<i>Pd-1</i> Gene Knockout Mouse Model Construction and Preliminary Phenotype Verification

doi:10.13523/j.cb.2106013

China Biotechnology

2021, Vol. 41

Issue (10): 1-11 DOI: 10.13523/j.cb.2106013

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-ling^1,^*(

)

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.

Key words： PD-1 PD-L1 CRISPR/Cas9 IL-6 IFN-γ

Received: 08 June 2021 Published: 08 November 2021

ZTFLH:

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Corresponding Authors: Ru-ling SHEN E-mail: shenruling@slarc.org.cn

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	Yang GUO
	Yan-juan CHEN
	Yi-chen LIU
	Hai-jie WANG
	Cheng-ji WANG
	Jue WANG
	Ying-han WAN
	Yu ZHOU
	Jun XI
	Ru-ling SHEN

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.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.2106013 OR https://manu60.magtech.com.cn/biotech/Y2021/V41/I10/1

Table 1 sgRNA sequence information

Table 2 Oligonucleotide chain sequence information

Table 3 Primer sequence information

Table 4 RT-PCR primer sequences

Fig.1 Construction strategy of Pd-1 knockout mice

Fig.2 Genotype results of Pd-1 gene knockout (a) Genotyping strategy of F0/1 Pd-1 knockout mouse (b) Pd-1^-/- mouse genotype identification electrophoresis results:wild type mouse PCR product has only a band of 2 032bp, Pd-1^-/+ mice with two bands of 2 032bp and 341bp, respectively, and Pd-1^-/- mice with single 341bp band.WT-wild type; HE- Pd-1^-/+; HO- Pd-1^-/-;M-GeneRuler 1 kb DNA

Table 5 The sequence information of Pd-1^-/- and WT mice

Fig.3 PD-1 expression using real-time PCR Statistics of PD-1 mRNA expression in the spleen, mesenteric lymph node, thymus and bone marrow of Pd-1^-/- mice (^**** P< 0.000 1)

Fig.4 PD-1 expressed in CD4⁺ T and CD8⁺ T cells using FACS before and after ConA stimulation (a) PD-1 expression in spleen (b) PD-1 expression in mesenteric lymph node (c) PD-1 expression in thymus (d) PD-1 expression in blood; WT-wild-type mice, Pd-1^-/--Pd-1^-/- mice, WT by ConA-wild type mice after stimulation, Pd-1^-/- by ConA -Pd-1^-/- mice after stimulation, ^* P<0.05, ^** P<0.01, ^**** P<0.000 1

Fig.5 Schematic diagram of scatter points of PD-1 expression in FACS detection before ConA stimulation (a) PD-1 expression in CD4⁺ or CD8⁺ T cells in spleen (b) PD-1 expression in CD4⁺ or CD8⁺ T cells in mesenteric lymph node (c) PD-1 expression in CD4⁺ or CD8⁺ T cells in thymus (d) PD-1 expression in CD4⁺ or CD8⁺ T cells in blood; 3 mice per group;WT-wild type mice, Pd-1^-/--Pd-1^-/- mice; FITC labeled fluorescent antibody anti-mouse CD4, PE labeled fluorescent antibody anti-mouse CD8, APC labeled fluorescent antibody anti-mouse CD279 detection

Fig.6 Schematic diagram of scatter points of PD-1 expression in FACS detection after ConA stimulation (a) PD-1 expression in CD4⁺ or CD8⁺ T cells in spleen (b) PD-1 expression in CD4⁺ or CD8⁺ T cells in mesenteric lymph node (c) PD-1 expression in CD4⁺ or CD8⁺ T cells in thymus (d) PD-1 expression in CD4⁺ or CD8⁺ T cells in blood; 3 mice per group; WT-wild type mice, Pd-1^-/--Pd-1^-/- mice; FITC labeled fluorescent antibody anti-mouse CD4, PE labeled fluorescent antibody anti-mouse CD8, APC labeled fluorescent antibody anti-mouse CD279 detection

Fig.7 IL-6, IFN-γ, IL12/IL23 and TNF-α level in serum of Pd-1^-/- mice before and after ConA stimulation (a) IL-6 level in serum before and after ConA stimulation (b) IFN-γ level in serum before and after ConA stimulation (c) IL12/IL23 level in serum before and after ConA stimulation (d) TNF-α level in serum before and after ConA stimulation; 5 mice per group: WT-wild type mice, Pd-1^-/--Pd-1^-/-mice, ConA-after ConA stimulation, ND-value is not detected, ^* P<0.05,^**** P<0.000 1


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