Toll样受体4(<i>TLR4</i>)基因剔除小鼠构建及初步表型分析

doi:10.13523/j.cb.2001010

中国生物工程杂志

2020, Vol. 40

Issue (6): 1-9 DOI: 10.13523/j.cb.2001010

研究报告

Toll样受体4(TLR4)基因剔除小鼠构建及初步表型分析

郭洋^1,³,万颖寒^2,³,王珏^2,³,龚慧^2,³,周宇⁴,慈磊⁴,万志鹏⁴,孙瑞林⁴,费俭^1,^3,^4,^*,沈如凌^2,^3,^*(

)

1 同济大学生命科学与技术学院上海 200092
2 上海实验动物研究中心上海 201203
3 模式生物及比较医学联合实验室上海实验动物研究中心同济大学生命科学与技术学院上海 201203
4 上海市模式动物工程技术研究中心上海 201318

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

GUO Yang^1,³,WAN Ying-han^2,³,WANG Jue^2,³,GONG Hui^2,³,ZHOU Yu⁴,CI Lei⁴,WAN Zhi-peng⁴,SUN Rui-lin⁴,FEI Jian^1,^3,^4,^*,SHEN Ru-ling^2,^3,^*(

)

1 School of Life Science and Technology, Tongji University, Shanghai 200092, China
2 Shanghai Laboratory Animal Research Center,Shanghai 201203, China
3 Joint Laboratory of Model Biology and Comparative Medical Research, Shanghai Laboratory Animal Research Center,Shanghai 201203,China
4 Shanghai Model Organisms Center Inc., Shanghai 201318, China

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摘要：

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。

关键词： Toll样受体4(TLR4); CRISPR/Cas9系统; 基因敲除; LPS

Abstract:

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.

Key words: TLR4 CRISPR/Cas9 gene Knock-out LPS

收稿日期: 2020-01-02 出版日期: 2020-06-23

ZTFLH:

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通讯作者: 费俭,沈如凌 E-mail: alieen_shen@163.com

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	郭洋
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	万志鹏
	孙瑞林
	费俭
	沈如凌

引用本文:

郭洋,万颖寒,王珏,龚慧,周宇,慈磊,万志鹏,孙瑞林,费俭,沈如凌. Toll样受体4(TLR4)基因剔除小鼠构建及初步表型分析[J]. 中国生物工程杂志, 2020, 40(6): 1-9.

GUO Yang,WAN Ying-han,WANG Jue,GONG Hui,ZHOU Yu,CI Lei,WAN Zhi-peng,SUN Rui-lin,FEI Jian,SHEN Ru-ling. Toll-like Receptor 4 (TLR4) Gene Knockout Mouse Model Construction and Preliminary Phenotypic Analysis. China Biotechnology, 2020, 40(6): 1-9.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2001010 或 https://manu60.magtech.com.cn/biotech/CN/Y2020/V40/I6/1

表1 sgRNA序列信息

表2 寡核苷酸链序列信息

表3 引物序列信息

表4 RT-PCR引物序列

图1 TLR4-/-小鼠基因敲除策略

图2 TLR4基因敲除小鼠基因型鉴定结果

表5 TLR4野生型和两种不同敲除类型的序列信息

图3 TLR4-/-小鼠TLR4及下游调控基因mRNA相对表达水平检测

图4 LPS刺激前后TLR4-/-小鼠iNOS,TNF-α和IL-6表达检测

图5 LPS给药刺激前后TLR4-/-小鼠肾功能情况

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