目的:制备Trim26/ZNF173条件性基因敲除小鼠,为研究Trim26基因在肝癌发生中的作用及其机制提供动物模型。方法:设计基因敲除策略,Trim26的外显子5~7缺失引起的移码突变会破坏蛋白结构域,进而导致Trim26蛋白功能丧失。为此,我们拟删除Tri...目的:制备Trim26/ZNF173条件性基因敲除小鼠,为研究Trim26基因在肝癌发生中的作用及其机制提供动物模型。方法:设计基因敲除策略,Trim26的外显子5~7缺失引起的移码突变会破坏蛋白结构域,进而导致Trim26蛋白功能丧失。为此,我们拟删除Trim26基因的外显子5~7,以借助Cre/lox P系统构建Trim26基因条件性敲除的小鼠。简单地说,先获得嵌合体小鼠,选择高嵌合率小鼠与B6/N小鼠交配获得F1代Trim26fl/wt基因型的杂合子小鼠。F1代杂合子小鼠自交得到F2代Trim26fl/fl基因型的纯合小鼠和对照野生型小鼠。提取鼠尾基因组DNA并行PCR鉴定基因型,在DNA水平确定小鼠的基因型。结果:成功构建了打靶载体,打靶载体经限制性内切酶线性化后,成功电转导入了B6/BLU ES 细胞中,ES克隆经过LR-PCR初筛及Southern blot进一步验证,确定得到了3株中靶ES细胞(即3D、6A和10F),将中靶ES细胞扩增后进行囊胚注射,获得了12只Trim26的嵌合体小鼠(嵌合率不小于50%)。选择性成熟后的高嵌合率小鼠与B6/N小鼠交配繁育,得到F1代小鼠,PCR鉴定基因型确认获得了Trim26fl/wt基因型的F1代杂合子小鼠。性成熟后的F1代杂合子小鼠合笼交配后得到F2代小鼠,PCR鉴定基因型确认获得了纯合的Trim26条件性基因敲除小鼠(基因型为Trim26fl/fl)。结论:成功制备了Trim26条件性基因敲除小鼠,为进一步解析Trim26在肝癌发生中的作用及机制打下了良好基础。展开更多
The transcription factors IRF3 and NF-kB are required for the expression of many genes involved in antiviral innate immune response,including type I interferons(IFNs)and proinflammatory cytokines.It is well establishe...The transcription factors IRF3 and NF-kB are required for the expression of many genes involved in antiviral innate immune response,including type I interferons(IFNs)and proinflammatory cytokines.It is well established thatTBK1 isan essential kinase engageddownstream of multiple pattern-recognition receptors(PRRs)to mediate IRF3 phosphorylation and activation,whereas the precise mechanisms of TBK1 activation have not been fully elucidated yet.Here,weidentified tripartite motif 26(TRIM26)as an important regulator for RNAvirus-triggered innate immune response.Knockdown of TRIM26 impaired virus-triggered IRF3,NF-kB activation,IFN-b induction,and cellular antiviral response.TRIM26 was physically associated with TBK1 independent of viral infection.As an E3 ligase,TRIM26 underwent autoubiquitination upon viral infection.Ubiquitinated TRIM26 subsequently associated with NEMO,thus bridging TBK1–NEMOinteraction,which is critical for the recruitment of TBK1 to the VISA signalsome and activation of TBK1.Our findings suggest that TRIM26 is an important regulator of innate immune responses against RNA viruses,which functions by bridging TBK1 to NEMO and mediating the activation of TBK1.展开更多
文摘目的:制备Trim26/ZNF173条件性基因敲除小鼠,为研究Trim26基因在肝癌发生中的作用及其机制提供动物模型。方法:设计基因敲除策略,Trim26的外显子5~7缺失引起的移码突变会破坏蛋白结构域,进而导致Trim26蛋白功能丧失。为此,我们拟删除Trim26基因的外显子5~7,以借助Cre/lox P系统构建Trim26基因条件性敲除的小鼠。简单地说,先获得嵌合体小鼠,选择高嵌合率小鼠与B6/N小鼠交配获得F1代Trim26fl/wt基因型的杂合子小鼠。F1代杂合子小鼠自交得到F2代Trim26fl/fl基因型的纯合小鼠和对照野生型小鼠。提取鼠尾基因组DNA并行PCR鉴定基因型,在DNA水平确定小鼠的基因型。结果:成功构建了打靶载体,打靶载体经限制性内切酶线性化后,成功电转导入了B6/BLU ES 细胞中,ES克隆经过LR-PCR初筛及Southern blot进一步验证,确定得到了3株中靶ES细胞(即3D、6A和10F),将中靶ES细胞扩增后进行囊胚注射,获得了12只Trim26的嵌合体小鼠(嵌合率不小于50%)。选择性成熟后的高嵌合率小鼠与B6/N小鼠交配繁育,得到F1代小鼠,PCR鉴定基因型确认获得了Trim26fl/wt基因型的F1代杂合子小鼠。性成熟后的F1代杂合子小鼠合笼交配后得到F2代小鼠,PCR鉴定基因型确认获得了纯合的Trim26条件性基因敲除小鼠(基因型为Trim26fl/fl)。结论:成功制备了Trim26条件性基因敲除小鼠,为进一步解析Trim26在肝癌发生中的作用及机制打下了良好基础。
基金supported by the Ministry of Science and Technology of China(2014CB542603,2015CB554302)the National Natural Science Foundation of China(31425010,31321001,31270932,31170792,31400742)the 10000 Talents Plan,and the WIV‘One-Three-Five’Strategic Programs.
文摘The transcription factors IRF3 and NF-kB are required for the expression of many genes involved in antiviral innate immune response,including type I interferons(IFNs)and proinflammatory cytokines.It is well established thatTBK1 isan essential kinase engageddownstream of multiple pattern-recognition receptors(PRRs)to mediate IRF3 phosphorylation and activation,whereas the precise mechanisms of TBK1 activation have not been fully elucidated yet.Here,weidentified tripartite motif 26(TRIM26)as an important regulator for RNAvirus-triggered innate immune response.Knockdown of TRIM26 impaired virus-triggered IRF3,NF-kB activation,IFN-b induction,and cellular antiviral response.TRIM26 was physically associated with TBK1 independent of viral infection.As an E3 ligase,TRIM26 underwent autoubiquitination upon viral infection.Ubiquitinated TRIM26 subsequently associated with NEMO,thus bridging TBK1–NEMOinteraction,which is critical for the recruitment of TBK1 to the VISA signalsome and activation of TBK1.Our findings suggest that TRIM26 is an important regulator of innate immune responses against RNA viruses,which functions by bridging TBK1 to NEMO and mediating the activation of TBK1.
