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Toll-like Receptor 4 (TLR4) Gene Knockout Mouse Model Construction and Preliminary Phenotypic Analysis |
GUO Yang1,3,WAN Ying-han2,3,WANG Jue2,3,GONG Hui2,3,ZHOU Yu4,CI Lei4,WAN Zhi-peng4,SUN Rui-lin4,FEI Jian1,3,4,*,SHEN Ru-ling2,3,*() |
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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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.
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Received: 02 January 2020
Published: 23 June 2020
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Corresponding Authors:
Jian FEI,Ru-ling SHEN
E-mail: alieen_shen@163.com
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Cite this article:
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.
URL:
https://manu60.magtech.com.cn/biotech/10.13523/j.cb.2001010 OR https://manu60.magtech.com.cn/biotech/Y2020/V40/I6/1
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