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                        血液細胞特異標記的轉基因斑馬魚

                        作者:zhangyun 發布時間:2020/7/6 12:00:00

                               斑馬魚具有研究血液系統的天然優勢:體小,繁殖力強,胚胎體外發育,胚胎透明,易于活體觀察,斑馬魚造血系統相關的轉錄因子同人類具有高度的同源性,因此斑馬魚已經成為研究體內造血系統發育、功能及相關疾病的理想選擇。


                               斑馬魚造血過程分為兩個階段:首先是原始造血。11hpf,源于中胚層的原始造血細胞主要產生紅系和髓系前體細胞,這些細胞可以進一步分化為紅系和髓系。30hpf,斑馬魚開始定向造血。斑馬魚的背部主動脈產生造血干細胞,并遷移至尾部造血組織。一部分遷移至胸腺分化成T淋巴細胞,另一部分遷移至腎臟中保持自我更新和分化生各類血細胞,如,紅系、淋系、中性粒細胞系等( 1 [1]


                        1 斑馬魚血液系統發育[2]


                               得益于各種特異性表達熒光蛋白的轉基因斑馬魚品系,幫助我們標記目標組織和細胞,可以清晰直觀地觀察到活體特異細胞的動態生物學過程。國家斑馬魚資源中心現有各類研究用品系1400多種,其中轉基因品系200多種, 本文為大家推薦其中分別標記紅細胞、血小板及免疫細胞(中性粒細胞、巨噬細胞、T細胞)等血液系統的標記品系


                               標記紅細胞(Erythrocytes)的品系首推CZ64 (sd2Tg,Tg(gata1a:DsRed))2,是紅細胞標記基因gata1a的啟動子驅動DsRed表達,在紅細胞中特異表達熒光蛋白[3]。該品系中熒光蛋白表達與gata1a基因高度一致,利用該品系可以持續追蹤觀察紅細胞。gata1a編碼的GATA結合蛋白1aGATAbinding protein 1a)具有序列特異性雙鏈DNA結合及轉錄活性,參與造血、基因表達調控、髓樣細胞分化等過程。在人類中,該基因的同源基因與唐氏綜合征(Down syndrome)、X-連鎖血小板減少癥伴β-地中海貧血(X-linked thrombocytopeniawith beta-thalassemia)、β-地中海貧血(beta thalassemia)和多發性骨髓癌(multiple bone marrow cancer)有關[4-6]。中心也保藏有gata1a基因的突變品系CZ67(gata1am651/+)[7]


                        圖2 CZ64 (sd2Tg,Tg(gata1a:DsRed))


                        標記血小板的品系,CZ60 (la2Tg,Tg(-6.0itga2b:EGFP))圖3是整合素α2bitga2b)的啟動子驅動EGFP在造血干祖細胞(Hematopoietic stem and progenitor cellsHSPCs)和血小板(thrombocytes)特異表達的轉基因品系[8]itga2b參與血管發生(angiogenesis)、凝血(blood coagulation)和內皮細胞增殖(endothelial cell proliferation)。在人類中,該基因的同源基因與Glanzmann血栓形成癥(Glanzmann's thrombasthenia)、血小板相關出血性疾病16platelet-type bleeding disorder 16)、血小板減少癥(thrombocytopenia)和血管1型性血友病1von Willebrand's disease1)相關[9-11]


                        圖3 CZ60 (la2Tg,Tg(-6.0itga2b:EGFP))


                               接下來再介紹幾種免疫細胞的標記品系。CZ58 (nz117Tg, Tg(lyz:EGFP))圖4CZ59(nz50Tg, Tg(lyz:DsRed2))圖5是中性粒細胞(Neutrophils)標記基因lyz的啟動子驅動EGFPDsRed2的表達,在中性粒細胞中特異表達熒光蛋白[12] lyz編碼的溶菌酶(Lysozyme)是一種陽離子抗菌酶,能夠水解細菌細胞壁內的特異性連接。在人類中,溶菌酶在粒細胞和單核細胞中合成,與家族性內臟淀粉樣變性疾病(familial visceral amyloidosis)相關[13]


                        圖4 CZ58 (nz117Tg,Tg(lyz:EGFP))


                        圖5 CZ59 (nz50Tg,Tg(lyz:DsRed2))


                               目前中心服務量最大的轉基因品系是CZ98ihb20Tg, Tg(mpeg1:EGFP)圖6,該品系由mpeg1啟動子驅動綠色熒光蛋白GFP的表達。mpeg1是編碼巨噬細胞特異表達基因1Macrophage expressed gene 1),該基因最初被鑒定為一個表達嚴格限于人和小鼠中巨噬細胞的基因,隨后被用作哺乳動物系統和斑馬魚中巨噬細胞特異性的標記基因圖7mpeg1啟動子驅動的轉基因則在巨噬細胞中特異表達,用于特異標記巨噬細胞。研究者們常通過尾鰭再生等損失實驗,利用多種轉基因品系,分析巨噬細胞和中性粒細胞等血細胞的不同動態變化、相互作用等,為炎癥、感染和白細胞生物學領域提供了新的資源[14]。此外,中心也有同時標記中性粒細胞和巨噬細胞的品系CZ61(hkz04tTg, Tg(coro1a:EGFP))圖8[15]


                        圖6 CZ98(ihb20Tg,Tg(mpeg1:EGFP)


                        圖7 尾鰭再生實驗(紅色標記中性粒細胞,綠色標記巨噬細胞)[14]


                        圖8 CZ61(hkz04tTg,Tg(coro1a:EGFP))


                               CZ65(zf411Tg,Tg(rag2:DsRed))圖9CZ71(zdf8Tg,Tg(rag2:GFP))10是重組激活基因2rag2)的啟動子驅動DsRed2EGFPT細胞特異表達的轉基因品系[16]rag2基因最早在嗅覺感覺神經元(olfactory sensory neuronsOSNs)中特異表達,斑馬魚OSNs最先發現的表達rag基因的非淋巴組織。Rag2具有DNA結合活性及脫氧核糖核酸內切酶活性,參與免疫系統發育和蛋白質DNA復合物組裝等過程。在人類中,該基因的同源基因與網膜綜合征(Omenn syndrome)、嚴重聯合免疫缺陷(severe combined immunodeficiency)、常染色體隱性遺傳疾病(autosomal recessive)相關[17-19]


                        圖9 CZ65(zf411Tg,Tg(rag2:DsRed))


                        圖10 CZ71(zdf8Tg,Tg(rag2:GFP))


                               除了這些經典的血液細胞標記的品系,CZRC還保藏了另外一些相關的品系及突變體(點擊下表中提供的CZ編號鏈接)。有問題歡迎隨時致電或發郵件到CZRC工作郵箱查詢。


                        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)


                        參考文獻:

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