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血液细胞特异标记的转基因斑马鱼

作者:zhangyun 发布时间:2020/7/6 12:00:00

       斑马鱼具有研究血液系统的天然优势:体小,繁殖力强,胚胎体外发育,胚胎透明,易于活体观察,斑马鱼造血系统相关的转录因子同人类具有高度的同源性,因此斑马鱼已经成为研究体内造血系统发育、功能及相关疾病的理想选择。


       斑马鱼造血过程分为两个阶段:首先是原始造血。11hpf,源于中胚层的原始造血细胞主要产生红系和髓系前体细胞,这些细胞可以进一步分化为红系和髓系。30hpf,斑马鱼开始定向造血。斑马鱼的背部主动脉产生造血干细胞,并迁移至尾部造血组织。一部分迁移至胸腺分化成T淋巴细胞,另一部分迁移至肾脏中保持自我更新和分化生各类血细胞,如,红系、淋系、中性粒细胞系等( 1 [1]


1 斑马鱼血液系统发育[2]


       得益于各种特异性表达荧光蛋白的转基因斑马鱼品系,帮助我们标记目标组织和细胞,可以清晰直观地观察到活体特异细胞的动态生物学过程。国家斑马鱼资源中心现有各类研究用品系1400多种,其中转基因品系200多种, 本文为大家推荐其中分别标记红细胞、血小板及免疫细胞(中性粒细胞、巨噬细胞、T细胞)等血液系统的标记品系


       标记红细胞(Erythrocytes)的品系首推CZ64 (sd2Tg,Tg(gata1a:DsRed))2,是红细胞标记基因gata1a的启动子驱动DsRed表达,在红细胞中特异表达荧光蛋白[3]。该品系中荧光蛋白表达与gata1a基因高度一致,利用该品系可以持续追踪观察红细胞。gata1a编码的GATA结合蛋白1aGATAbinding protein 1a)具有序列特异性双链DNA结合及转录活性,参与造血、基因表达调控、髓样细胞分化等过程。在人类中,该基因的同源基因与唐氏综合征(Down syndrome)、X-连锁血小板减少症伴β-地中海贫血(X-linked thrombocytopeniawith beta-thalassemia)、β-地中海贫血(beta thalassemia)和多发性骨髓癌(multiple bone marrow cancer)有关[4-6]。中心也保藏有gata1a基因的突变品系CZ67(gata1am651/+)[7]


图2 CZ64 (sd2Tg,Tg(gata1a:DsRed))


标记血小板的品系,CZ60 (la2Tg,Tg(-6.0itga2b:EGFP))图3是整合素α2bitga2b)的启动子驱动EGFP在造血干祖细胞(Hematopoietic stem and progenitor cellsHSPCs)和血小板(thrombocytes)特异表达的转基因品系[8]itga2b参与血管发生(angiogenesis)、凝血(blood coagulation)和内皮细胞增殖(endothelial cell proliferation)。在人类中,该基因的同源基因与Glanzmann血栓形成症(Glanzmann's thrombasthenia)、血小板相关出血性疾病16platelet-type bleeding disorder 16)、血小板减少症(thrombocytopenia)和血管1型性血友病1von Willebrand's disease1)相关[9-11]


图3 CZ60 (la2Tg,Tg(-6.0itga2b:EGFP))


       接下来再介绍几种免疫细胞的标记品系。CZ58 (nz117Tg, Tg(lyz:EGFP))图4CZ59(nz50Tg, Tg(lyz:DsRed2))图5是中性粒细胞(Neutrophils)标记基因lyz的启动子驱动EGFPDsRed2的表达,在中性粒细胞中特异表达荧光蛋白[12] lyz编码的溶菌酶(Lysozyme)是一种阳离子抗菌酶,能够水解细菌细胞壁内的特异性连接。在人类中,溶菌酶在粒细胞和单核细胞中合成,与家族性内脏淀粉样变性疾病(familial visceral amyloidosis)相关[13]


图4 CZ58 (nz117Tg,Tg(lyz:EGFP))


图5 CZ59 (nz50Tg,Tg(lyz:DsRed2))


