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

doi:10.13523/j.cb.2001010

China Biotechnology

2020, Vol. 40

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

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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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

Received: 02 January 2020 Published: 23 June 2020

ZTFLH:

Q291

Corresponding Authors: Jian FEI,Ru-ling SHEN E-mail: alieen_shen@163.com

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	Yang GUO
	Ying-han WAN
	Jue WANG
	Hui GONG
	Yu ZHOU
	Lei CI
	Zhi-peng WAN
	Rui-lin SUN
	Jian FEI
	Ru-ling SHEN

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.

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

Table 1 sgRNA sequence information

Table 2 Oligonucleotide chain sequence information

Table 3 Primer sequence information

Table 4 RT-PCR primer sequences

Fig.1 Knockout strategy of TLR4^-/- mice

Fig.2 Genotype results of TLR4 gene knockout (a) Genotyping strategy of TLR4 gene knockout mouse (b) F1 generation of TLR4 gene knockout mouse genotyping representative result: wild type mouse PCR product has only a band of 1 491bp, TLR4 gene knockout heterozygotes In addition to the wild type 1 491bp band, PCR product has another 674 bp band (c) The genotyping representative result of the offspring mice: Wild type mice with a 1 491bp band, the homozygous mice with a 674bp band; the heterozygous mice with two bands of 1 491bp and 674bp, respectively. WT: wild type; HE: heterozygous; HO: homozygous; Con: wild type control; M: 1kb DNA marker

Table 5 The sequence information of TLR4 wild type and two knockout genotype

Fig.3 Analysis of relative mRNA expression level in TLR4 and its targets TLR4 (a) and IL1b、iNOS、MyD88、IL6、TNFα (b)were determined by Real-time PCR in WT and TLR4^-/- mice LPS induced

Fig.4 Expression of iNOS, TNF-α and IL-6 in mice induced by LPS before and after (a) Expression of iNOS in liver used by Western-blot:1-WT;2-WT by LPS induced;3-TLR4 ^-/-;4-TLR4^-/- by LPS induced, n=3 (b) Level of serum TNF-α and IL-6 induced by LPS before and after using ELISA,NS means not be detected

Fig.5 The function of kidney in mice induced by LPS before and after (a) Serum level of Urea and Cre,n=3(^* P<0.05, ^*** P<0.001) (b) LPS induced Kidney injury in WT and TLR4^-/- mice,black arrows mean brushfringe of renal tubule


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