<i>Pd-1</i>基因敲除小鼠构建及初步表型验证

doi:10.13523/j.cb.2106013

中国生物工程杂志

2021, Vol. 41

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

研究报告

Pd-1基因敲除小鼠构建及初步表型验证

郭洋¹,陈艳娟¹,刘怡辰¹,王海杰¹,王成稷¹,王珏¹,万颖寒¹,周宇²,奚骏²,沈如凌^1,^*(

)

1 上海实验动物研究中心上海 201203
2 上海市模式动物工程技术研究中心上海 201318

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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摘要：

目的:细胞程序性死亡蛋白(programmed death ligand-1,PD-1)是机体T细胞的免疫检查点,也是肿瘤治疗的重要靶点。采用CRISPR/Cas9技术,利用非同源重组修复引入突变的方式,使基因蛋白读码框移码造成PD-1功能缺失,建立Pd-1基因敲除小鼠模型,为深入探究Pd-1基因功能及作用机制提供基础。方法:针对Pd-1基因2-4号外显子设计并合成2对sgRNA片段,与编码Cas9片段共同体外转录,通过受精卵显微注射方法将两者mRNA混合注射到C57BL/6小鼠受精卵中,经PCR产物测序鉴定获得F0代小鼠,之后与野生型C57BL/6小鼠交配获得F1代杂合子小鼠,F1代小鼠自交即获得F2代纯合子小鼠品系(Pd-1^-/-)。刀豆蛋白(concanavalin A,ConA)刺激Pd-1^-/-小鼠后,通过实时荧光定量PCR和流式细胞技术在mRNA和蛋白水平上分别检测Pd-1^-/-小鼠中Pd-1基因在转录和翻译过程中的表达情况,并通过ELISA方法检测Pd-1^-/-小鼠血清中IL-6、IFN-γ、IL12/IL23及TNF-α等因子的表达水平,初步分析Pd-1通路在T细胞反应调控中的作用机制及对免疫刺激的响应情况。结果:PCR及测序结果表明在小鼠基因组中Pd-1基因2-4号外显子被成功敲除;Real-Time PCR实验和流式检测结果显示:与野生型小鼠相比,Pd-1^-/-小鼠脾、肠系膜淋巴结、胸腺和血液各组织中Pd-1表达水平均显著降低;双抗夹心ELISA测定结果显示:Pd-1敲除后经ConA刺激,血清中IL-6和IFN-γ表达上调。结论:成功构建Pd-1基因敲除小鼠模型。Pd-1缺失能够上调IL-6和IFN-γ对ConA刺激的响应,增加ConA引起的炎症反应,为Pd-1的体内基因功能研究提供了新的小鼠模型和研究思路。

关键词： Pd-1; PD-L1; CRISPR/Cas9; IL-6; IFN-γ

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

收稿日期: 2021-06-08 出版日期: 2021-11-08

ZTFLH:

Q819

通讯作者: 沈如凌 E-mail: shenruling@slarc.org.cn

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	郭洋
	陈艳娟
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	王海杰
	王成稷
	王珏
	万颖寒
	周宇
	奚骏
	沈如凌

引用本文:

郭洋,陈艳娟,刘怡辰,王海杰,王成稷,王珏,万颖寒,周宇,奚骏,沈如凌. Pd-1基因敲除小鼠构建及初步表型验证[J]. 中国生物工程杂志, 2021, 41(10): 1-11.

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.

链接本文:

