CN110055277A - 一种肝脏特异性聚集巨噬细胞的转基因斑马鱼模型 - Google Patents
一种肝脏特异性聚集巨噬细胞的转基因斑马鱼模型 Download PDFInfo
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Abstract
本发明公开了一种肝脏特异性聚集巨噬细胞的转基因斑马鱼模型。该斑马鱼为Tg (fabp10a:il34)转基因的斑马鱼。本发明通过以下步骤实现:(1)斑马鱼养殖;(2)斑马鱼胚胎整体原位杂交和抗体染色;(3)激光扫描共聚焦荧光显微镜活体成像。本发明首次证实了在肝脏异位表达、过表达的白细胞介素34可以诱导巨噬细胞向肝脏富集,并运用活体显像技术提供了坚实而直接的证据。另外,本发明中Tg(fabp10a: il34;mpeg1:GFP)成鱼具有以下表型:①巨噬细胞数目仅特异性的在肝脏区域增高;②其他组织区域如尾部造血组织(CHT)区域巨噬细胞数目不变;③粒细胞数目不变。
Description
技术领域
本发明属于生物技术领域,具体涉及一种肝脏特异性聚集巨噬细胞的转基因斑马鱼模型。
背景技术
巨噬细胞是髓系细胞的一种,也是免疫系统的重要组成部分,在清除生物体中的外来物质(包括细胞碎片、微生物和癌细胞)过程中发挥着关键作用。在成年哺乳动物中,这些大型吞噬细胞存在于它们进行免疫监测的所有组织中,能够吞没、破坏受损伤组织,有助于启动康复过程。巨噬细胞可以根据其在不同组织中的位置分为库普弗细胞、小胶质细胞和朗格汉斯细胞等几种类型,但一般来说,它们都是单核吞噬细胞系统的一部分。
白细胞介素34(IL-34)是一类细胞因子,也是一种由多种细胞分泌的蛋白质,包括巨噬细胞、内皮细胞、神经元和成纤维细胞。IL-34广泛表达于脑、心、肝、脾、肾、结肠等多种组织,主要分布于皮肤和中枢神经系统(CNS)。在最近的研究中,IL-34被认为是巨噬细胞集落刺激因子受体(M-CSFR;又称CSF-1R)的另一种有功能的配体,并被认为参与巨噬细胞的增殖、分化和存活。除此之外,IL-34还被认为能诱导髓系细胞(THP-1和M2a巨噬细胞)迁移,这一过程主要通过Syndecan-1依赖的机制。此外,IL-34通过增加黏着斑激酶(FAK)和细胞外信号相关激酶1和2(ERK 1/2)的磷酸化水平,参与诱导巨噬细胞的浓度依赖性的趋化过程,但在体内缺乏IL-34诱导巨噬细胞聚集的直接证据,现有技术大多是在体外细胞系实验中进行验证,尚缺乏在体内实验中的证据,也无在体内环境中实时观察巨噬细胞聚集在肝脏的直接证据。
脂肪酸结合蛋白10a(fabp10a)是一种小蛋白质,对肝内长链脂肪酸的胞内结合和转运起重要作用。2.8kb的启动子序列可以有效地驱动GFP(绿色荧光蛋白)在肝脏中的表达,在受精后2天可首次观察到荧光,并可持续到成年期。
发明内容
本发明的目的在于克服现有技术的缺点与不足,提供一种肝脏特异性聚集巨噬细胞的转基因斑马鱼模型。
本发明的目的通过下述技术方案实现。
肝脏特异性聚集巨噬细胞的转基因斑马鱼模型,具体步骤为:
(1)通过聚合酶链式反应利用野生型斑马鱼基因组DNA得到斑马鱼肝脏细胞特异性启动子——脂肪酸转运蛋白10a,同时通过聚合酶链式反应利用野生型斑马鱼cDNA得到白细胞介素34基因,将这两部分片段利用DNA连接酶分段连接进入空载pBLK-sv40构建pBLK-fabp10a-il34-sv40质粒,利用显微注射技术将此质粒注射入已标记了的转基因家系Tg(mpeg1:GFP)(即利用巨噬细胞特异性的启动子——巨噬细胞表达1来驱动绿色荧光蛋白在巨噬细胞中表达的转基因斑马鱼)斑马鱼卵中,然后用白细胞介素34的RNA探针进行原位杂交技术鉴定,观察在瞬时表达情况下白细胞介素34在斑马鱼体内的特异性表达情况,在肝脏瞬时表达白细胞介素34情况下,通过抗体染色鉴定巨噬细胞在肝脏部位特意性聚集情况;
