Construction and Preliminary Phenotypic Verification of PD-L1 Knockout Mice

doi:10.13523/j.cb.20191206

China Biotechnology

2019, Vol. 39

Issue (12): 42-49 DOI: 10.13523/j.cb.20191206

Orginal Article

Construction and Preliminary Phenotypic Verification of PD-L1 Knockout Mice

WAN Ying-han¹,CI Lei^3,^*,WANG Jue¹,GONG Hui¹,LI Jun¹,DONG Ru¹,SUN Rui-lin³,FEI Jian^2,³,SHEN Ru-ling^1,^**(

)

1 Joint Laboratory of Model Biology and Comparative Medical Research,Shanghai Laboratory Animal Research Center,Shanghai 201210,China
2 School of Life Science and Technology, Tongji University, Shanghai 200092, China
3 Shanghai Model Organisms Center,Shanghai 201318,China

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Abstract

Objective: Programmed death ligand-1 (PD-L1) is an important factor in the immunoregulatory pathway and is one of the important targets in anti-tumor immunotherapy. The PD-L1 knockout mouse model was successfully constructed using CRISPR/Cas9 technology, and its phenotype was initially analyzed.Methods: Cas9 and sgRNA vectors were constructed and transcribed to obtain RNA. RNA was injected into C57BL/6 mouse fertilized eggs by microinjection, and F₀ generation positive mice were obtained. F₀ generation mice were mated with wild type C57BL/6 mice to obtain F₁ generation heterozygous mice, and F₂ generation homozygous mouse strains were obtained by self-crossing of F₁ generation mice. Subsequently, the expression of PD-L1 gene at the mRNA and protein levels was detected by Real-Time PCR and flow cytometry, respectively.Results: Real-Time PCR and flow cytometry showed that the PD-L1 mRNA expression level and protein expression on PD-L1 homozygous mice with only background signals were significantly decreased, compared with wild-type C57 mice. It was confirmed that the PD-L1 knockout mouse strain was successfully constructed, which provided a new mouse model for PD-L1 gene function research in vivo.

Key words： PD-L1 PD-1 CRISPR/Cas9 Gene knockout

Received: 10 May 2019 Published: 15 January 2020

ZTFLH:

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

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	Ying-han WAN
	Lei CI
	Jue WANG
	Hui GONG
	Jun LI
	Ru DONG
	Rui-lin SUN
	Jian FEI
	Ru-ling SHEN

Cite this article:

WAN Ying-han,CI Lei,WANG Jue,GONG Hui,LI Jun,DONG Ru,SUN Rui-lin,FEI Jian,SHEN Ru-ling. Construction and Preliminary Phenotypic Verification of PD-L1 Knockout Mice. China Biotechnology, 2019, 39(12): 42-49.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20191206 OR https://manu60.magtech.com.cn/biotech/Y2019/V39/I12/42

Fig.1 Construction strategy of PD-L1 knockout mice Cas9/sgRNA complex recognized the target sequence in PD-L1 gene and cleaved off exon3, 4, 5 of the gene

Fig.2 F₀ generation mouse genotype identification (a) Identification strategy of F₀ PD-L1 knockout mice (b) F₀ generation mouse genotype identification electrophoresis results

Table 1 F₀ generation mouse sequence mutation

Fig.3 F₂ generation mouse genotype identification (a) Identification strategy of F₂ PD-L1 knockout mice (b)F₂ generation mouse genotype identification electrophoresis results The primer pair P3/P4 amplify a 642bp wild type band for the wild type gene; the primer pair P5/P6 amplify the 1 026bp band for knockout positive pattern. The wild type mouse (WT) can only amplify the 642bp product by the P3/P4 primer pair; the heterozygous mouse (HE) P3/P4 primer pair and the P5/P6 primer pair can amplify the 642bp and 1 026bp products, respectively; the homozygous mouse (HO) can only amplify a 1 026bp product by P5/P6 primer. M:DL2000 DNA marker

Fig.4 Detection of relative expression levels of PD-L1 mRNA in mouse tissues of different genotype (1)Expression of PD-L1 gene in each major tissue (b) Detection of relative expression of PD-L1 mRNA in heart, spleen and peritoneal macrophages of each genotype mouse (c) Electrophoresis identification results WT:Wild type mice;HE:Heterozygous mice;HO:Homozygous mice;3 mice per group, each experiment was repeated three times (*** P<0.001)

Fig.5 Flow cytometry analysis of PD-L1 expression in peritoneal macrophages and spleen cells of PD-L1 knockout mice (a) Schematic diagram of PD-L1 expression in peritoneal macrophages and spleen cells of different genotype mice; PE-labeled fluorescent antibody anti-CD274 (b)Histogram of PD-L1 percentage in peritoneal macrophages and spleen cells of different genotype mice; WT-wild type mice, HE-heterozygous mice, HO-homozygous mice; 3 mice per group, each experiment was repeated three times (*** P<0.001)


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