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

表1 sgRNA序列信息

表2 寡核苷酸链序列信息

表3 引物序列信息

表4 RT-PCR引物序列

图1 Pd-1基因敲除小鼠构建策略

图2 Pd-1-/-基因型鉴定

表5 Pd-1-/-和WT小鼠的序列信息

图3 Real-Time PCR检测PD-1 mRNA表达情况

图4 流式检测ConA刺激前后PD-1 在CD4+ T细胞和CD8+ T细胞上表达情况

图5 ConA刺激前流式检测PD-1表达情况散点示意图

图6 经ConA刺激后流式检测PD-1表达情况散点示意图

图7 ConA刺激前后Pd-1-/-小鼠血清中IL-6、IFN-γ、IL12/IL23和TNF-α的表达情况

[1]	Yamazaki T, Akiba H, Iwai H, et al. Expression of programmed death 1 ligands by murine T cells and APC. Journal of Immunology, 2002, 169(10): 5538-5545. doi: 10.4049/jimmunol.169.10.5538
[2]	Nishimura H, Agata Y, Kawasaki A, et al. Developmentally regulated expression of the PD-1 protein on the surface of double-negative(CD4-CD8-) thymocytes. International Immunology, 1996, 8(5): 773-780. pmid: 8671666
[3]	Nishimura H, Honjo T, Minato N. Facilitation of β selection and modification of positive selection in the Thymus of Pd-1-deficient mice. Journal of Experimental Medicine, 2000, 191(5): 891-898. doi: 10.1084/jem.191.5.891
[4]	Zhang X W, Schwartz J C D, Guo X L, et al. Structural and functional analysis of the costimulatory receptor programmed death-1. Immunity, 2004, 20(3): 337-347. doi: 10.1016/S1074-7613(04)00051-2
[5]	Ahmadzadeh M, Johnson L A, Heemskerk B, et al. Tumor antigen-specific CD8 T cells infiltrating the tumor express high levels of PD-1 and are functionally impaired. Blood, 2009, 114(8): 1537-1544. doi: 10.1182/blood-2008-12-195792 pmid: 19423728
[6]	Latchman Y, Wood C R, Chernova T, et al. PD-L2 is a second ligand for PD-1 and inhibits T cell activation. Nature Immunology, 2001, 2(3): 261-268. pmid: 11224527
[7]	Keir M E, Butte M J, Freeman G J, et al. PD-1 and its ligands in tolerance and immunity. Annual Review of Immunology, 2008, 26: 677-704. doi: 10.1146/immunol.2008.26.issue-1
[8]	Cho H Y, Choi E K, Lee S W, et al. Programmed death-1 receptor negatively regulates LPS-mediated IL-12 production and differentiation of murine macrophage RAW264.7 cells. Immunology Letters, 2009, 127(1): 39-47. doi: 10.1016/j.imlet.2009.08.011
[9]	Said E A, Dupuy F P, Trautmann L, et al. Programmed death-1-induced interleukin-10 production by monocytes impairs CD4+ T cell activation during HIV infection. Nature Medicine, 2010, 16(4): 452-459. doi: 10.1038/nm.2106
[10]	Xiao G, Deng A Q, Liu H F, et al. Activator protein 1 suppresses antitumor T-cell function via the induction of programmed death 1. PNAS, 2012, 109(38): 15419-15424. doi: 10.1073/pnas.1206370109
[11]	Salmaninejad A, Khoramshahi V, Azani A, et al. PD-1 and cancer: molecular mechanisms and polymorphisms. Immunogenetics, 2018, 70(2): 73-86. doi: 10.1007/s00251-017-1015-5 pmid: 28642997
[12]	Okazaki T, Tanaka Y, Nishio R, et al. Autoantibodies against cardiac troponin I are responsible for dilated cardiomyopathy in PD-1-deficient mice. Nature Medicine, 2003, 9(12): 1477-1483. pmid: 14595408
[13]	Wang J, Okazaki I M, Yoshida T, et al. PD-1 deficiency results in the development of fatal myocarditis in MRL mice. International Immunology, 2010, 22(6): 443-452. doi: 10.1093/intimm/dxq026
[14]	Kasprowicz V, Schulze zur Wiesch J, Kuntzen T, et al. High level of PD-1 expression on hepatitis C virus (HCV)-specific CD8+ and CD4+ T cells during acute HCV infection, irrespective of clinical outcome. Journal of Virology, 2008, 82(6): 3154-3160. pmid: 18160439
[15]	Nakamoto N, Kaplan D E, Coleclough J, et al. Functional restoration of HCV-specific CD8 T cells by PD-1 blockade is defined by PD-1 expression and compartmentalization. Gastroenterology, 2008, 134(7): 1927-37, 1937.e1-2. doi: 10.1053/j.gastro.2008.02.033
[16]	Borkner L, Kaiser A, Kasteele W, et al. RNA interference targeting programmed death receptor-1 improves immune functions of tumor-specific T cells. Cancer Immunology, Immunotherapy, 2010, 59(8): 1173-1183. doi: 10.1007/s00262-010-0842-0
[17]	Hino R, Kabashima K, Kato Y, et al. Tumor cell expression of programmed cell death-1 ligand 1 is a prognostic factor for malignant melanoma. Cancer, 2010, 116(7): 1757-1766. doi: 10.1002/cncr.v116:7
[18]	Iwai Y, Hamanishi J, Chamoto K, et al. Cancer immunotherapies targeting the PD-1 signaling pathway. Journal of Biomedical Science, 2017, 24(1): 26. doi: 10.1186/s12929-017-0329-9