(2)构建Tg(fabp10a:il34)(即利用肝脏细胞特异性启动子——脂肪酸转运蛋白10a来驱动白细胞介素34在肝脏细胞中表达的转基因斑马鱼)稳定遗传的转基因家系的斑马鱼,即本专利所述可在肝脏中特异性聚集巨噬细胞的动物模型,与Tg(mpeg1:GFP)(即利用巨噬细胞特异性的启动子——巨噬细胞表达1来驱动绿色荧光蛋白在巨噬细胞中表达的转基因斑马鱼)转基因斑马鱼杂交后,利用白细胞介素34的RNA探针进行原位杂交技术鉴定,在稳定表达情况下白细胞介素34在斑马鱼体内特异性表达情况,然后在肝脏稳定表达白细胞介素34时,用抗体染色鉴定巨噬细胞在肝脏部位特意性聚集情况;
(3)将Tg(fabp10a:il34;mpeg1:GFP)(即利用肝脏细胞特异性启动子——脂肪酸转运蛋白10a来驱动白细胞介素34在肝脏细胞中表达的转基因斑马鱼与利用巨噬细胞特异性的启动子——巨噬细胞表达1来驱动绿色荧光蛋白在巨噬细胞中表达的转基因斑马鱼杂交后得到的双转基因斑马鱼)与已做标记的转基因家系Tg(fabp10a:dsRed)(即利用肝脏细胞特异性启动子——脂肪酸转运蛋白10a来驱动红色荧光蛋白在肝脏细胞中表达的转基因斑马鱼)杂交,获得受精卵,用激光扫描共聚焦显微镜实时活体成像技术观察在受精后3天到3.5天内巨噬细胞向肝脏区域迁移的过程。
进一步地,所述肝脏特异性聚集巨噬细胞的转基因斑马鱼模型具体步骤中,步骤(1)所述已标记了转基因家系Tg(mpeg1:GFP)(即利用巨噬细胞特异性的启动子——巨噬细胞表达1来驱动绿色荧光蛋白在巨噬细胞中表达的转基因斑马鱼)斑马鱼卵是用绿色荧光蛋白GFP标记了转基因家系Tg(mpeg1:GFP)(即利用巨噬细胞特异性的启动子——巨噬细胞表达1来驱动绿色荧光蛋白在巨噬细胞中表达的转基因斑马鱼)斑马鱼卵的巨噬细胞。
进一步地,所述肝脏特异性聚集巨噬细胞的转基因斑马鱼模型的具体步骤中,步骤(1)中所述已标记了转基因家系Tg(mpeg1:GFP)(即利用巨噬细胞特异性的启动子——巨噬细胞表达1来驱动绿色荧光蛋白在巨噬细胞中表达的转基因斑马鱼)斑马鱼卵为单细胞期。
进一步地,所述的肝脏特异性聚集巨噬细胞的转基因斑马鱼模型的具体步骤中,步骤(2)中所述已做标记的转基因家系Tg(fabp10a:dsRed)(即利用肝脏细胞特异性启动子——脂肪酸转运蛋白10a来驱动红色荧光蛋白在肝脏细胞中表达的转基因斑马鱼)是用红色荧光蛋白dsRed标记转基因家系Tg(fabp10a:dsRed)(即利用肝脏细胞特异性启动子——脂肪酸转运蛋白10a来驱动红色荧光蛋白在肝脏细胞中表达的转基因斑马鱼)的肝脏细胞。
进一步地,所述肝脏特异性聚集巨噬细胞的转基因斑马鱼模型中,所述特异性肝脏中聚集巨噬细胞的表型是由于白细胞介素34在肝脏中过表达引起。
进一步地,所述肝脏特异性聚集巨噬细胞的转基因斑马鱼模型中,所述斑马鱼为Tg(fabp10a:il34)(即利用肝脏细胞特异性启动子——脂肪酸转运蛋白10a来驱动白细胞介素34在肝脏细胞中表达的转基因斑马鱼)转基因斑马鱼,即fabp10a(脂肪酸转运蛋白10a)基因启动子特异性调控白细胞介素34基因在肝脏中过表达的转基因斑马鱼。
进一步地,所述肝脏特异性聚集巨噬细胞的转基因斑马鱼模型中,所述特异性肝脏中聚集巨噬细胞,表现为巨噬细胞数目仅特异性的在肝脏区域增高,其他组织区域如尾部造血组织CHT区域(大约在第13和第17梭形体之间)中的巨噬细胞数目不变,同时粒细胞数目不变。
与现有技术相比,本发明具有如下优点和有益效果:
(1)本发明首次证实了在肝细胞中过表达IL-34,足以诱导巨噬细胞向肝脏富集;
(2)本发明通过活体显像显示,稳定的转基因鱼在肝脏中高表达IL-34可直接诱导巨噬细胞向肝脏迁移;
