 bp)were obtained. RAW264.7 cells were transfected with the TALEN2-TLR3,and then digested with pancreatic enzymes after 24 h,and were screened by adding 800μg/mL G418,a single clone was obtained after 7 d. The positive cell clones were chosen for identification and sequencing by T7 endonuclease digestion. The results showed that No.4-1 and 4-40 clone cells were double knockout cell lines,they all missed 7 bp nucleotide bases,as non-three inte-ger times base deletion. It could cause subsequent frame shift mutations,and inactivated gene function of the cell.Con-clusionRAW264.7TLR3-/-cell line from macrophages of mice with the double knockout of TLR3 gene was constructed successfully by TALEN technology. This can be applied for further study on the relationship between cytokines and TLR3 after rabies virus infects cells.