[19]	万颖寒, 慈磊, 王珏, 等. 淋巴细胞活化基因-3敲除(Lag-3-/-)小鼠构建及初步表型分析. 中国实验动物学报, 2020, 28(1): 49-57.
	Wan Y H, Ci L, Wang J, et al. Construction and preliminary phenotypic analysis of Lag-3-/- mice. Acta Laboratorium Animalis Scientia Sinica, 2020, 28(1): 49-57.
[20]	Hoejberg L, Bastholt L, Schmidt H. Interleukin-6 and melanoma. Melanoma Research, 2012, 22(5): 327-333. doi: 10.1097/CMR.0b013e3283543d72 pmid: 22713796
[21]	Wang F, Xu J, Zhu Q, et al. Downregulation of IFNG in CD4(+) T cells in lung cancer through hypermethylation: a possible mechanism of tumor-induced immunosuppression. PLoS One, 2013, 8(11): e79064. doi: 10.1371/journal.pone.0079064
[22]	Tanaka T, Narazaki M, Kishimoto T. IL-6 in inflammation, immunity, and disease. Cold Spring Harbor Perspectives in Biology, 2014, 6(10): a016295. doi: 10.1101/cshperspect.a016295
[23]	Rossi J F, Lu Z Y, Jourdan M, et al. Interleukin-6 as a therapeutic target. Clinical Cancer Research, 2015, 21(6): 1248-1257. doi: 10.1158/1078-0432.CCR-14-2291
[24]	Tsukamoto H, Fujieda K, Hirayama M, et al. Soluble IL6R expressed by myeloid cells reduces tumor-specific Th1 differentiation and drives tumor progression. Cancer Research, 2017, 77(9): 2279-2291. doi: 10.1158/0008-5472.CAN-16-2446 pmid: 28235765
[25]	Rotz S J, Leino D, Szabo S, et al. Severe cytokine release syndrome in a patient receiving PD-1-directed therapy. Pediatric Blood & Cancer, 2017, 64(12): e26642. doi: 10.1002/pbc.26642
[26]	Grupp S A, Kalos M, Barrett D, et al. Chimeric antigen receptor-modified T cells for acute lymphoid leukemia. The New England Journal of Medicine, 2013, 368(16): 1509-1518. doi: 10.1056/NEJMoa1215134
[27]	Tanaka R, Okiyama N, Okune M, et al. Serum level of interleukin-6 is increased in nivolumab-associated psoriasiform dermatitis and tumor necrosis factor-α is a biomarker of nivolumab recativity. Journal of Dermatological Science, 2017, 86(1): 71-73. doi: S0923-1811(16)31098-2 pmid: 28069323
[28]	Leplina O, Smetanenko E, Tikhonova M, et al. Binding of the placental growth factor to VEGF receptor type 1 modulates human T cell functions. Journal of Leukocyte Biology, 2020, 108(3): 1013-1024. doi: 10.1002/jlb.v108.3
[29]	Lee S J, Jang B C, Lee S W, et al. Interferon regulatory factor-1 is prerequisite to the constitutive expression and IFN-γ-induced upregulation of B7-H1 (CD274). FEBS Letters, 2006, 580(3): 755-762. doi: 10.1016/j.febslet.2005.12.093
[30]	Oestreich K J, Yoon H, Ahmed R, et al. NFATc1 regulates PD-1 expression upon T cell activation. Journal of Immunology, 2008, 181(7): 4832-4839. pmid: 18802087
[31]	Melssen M, Slingluff C L Jr. Vaccines targeting helper T cells for cancer immunotherapy. Current Opinion in Immunology, 2017, 47: 85-92. doi: S0952-7915(17)30050-X pmid: 28755541
[32]	Spitzer M H, Carmi Y, Reticker-Flynn N E, et al. Systemic immunity is required for effective cancer immunotherapy. Cell, 2017, 168(3): 487-502.e15. doi: 10.1016/j.cell.2016.12.022
[33]	Li J, Jie H B, Lei Y, et al. PD-1/SHP-2 inhibits Tc1/Th1 phenotypic responses and the activation of T cells in the tumor microenvironment. Cancer Research, 2015, 75(3): 508-518. doi: 10.1158/0008-5472.CAN-14-1215
[34]	McAlees J W, Lajoie S, Dienger K, et al. Differential control of CD4(+) T-cell subsets by the PD-1/PD-L1 axis in a mouse model of allergic asthma. European Journal of Immunology, 2015, 45(4): 1019-1029. doi: 10.1002/eji.201444778 pmid: 25630305
[35]	Karachaliou N, Gonzalez-Cao M, Crespo G, et al. Interferon gamma, an important marker of response to immune checkpoint blockade in non-small cell lung cancer and melanoma patients. Therapeutic Advances in Medical Oncology, 2018, 10: 1758834017749748.
[36]	Kochupurakkal B S, Wang Z C, Hua T, et al. RelA-induced interferon response negatively regulates proliferation. PLoS One, 2015, 10(10): e0140243. doi: 10.1371/journal.pone.0140243
[37]	Gordon S R, Maute R L, Dulken B W, et al. PD-1 expression by tumour-associated macrophages inhibits phagocytosis and tumour immunity. Nature, 2017, 545(7655): 495-499. doi: 10.1038/nature22396

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