(3)在以往的研究对IL-34诱导巨噬细胞迁移多集中在体外细胞系实验,本发明提供了直接的证据证明在肝脏异位表达IL-34可以诱导巨噬细胞迁移向肝脏;
(4)本发明中Tg(fabp10a:il34;mpeg1:GFP)成鱼具有以下表型:①巨噬细胞数目仅特异性的在肝脏区域增高;②其他组织区域如尾部造血组织(CHT)区域巨噬细胞数目不变;③粒细胞数目不变。
附图说明
图1是在肝脏中瞬时过表达IL-34导致巨噬细胞在肝脏中富集的原位杂交和抗体染色图(比例尺:100μm(白线);50μm(灰线));
图2是定量分析未注射质粒胚胎和注射质粒胚胎中肝脏区域和CHT尾部区域巨噬细胞或粒细胞的数量柱状图(均值±标准误,*p<0.05,*p<0.01,*p<0.001,n=5);
图3是在肝脏中稳定高表达IL-34导致巨噬细胞在肝脏中富集的原位杂交和抗体染色显微镜图(插图是相应的白色虚线框状区域的高放大率图(20×),比例尺:100μm(白线);50μm(灰线));
图4是定量分析Tg(mpeg 1:GFP)和Tg(fap10a:il34;mpeg 1:GFP)胚胎肝脏和尾部CHT区域巨噬细胞或粒细胞的数量柱状图(均值±标准误,*p<0.05,*p<0.01,*p<0.001;n=5);
图5是IL-34的过表达导致巨噬细胞向肝脏的迁移的活体成像显微镜下图(比例尺:40μm(白线))。
具体实施方式
下面结合实施例对本发明作进一步详细的描述,但本发明的实施方式不限于此。下述实施例中所使用的各原料,除特别指出的以外,均可由市售获得。
本发明中所使用的术语“野生型”是指野生型斑马鱼。
实施例1
1、材料和方法:
(1)斑马鱼养殖
斑马鱼的养殖如文献(Westerfield M:The zebrafish:guide for thelaboratory use of zebrafish(Brachdaniorerio).Edition by Eugene,OR,M.Westerfield,1993)所述。
本发明中用到如下的品系:AB野生型斑马鱼、Tg(mpeg 1:GFP)(即利用巨噬细胞特异性的启动子——巨噬细胞表达1来驱动绿色荧光蛋白在巨噬细胞中表达的转基因斑马鱼)、Tg(lyz:eGFP)(即利用中性粒细胞特异性的启动子——溶菌酶来驱动绿色荧光蛋白在巨噬细胞中表达的转基因斑马鱼)、Tg(fabp10a:il34)(即利用肝脏细胞特异性启动子——脂肪酸转运蛋白10a来驱动白细胞介素34在肝脏细胞中表达的转基因斑马鱼)、Tg(fabp10a:dsRed)(即利用肝脏细胞特异性启动子——脂肪酸转运蛋白10a来驱动红色荧光蛋白在肝脏细胞中表达的转基因斑马鱼)。
转基因斑马鱼Tg(mpeg 1:GFP),来源Zilong Wen实验室(未发表);Tg(lyz:eGFP)(即利用中性粒细胞特异性的启动子——溶菌酶来驱动绿色荧光蛋白在巨噬细胞中表达的转基因斑马鱼),来源于Phil Crosier实验室(Hall,C.,Flores,M.V.,Storm,T.,Crosier,K.,Crosier,P.(2007).The zebrafish lysozyme C promoter drives myeloid-specificexpression in transgenic fish.BMC Dev Biol,7,42.);Tg(fabp10a:dsRed)(即利用肝脏细胞特异性启动子——脂肪酸转运蛋白10a来驱动红色荧光蛋白在肝脏细胞中表达的转基因斑马鱼),来源于Jinrong Peng实验室(Her,G.M.,Chiang,C.C.,Chen,W.Y.,&Wu,J.L.(2003).In vivo studies of liver-type fatty acid binding protein(L-FABP)geneexpression in liver of transgenic zebrafish(Daniorerio).FEBS Lett,538(1-3),125-133.);Tg(fabp10a:il34)(即利用肝脏细胞特异性启动子——脂肪酸转运蛋白10a来驱动白细胞介素34在肝脏细胞中表达的转基因斑马鱼)来源于本实验室。
