Toll样受体4(TLR4)基因剔除小鼠构建及初步表型分析

目的 利用CRISPR/Cas9技术构建Toll样受体4(TLR4)基因敲除小鼠模型,并观察突变小鼠对革兰氏阴性细菌脂多糖(LPS)刺激响应的变化。方法 针对TLR4基因外显子2设计并合成1对sgRNA片段,与编码Cas9的mRNA混合后通过受精卵显微注射方法,建立TLR4基因敲除小鼠,通过繁育获得基因敲除纯合子小鼠(TLR4^-/-小鼠);通过LPS刺激,分析TLR4^-/-小鼠对炎症应激的反应情况,并在分子和病理水平上和野生型对照(WT)进行比较。结果 PCR及测序检测表明TLR4基因外显子2在小鼠基因中被成功敲除;给予LPS刺激后,IL1β、IL6、MyD88、iNOS及TNFa等炎症因子的表达在野生型小鼠的心、肝和肺组织中显著上调,而在TLR4^-/-小鼠中则几乎没有变化;血生化指标显示LPS刺激后WT小鼠血清中的尿素(Urea)和肌酐(Cre)水平显著升高,而TLR4^-/-小鼠刺激前后无显著变化,病理分析同样发现TLR4^-/-小鼠能够抵抗LPS对肾组织的损伤。结论 利用CRISPR/Cas9技术成功构建了TLR4基因剔除小鼠模型,TLR4的缺失能够降低IL1β、IL6、MyD88、iNOS及TNFa炎症因子对LPS刺激的响应,抑制LPS引起的炎症反应及对组织的损伤。

Toll-like Receptor 4 (TLR4) Gene Knockout Mouse Model Construction and Preliminary Phenotypic Analysis

Objective: To construct a Toll-like receptor 4 (TLR4) gene knockout mouse model using CRISPR / Cas9 technology, and observe the changes in mutant mice’s response to gram-negative bacterial lipopolysaccharide (LPS) stimulation.Methods: One pair of sgRNA fragments were designed and synthesized for exon 2 of TLR4 gene, mixed with mRNA encoding Cas9 and then injected with TLR4 gene knockout mice through fertilized egg microinjection method, and gene knockout homozygous mice were obtained by breeding (TLR4 ^-/-mice); The response of TLR4^-/-mice to inflammatory stress was analyzed by LPS stimulation, and compared with wild-type control (WT) at the molecular and pathological levels.Results: PCR and sequencing showed that exon 2 of TLR4 gene was successfully knocked out in mouse genes. After LPS stimulation, the expressions of inflammatory factors such as IL1β, IL6, MyD88, iNOS and TNFa were detected in the heart of wild-type mice. Significantly up-regulated in liver and lung tissues, but almost no change in TLR4 ^-/-mice; blood biochemical indicators showed that urea (Crea) and creatinine (Cre) levels in WT mice were significantly increased after LPS stimulation, and TLR4^-/-mice had no significant changes before and after stimulation. Pathological analysis also found that TLR4^-/-mice were able to resist LPS damage to kidney tissue.Conclusion: TLR4 gene knockout mouse model was successfully constructed using CRISPR / Cas9 technology. The deletion of TLR4 can reduce the response of IL1β, IL6, MyD88, iNOS and TNFa inflammatory factors to LPS stimulation, inhibit LPS-induced inflammatory response and tissue damage.