小鼠TLR3基因双敲除打靶载体构建及巨噬细胞RAW264.7^(TLR3-/-)细胞系的建立

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

【目的】建立Toll样受体3(TLR3)基因缺失的小鼠巨噬细胞RAW264.7细胞系,为探索狂犬病毒感染机体过程中TLR3在固有免疫反应中的作用机制提供理论依据。【方法】采用Golden Gate Kit试剂盒组装转录激活样效应因子核酸酶(TALEN)打靶载体p TALEN-TLR3,经酶切和测序验证其连接正确后,通过脂质体瞬时转染RAW264.7细胞,转染后提取细胞DNA,用T7核酸内切酶酶切验证TALEN质粒剪切活性。【结果】TALENs左右臂分两部分连接,首先完成A、B部分的各自连接,然后分别将T1LA与T1LB、T1RA与T1RB、T2LA与T2LB、T2RA与T2RB连接,TALEN模块经过两次连接后的PCR鉴定结果显示,T1L、T1R和T2L的4个克隆均呈阳性,T2R有3个克隆呈阳性。T2L和T2R质粒共转染RAW264.7细胞后提取DNA为模板,经PCR扩增后用T7核酸内切酶进行酶切,酶切后的DNA电泳结果显示TALEN2剪切活性较强,共获得3条条带(931、555和376 bp)。TALEN打靶载体p TALEN-TLR3转染RAW264.7细胞24 h后用胰酶进行消化,并加入800μg/m L G418进行筛选,7 d后获得细胞单克隆;挑选阳性细胞克隆进行T7核酸内切酶酶切鉴定及测序,结果发现4-1和4-40号细胞克隆为双敲细胞系,均缺失7 bp的核苷酸碱基,为非3整数倍碱基缺失,可造成后续基因移码突变,使细胞基因功能失活。【结论】通过TALEN技术可成功构建TLR3基因双敲除的小鼠巨噬细胞RAW264.7TLR3-/-细胞系,且可用于狂犬病毒感染细胞后细胞因子和TLR3间的关系研究。

【Objective】The 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.【Method】The Golden Gate Kit was used for constructing transcription activation effect factor nuclease（TALEN）targeting vector p TALEN-TLR3,and correctness was verified by enzyme digestion and sequencing,RAW264.7 cells by were transiently transfected liposome,and the cells DNA was extracted after transfection,then the shear activity of p TALEN-TLR3 was verified with T7 nucleic acid enzyme.【Result】The 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 T1 LA and T1 LB,T1 RA and T1 RB,T2 LA and T2 LB,T2 RA and T2 RB were connected separately. After the two steps,TALEN module was identified by PCR,the results show that all the four clones of T1 L,T1 R and T2 L were positive,three clones of T2 R were positive. The T2 L and T2 R 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 800 μg/m L 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 integer times base deletion. It could cause subsequent frame shift mutations,and inactivated gene function of the cell.【Conclusion】RAW264.7^ TLR3-/-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.