Tg(fabp10a:il34)(即利用肝脏细胞特异性启动子——脂肪酸转运蛋白10a来驱动白细胞介素34在肝脏细胞中表达的转基因斑马鱼)转基因斑马鱼是由斑马鱼fabp10a(脂肪酸转运蛋白10a)基因启动子特异性调控白细胞介素34基因在肝脏中过表达,诱导巨噬细胞特异性在肝脏区域聚集的转基因斑马鱼。
(2)斑马鱼胚胎整体原位杂交和抗体染色
收集受精后4天的斑马鱼胚胎(40条)置于1.5ml EP管(小型的离心管)中,1×PBST(1倍浓度的磷酸盐吐温缓冲液)润洗两次后加入1ml 4%多聚甲醛(将4g多聚甲醛粉末在60度溶解于96g去离子水中)室温固定2h;用1×PBST(1倍浓度的磷酸盐吐温缓冲液)室温下漂洗胚胎,2次,每次漂洗5分钟;分别用1ml50%甲醇溶液(1ml无水甲醇液体与1ml 1×PBST混合)、1ml无水甲醇液体梯度脱水(分别脱水5分钟),最后更换新的1ml无水甲醇液体,-20℃静置4h以上。分别用无水甲醇:1×PBST为3:1;1:1;1:3的混合溶液(体积比)漂洗胚胎复水各一次,每次漂洗5分钟;室温下用1×PBST漂洗胚胎2次,每次漂洗5分钟;1ml 10μg/mL蛋白酶K(事先用1×PBST配制而成)处理胚胎20分钟后用4%多聚甲醛(将4g多聚甲醛粉末在60度溶解于96g去离子水中)室温固定20分钟;用1×PBST室温下漂洗胚胎,3次,每次漂洗5分钟;加入1ml核酸杂交缓冲液,65℃孵育30分钟后更换新鲜核酸杂交缓冲液,65℃孵育3小时;从-20℃冰箱取出dig标记(地高辛标记探针)的反义RNA探针(以mRNA为模板做的反义RNA),68℃变性至少8分钟,变性后取出迅速置于冰上;加入反义RNA探针后65℃孵育12小时;回收探针,分别用事先65℃预热的50%甲酰胺/2×SSCT(1ml甲酰胺溶液与1ml二倍浓度的生理盐水柠檬酸钠吐温缓冲溶液混合配制而成)、2×SSCT(二倍浓度的生理盐水柠檬酸钠吐温缓冲溶液)、0.2×SSCT(零点二倍浓度的生理盐水柠檬酸钠吐温缓冲溶液)溶液漂洗胚胎各两次,每次漂洗20分钟;用1×PBST室温下漂洗胚胎,3次,每次漂洗5分钟,然后加入5%(5ml胎牛血清与95ml 1×PBST混合配制而成)FBS封闭液(胎牛血清),室温下孵育1小时;弃去FBS封闭液,换成1:2000Anti-digoxigenin-HRP(1μl与辣根过氧化物酶共轭的抗地高辛抗体混合2ml FBS封闭液)的抗体,4℃孵育12小时;用1×PBST室温下漂洗胚胎,6次,每次漂洗20分钟;用1X Plus Amplification Diluent(一倍浓度的放大稀释剂)润洗胚胎5分钟后加入Cyanine 3Plus Amplification Reagent(花菁3扩增试剂)(1:50)(1μl花菁3扩增试剂与49μl一倍浓度的放大稀释剂混合配制),避光染色10分钟;用1×PBST室温下漂洗胚胎,3次,每次漂洗20分钟;更换含有1:400Anti-GFP-Goat antibody(1μl羊属抗绿色荧光蛋白抗体与399μlFBS封闭液混合配制)的抗体,4℃孵育12小时;用1×PBST室温下漂洗胚胎,6次,每次漂洗20分钟;更换含有1:400Alexa anti-goat-488antibody(1μl与488共轭的抗羊属抗体与399μlFBS封闭液混合配制)的抗体,4℃孵育12小时;用1×PBST室温下漂洗胚胎,6次,每次漂洗20分钟;去掉漂洗液,然后加入1ml 70%甘油(70μl甘油混合30μl去离子水),-20℃长期保存,拍照结果见图1、图2、图3和图4。