       目前中心服务量最大的转基因品系是CZ98ihb20Tg, Tg(mpeg1:EGFP)图6,该品系由mpeg1启动子驱动绿色荧光蛋白GFP的表达。mpeg1是编码巨噬细胞特异表达基因1Macrophage expressed gene 1),该基因最初被鉴定为一个表达严格限于人和小鼠中巨噬细胞的基因,随后被用作哺乳动物系统和斑马鱼中巨噬细胞特异性的标记基因图7mpeg1启动子驱动的转基因则在巨噬细胞中特异表达,用于特异标记巨噬细胞。研究者们常通过尾鳍再生等损失实验,利用多种转基因品系,分析巨噬细胞和中性粒细胞等血细胞的不同动态变化、相互作用等,为炎症、感染和白细胞生物学领域提供了新的资源[14]。此外,中心也有同时标记中性粒细胞和巨噬细胞的品系CZ61(hkz04tTg, Tg(coro1a:EGFP))图8[15]


图6 CZ98(ihb20Tg,Tg(mpeg1:EGFP)


图7 尾鳍再生实验(红色标记中性粒细胞,绿色标记巨噬细胞)[14]


图8 CZ61(hkz04tTg,Tg(coro1a:EGFP))


       CZ65(zf411Tg,Tg(rag2:DsRed))图9CZ71(zdf8Tg,Tg(rag2:GFP))10是重组激活基因2rag2)的启动子驱动DsRed2EGFPT细胞特异表达的转基因品系[16]rag2基因最早在嗅觉感觉神经元(olfactory sensory neuronsOSNs)中特异表达,斑马鱼OSNs最先发现的表达rag基因的非淋巴组织。Rag2具有DNA结合活性及脱氧核糖核酸内切酶活性,参与免疫系统发育和蛋白质DNA复合物组装等过程。在人类中,该基因的同源基因与网膜综合征(Omenn syndrome)、严重联合免疫缺陷(severe combined immunodeficiency)、常染色体隐性遗传疾病(autosomal recessive)相关[17-19]


图9 CZ65(zf411Tg,Tg(rag2:DsRed))


图10 CZ71(zdf8Tg,Tg(rag2:GFP))


       除了这些经典的血液细胞标记的品系,CZRC还保藏了另外一些相关的品系及突变体(点击下表中提供的CZ编号链接)。有问题欢迎随时致电或发邮件到CZRC工作邮箱查询。


CZ ID

Genotype

Construct

CZ13

rj30Tg/+

Tg(mpx:EGFP)

CZ58

nz117Tg/+

Tg(lyz:EGFP)

CZ59

nz50Tg/+

Tg(lyz:DsRed2)

CZ60

la2Tg/+

Tg(-6.0itga2b:EGFP)

CZ61

hkz04tTg/+

Tg(coro1a:EGFP)

CZ64

sd2Tg/+

Tg(gata1a:DsRed)

CZ65

zf411Tg/+

Tg(rag2:DsRed)

CZ71

zdf8Tg/+

Tg(rag2:EGFP)

CZ74

cz2Tg/+

Tg(lck:lck-EGFP)

CZ98

ihb20Tg/+

Tg2(mpeg1:EGFP)

CZ273

zf169Tg/+

TgPAC(myb:2xmyb-EGFP)


参考文献:

1. Traver D, Herbomel P, Patton EE, Murphey RD, Yoder JA, Litman GW, Catic A, Amemiya CT, Zon LI, Trede NS: The zebrafish as a model organism to study development of the immune system. Adv Immunol 2003, 81:253-330.
2. Jing L, Zon LI: Zebrafish as a model for normal and malignant hematopoiesis. Dis Model Mech 2011, 4(4):433-438.
3. Bresciani E, Confalonieri S, Cermenati S, Cimbro S, Foglia E, Beltrame M, Di Fiore PP, Cotelli F: Zebrafish numb and numblike are involved in primitive erythrocyte differentiation. PLoS One 2010, 5(12):e14296.
4. Ahmed M, Sternberg A, Hall G, Thomas A, Smith O, O'Marcaigh A, Wynn R, Stevens R, Addison M, King D et al: Natural history of GATA1 mutations in Down syndrome. Blood 2004, 103(7):2480-2489.
5. Yu C, Niakan KK, Matsushita M, Stamatoyannopoulos G, Orkin SH, Raskind WH: X-linked thrombocytopenia with thalassemia from a mutation in the amino finger of GATA-1 affecting DNA binding rather than FOG-1 interaction. Blood 2002, 100(6):2040-2045.
6. Tubman VN, Levine JE, Campagna DR, Monahan-Earley R, Dvorak AM, Neufeld EJ, Fleming MD: X-linked gray platelet syndrome due to a GATA1 Arg216Gln mutation. Blood 2007, 109(8):3297-3299.
7. Lyons SE, Lawson ND, Lei L, Bennett PE, Weinstein BM, Liu PP: A nonsense mutation in zebrafish gata1 causes the bloodless phenotype in vlad tepes. Proc Natl Acad Sci U S A 2002, 99(8):5454-5459.
8. Lin HF, Traver D, Zhu H, Dooley K, Paw BH, Zon LI, Handin RI: Analysis of thrombocyte development in CD41-GFP transgenic zebrafish. Blood 2005, 106(12):3803-3810.
9. Prandini MH, Denarier E, Frachet P, Uzan G, Marguerie G: Isolation of the human platelet glycoprotein IIb gene and characterization of the 5' flanking region. Biochem Biophys Res Commun 1988, 156(1):595-601.
10. Hardisty R, Pidard D, Cox A, Nokes T, Legrand C, Bouillot C, Pannocchia A, Heilmann E, Hourdille P, Bellucci S et al: A defect of platelet aggregation associated with an abnormal distribution of glycoprotein IIb-IIIa complexes within the platelet: the cause of a lifelong bleeding disorder. Blood 1992, 80(3):696-708.
11. Peyruchaud O, Nurden AT, Milet S, Macchi L, Pannochia A, Bray PF, Kieffer N, Bourre F: R to Q amino acid substitution in the GFFKR sequence of the cytoplasmic domain of the integrin IIb subunit in a patient with a Glanzmann's thrombasthenia-like syndrome. Blood 1998, 92(11):4178-4187.
12. Hall C, Flores MV, Storm T, Crosier K, Crosier P: The zebrafish lysozyme C promoter drives myeloid-specific expression in transgenic fish. BMC Dev Biol 2007, 7:42.
13. Yazaki M, Farrell SA, Benson MD: A novel lysozyme mutation Phe57Ile associated with hereditary renal amyloidosis. Kidney Int 2003, 63(5):1652-1657.
14. Ellett F, Pase L, Hayman JW, Andrianopoulos A, Lieschke GJ: mpeg1 promoter transgenes direct macrophage-lineage expression in zebrafish. Blood 2011, 117(4):e49-56.
15. Li L, Yan B, Shi YQ, Zhang WQ, Wen ZL: Live imaging reveals differing roles of macrophages and neutrophils during zebrafish tail fin regeneration. J Biol Chem 2012, 287(30):25353-25360.
16. Jessen JR, Jessen TN, Vogel SS, Lin S: Concurrent expression of recombination activating genes 1 and 2 in zebrafish olfactory sensory neurons. Genesis 2001, 29(4):156-162.
17. Schwarz K, Gauss GH, Ludwig L, Pannicke U, Li Z, Lindner D, Friedrich W, Seger RA, Hansen-Hagge TE, Desiderio S et al: RAG mutations in human B cell-negative SCID. Science 1996, 274(5284):97-99.
18. Marrella V, Poliani PL, Casati A, Rucci F, Frascoli L, Gougeon ML, Lemercier B, Bosticardo M, Ravanini M, Battaglia M et al: A hypomorphic R229Q Rag2 mouse mutant recapitulates human Omenn syndrome. J Clin Invest 2007, 117(5):1260-1269.
19. Corneo B, Moshous D, Gungor T, Wulffraat N, Philippet P, Le Deist FL, Fischer A, de Villartay JP: Identical mutations in RAG1 or RAG2 genes leading to defective V(D)J recombinase activity can cause either T-B-severe combined immune deficiency or Omenn syndrome. Blood 2001, 97(9):2772-2776.


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