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Pd-1 Gene Knockout Mouse Model Construction and Preliminary Phenotype Verification |
GUO Yang1,CHEN Yan-juan1,LIU Yi-chen1,WANG Hai-jie1,WANG Cheng-ji1,WANG Jue1,WAN Ying-han1,ZHOU Yu2,XI Jun2,SHEN Ru-ling1,*() |
1 Shanghai Laboratory Animal Research Center, Shanghai 201203, China 2 Shanghai Model Organisms Center Inc., Shanghai 201318, China |
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Abstract Objective: Programmed cell death protein (PD-1) is a T cell immune checkpoint and an important target for tumor therapy. This article used CRISPR/Cas9 technology to repair the introduced mutations by non-homologous recombination, causing the frame shift of the gene protein reading frame and the loss of PD-1 function. Estabilishment of Pd-1 gene knockout mouse model provides the basis for in-depth exploration of Pd-1 gene function and mechanism. Methods: We designed and synthesized 2 pairs of sgRNA fragments for exons 2-4 of the Pd-1 gene, and transcribed them in vitro together with the Cas9 fragments encoding them. The two mRNAs were mixed into C57BL/6 mouse fertilized eggs by microinjection. F0 generation mice were obtained by PCR product sequencing and then mated with wild-type C57BL/6 mice to obtain F1 generation heterozygous mice. F1 generation mice were intercoursed to obtain F2 generation homozygous mouse strains (Pd-1-/-). After it was stimulated with concanavalin (ConA), PD-1 in Pd-1-/- mice was detected by Real-Time fluorescent quantitative PCR and flow cytometry at the mRNA and protein levels, respectively. The expression levels of IL-6, IFN-γ, IL12/IL23 and TNF-α in the serum of Pd-1-/- mice were detected by the ELISA method, and the mechanism of Pd-1 pathway in the regulation of T cell response and its countermeasures were preliminarily analyzed. Results: PCR and sequencing results showed that exons 2-4 of the Pd-1 gene in the mouse genome were successfully knocked out; Real-Time PCR experiments and flow cytometry results showed that the expression of PD-1 was significantly reduced in Pd-1-/- spleen, mesenteric lymph nodes, thymus and blood tissues compared with wild-type mice; the double-antibody sandwich ELISA test results showed that the expression of serum IL-6 and IFN-γ is up-regulated stimulated by ConA after Pd-1 gene was knocked out. Conclusion: The Pd-1 gene knockout mouse model has been successfully constructed. Preliminary analysis shows that Pd-1 deletion can upregulate the response of IL-6 and IFN-γ to ConA stimulation, increase the inflammatory response caused by ConA, and provide a new mouse model for the study of Pd-1 in vivo gene function and research ideas.
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Received: 08 June 2021
Published: 08 November 2021
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Corresponding Authors:
Ru-ling SHEN
E-mail: shenruling@slarc.org.cn
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Cite this article:
GUO Yang,CHEN Yan-juan,LIU Yi-chen,WANG Hai-jie,WANG Cheng-ji,WANG Jue,WAN Ying-han,ZHOU Yu,XI Jun,SHEN Ru-ling. Pd-1 Gene Knockout Mouse Model Construction and Preliminary Phenotype Verification. China Biotechnology, 2021, 41(10): 1-11.
URL:
https://manu60.magtech.com.cn/biotech/10.13523/j.cb.2106013 OR https://manu60.magtech.com.cn/biotech/Y2021/V41/I10/1
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