(3)激光扫描共聚焦荧光显微镜活体成像
将受精后3天的Tg(fabp10a:dsRed;mpeg1-GFP)(即利用肝脏细胞特异性启动子——脂肪酸转运蛋白10a来驱动红色荧光蛋白在肝脏细胞中表达的转基因斑马鱼与利用巨噬细胞特异性的启动子——巨噬细胞表达1来驱动绿色荧光蛋白在巨噬细胞中表达的转基因斑马鱼杂交后得到的双转基因斑马鱼)和Tg(fabp10a:il34;fabp10a:dsRed;mpeg1-GFP)(即利用肝脏细胞特异性启动子——脂肪酸转运蛋白10a来驱动白细胞介素34在肝脏细胞中表达的转基因斑马鱼与利用巨噬细胞特异性的启动子——巨噬细胞表达1来驱动绿色荧光蛋白在巨噬细胞中表达的转基因斑马鱼杂交后得到的双转基因斑马鱼再与利用肝脏细胞特异性启动子——脂肪酸转运蛋白10a来驱动红色荧光蛋白在肝脏细胞中表达的转基因斑马鱼杂交得到的三转基因鱼)转基因斑马鱼胚胎在1%低熔点琼脂糖(1g低熔点琼脂糖粉末在60℃时溶解于99g去离子水中)中固定位置,利用激光扫描共聚焦荧光显微镜拍摄观察受精后3-3.5天内胚胎中巨噬细胞向肝脏中迁移的情况,如图5所示。
结果分析:(请在具体实施例后补充对结果的分析,说明结果图以及从结果图上得到怎样的结论)
在肝脏中瞬时过表达IL-34导致巨噬细胞在肝脏中富集的情况见图1。共将1.8nl(30ng/μ1)的pBLK-fabp10a-il34-sv40基因微注射到单细胞期用绿色荧光蛋白GFP标记巨噬细胞的转基因家系Tg(mpeg 1:GFP)和用绿色荧光蛋白eGFP标记粒细胞的转基因家系Tg(lyz:eGFP)斑马鱼胚胎中,图中,巨噬细胞为白色,IL-34为灰色。图1的A部分是在受精后4天的胚胎中对il34的表达分别进行原位杂交和抗体染色的结果。图1的B部分是在受精后4天的胚胎中对GFP的表达分别进行原位杂交和抗体染色的结果。插图是相应的框状区域(虚线区域)的高放大率图(20×)。由图1可得,在肝脏中瞬时过表达IL-34导致巨噬细胞在肝脏中富集,粒细胞不变。
定量分析未注射质粒胚胎和注射质粒胚胎中肝脏区域(以白点区域表示)和CHT尾部区域(大约在第13和第17梭形体之间,在两条白点线之间)巨噬细胞或粒细胞的数量对比结果见于图2(均值±标准误,*p<0.05,*p<0.01,*p<0.001,n=5)。由图2可得,在肝脏中瞬时过表达IL-34导致巨噬细胞在肝脏中显著性富集,而肝脏中粒细胞并没有显著性数目变化。
在肝脏中稳定高表达IL-34导致巨噬细胞在肝脏中富集的原位杂交和抗体染色结果见于图3。在受精后4天的胚胎中对il34和GFP的表达分别进行原位杂交和抗体染色。插图是相应的框状区域(虚线区域)的高放大率图(20×),图中巨噬细胞为白色,IL-34为灰色;由图3可得,在肝脏中稳定高表达IL-34导致巨噬细胞在肝脏中富集。
定量分析Tg(mpeg 1:GFP)和Tg(fap10a:il34;mpeg 1:GFP)胚胎肝脏(以图3白点区域表示)和尾部CHT区域(大约在第13和第17梭形体之间,以图3两条白点线之间)巨噬细胞或粒细胞的数量对比见于图4(均值±标准误,*p<0.05,*p<0.01,*p<0.001;n=5)由图4可得,在肝脏中稳定高表达IL-34导致巨噬细胞在肝脏中富集是有统计学意义的。
IL-34的过表达导致巨噬细胞向肝脏的迁移的活体成像结果见于图5。其中图5的A部分是对照组中巨噬细胞(白色,由白色箭头标记)在28分钟内通过肝脏(灰色)的过程,图5的B部分是实验组中巨噬细胞(白色,白色箭头标记)在28分钟内迁移到肝脏(灰色)的过程。比例尺:40μm(白线)。由图5可得,在肝脏中稳定高表达IL-34导致巨噬细胞向肝脏中迁移增加。
上述实施例为本发明较佳的实施方式,但本发明的实施方式并不受上述实施例的限制,其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,都包含在本发明的保护范围之内。
Claims (7)
1.一种肝脏特异性聚集巨噬细胞的转基因斑马鱼模型,其特征在于,包括如下步骤:
(1)通过聚合酶链式反应利用野生型斑马鱼基因组DNA得到斑马鱼肝脏细胞特异性启动子——脂肪酸转运蛋白10a,同时通过聚合酶链式反应利用野生型斑马鱼cDNA得到白细胞介素34基因,将这两部分片段利用DNA连接酶分段连接进入空载pBLK-sv40构建pBLK-fabp10a-il34-sv40质粒,利用显微注射技术将此质粒注射入已标记了的转基因家系Tg(mpeg1: GFP)斑马鱼卵中,然后用白细胞介素34的RNA探针进行原位杂交技术鉴定,观察在瞬时表达情况下白细胞介素34在斑马鱼体内的特异性表达情况,在肝脏瞬时表达白细胞介素34情况下,通过抗体染色鉴定巨噬细胞在肝脏部位特意性聚集情况;
(2)构建Tg (fabp10a:il34)稳定遗传的转基因家系的斑马鱼,即所述可在肝脏中特异性聚集巨噬细胞的动物模型,与Tg (mpeg1: GFP)转基因斑马鱼杂交后,利用白细胞介素34的RNA探针进行原位杂交技术鉴定,在稳定表达情况下白细胞介素34在斑马鱼体内特异性表达情况,然后在肝脏稳定表达白细胞介素34时,用抗体染色鉴定巨噬细胞在肝脏部位特意性聚集情况;
(3)将Tg (fabp10a: il34; mpeg1: GFP)与已做标记的转基因家系Tg( fabp10a:dsRed)杂交,获得受精卵,用激光扫描共聚焦显微镜实时活体成像技术观察在受精后3天到3.5天内巨噬细胞向肝脏区域迁移的过程。
2.根据权利要求1所述肝脏特异性聚集巨噬细胞的转基因斑马鱼模型,其特征在于,所述肝脏中特异性聚集巨噬细胞的表型是由于白细胞介素34在肝脏中过表达引起。
3.根据权利要求1所述肝脏特异性聚集巨噬细胞的转基因斑马鱼模型,其特征在于,所述斑马鱼为Tg (fabp10a: il34)转基因斑马鱼,即fabp10a基因启动子特异性调控白细胞介素34基因在肝脏中过表达的转基因斑马鱼。
4.根据权利要求1所述肝脏特异性聚集巨噬细胞的转基因斑马鱼模型,其特征在于,所述特异性肝脏中聚集巨噬细胞,表现为巨噬细胞数目仅特异性的在肝脏区域增高,其他组织区域造血组织CHT区域中的巨噬细胞数目不变,同时粒细胞数目不变。
5.根据权利要求1所述肝脏特异性聚集巨噬细胞的转基因斑马鱼模型,其特征在于,步骤(1)中所述已标记了转基因家系Tg (mpeg1: GFP)斑马鱼卵是用绿色荧光蛋白GFP标记了转基因家系Tg (mpeg1: GFP)斑马鱼卵的巨噬细胞。
6.根据权利要求1所述肝脏特异性聚集巨噬细胞的转基因斑马鱼模型,其特征在于,步骤(1)中所述已标记了转基因家系Tg (mpeg1: GFP)斑马鱼卵为单细胞期。
7.根据权利要求1所述的肝脏特异性聚集巨噬细胞的转基因斑马鱼模型,其特征在于,步骤(2)中所述已做标记的转基因家系Tg( fabp10a: dsRed)是用红色荧光蛋白dsRed标记了肝脏细胞的转基因家系Tg( fabp10a: dsRed)。
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CN112997926A (zh) * | 2020-12-17 | 2021-06-22 | 中国科学院水生生物研究所 | 基于斑马鱼幼鱼成像模型评价缓解食源性肠炎成分的方法 |
CN112997926B (zh) * | 2020-12-17 | 2021-12-28 | 中国科学院水生生物研究所 | 基于斑马鱼幼鱼成像模型评价缓解食源性肠炎成分的方法 |
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