作者感谢克里斯塔·哈泽，多丽丝·韦伯和布里吉特科特技术援助。在Weidinger实验室工作是由德意志研究联合会WE 4223 / 3-1的补助，我们4223 / 4-1和德意志GESELLSCHAFT献给Kardiologie通过奥斯卡 - 拉普 - Stipendium和克劳斯 - 乔治·UND-Sigrid- Hengstberger-Forschungsstipendium。

1.代转基因鱼TetActivator的行<ol><li> TetActivator结构的生成<ol><li>在TetActivator盒兴趣上游的矢量＃克隆的调控序列1247采用标准技术。可替代地，可使用重组技术来生成的BAC，其中TetActivator盒（矢量＃1180）被插入靶基因的第一外显子，并且其中所述TOL2反向重复被引入BAC骨干（详细重组工程协议见7,8）。 </li><li>准备无毒素分解质粒DNA或用市售试剂盒BAC DNA的准备工作。 </li></ol></li><li>转基因鱼TetActivator代线<ol><li>根据标准程序9执行TetActivator构建体的显微注射。 <ol><li>注入25-50皮克的质粒DNA或高达250皮克BAC DNA导入单细胞阶段的胚胎一起封义RNA编码TOL2 TRAnsposase转座子介导的转基因，或I-SceI的蛋白I-SceI的介导的转基因。 注:考虑对转基因方法的选择可以在讨论部分。 注:优选注入到细胞质，而不是蛋黄中，由于当DNA不传递到细胞质转基因的效率可能会降低。 </li></ol></li><li>提高注入胚胎G0到成年。可选，屏幕G0胚胎AmCyan荧光和优先发展的胚胎显示所需的表达模式。这可能增加了种系传递的速率，特别是用于TOL2介导的转基因。 </li><li>通过使用荧光立体显微镜在F1中的胚胎或幼虫的预期表达模式异交个体G0鱼与野生型鱼和外观AmCyan荧光的（实施例示于图1D）评估成功种系整合。 </li><li>提高荧光阳性F1胚胎到成年和队友它们与野生型鱼类，以获得稳定的F2转基因鱼。 </li><li>提高和表征不同G0创始人鱼的至少3个不同的亚系，由于个别线路可能会有所不同的表达水平，表达模式，能够诱发TetResponder线，泄漏，他们易患沉默。 注:如果强劲表现在胚胎许多转基因是部分或完全沉默的后期幼体或成鱼。因此，如果线是指在成鱼中使用，在成年期表征为其TetActivator表达和功能几个独立的行。 注意:我们已经产生的TetActivator线为TetActivator在两个胚胎和成鱼，其列于表1组织特异性表达一个面板，这些线可从根据请求Weidinger实验室。 </li></ol></li></ol> 2.代转基因TetResponder鱼线注:我们已经产生了TetResponder结构允许I-SceI的或TOL2介导产生稳定TetResponder线，这是根据要求可从Weidinger实验室（Weidinger实验室质粒数据库中没有1444; 图1E）。此构建体含有一个四环素算子，其后为多接头区有利于感兴趣的基因和YFP - 衍生物YPet编码序列的编码序列（CDS）的插入。因此，该构建体被设计为与YPet目的蛋白质的C-末端融合的TETA介导的表达。如果已要避免一个标签的融合蛋白的表达，一个P2A或T2A肽可与感兴趣的CDS的基因，这有利于共表达的兴趣和YPet蛋白作为单独的多肽6,10引入。 <ol><li> TetResponder结构的生成<ol><li>感兴趣3基因的克隆CDS"四环素运营商和5&#39;YPet CDS的根据标准方法。确保该终止密码子已被来自感兴趣的CDS的基因去除。 </li><li>制备使用市售的试剂盒毒素无质粒DNA制备。 </li></ol></li><li>转基因鱼TetResponder代线<ol><li>执行根据标准程序9 TetResponder的显微注射构建。 <ol><li>注入25-50皮克质粒DNA的成一细胞期胚胎的细胞质连同封义RNA编码TOL2转座转座子介导的转基因或与Ⅰ-SceⅠ限制性酶切位酶为大范围核酸酶辅助转基因。 注:考虑对转基因方法的选择可以在讨论部分。 注:优选注入到细胞质，而不是蛋黄中，由于当DNA不传递到细胞质转基因的效率可能会降低。 </li></ol></li><li>提高注入胚胎G0到dulthood。 </li><li>通过与先前建立TetActivator线后跟强力霉素治疗的F1胚或F1的幼虫配合个别G0鱼评估成功种系整合的转基因和诱导能力。 注:通常我们使用泛素启动子驱动TetActivator线（ 泛素 :irtTAM2（3F）-p2a-AmCyan ulm2，短泛素 :TETA AmCyan 3）筛选功能TetResponder运营商，因为这条线在胚胎和成人相当驱动无处不体现鱼，因此非常适合于评估在所有感兴趣的组织TetResponder诱导能力。这条线路是从可应要求Weidinger实验室。 </li><li>收集各离合器的胚在含有E3胚胎培养基一个单独的10cm培养皿（见材料表）和孵化菜在28.5℃。 </li><li>地方G0鱼胚胎了成单独编号的坦克（箱养殖;见材料表），直到胚胎已屏幕ED。这允许对G0鱼，显示转基因的种系传递恢复。 </li><li>如果预计只有一半的胚胎携带TetActivator转基因（如遇杂TetActivator载体进行杂交到一个TetResponder创始人鱼），选择用于包含TetActivator转基因由外观AmCyan荧光的在适当的发育阶段的胚胎。 </li><li>通过在2.3节所述治疗用胚胎多西环素（DOX）诱导TetActivator转录活性。 </li><li>屏幕F1胚胎YPet荧光出现。偏爱线（G0鱼）产生均匀YPet表达，即在离合器胚胎应预期表达结构域和单个胚胎之间很少变化内显示小嵌合体。见箭头在图1F和图1G为代表性实例。 </li><li>队友G0鱼在传输的标识诱导TetResponder转基因步骤2.2.8与野生型鱼类，并提高所有F1胚胎到成年。 </li><li>确定成人F1转基因鱼或者通过基于PCR的基因分型（见或个人渡鱼F1到TetActivator线之后DOX处理F2胚胎和YPet荧光（参见2.5节）的出现。 </li><li>建立和特点从不同的G0创始人鱼的至少3个不同的TetResponder亚系，因为诱导性可能会有所不同的个别线路。特别是可实现的表达水平，以及是否响应可以在所有类型的细胞被​​激活可能会有所不同。此外，易患沉默将行之间不同。最后，在某些情况下，可能优选选择介导感兴趣的蛋白质的唯一中等表达水平，尤其是如果与在不存在强力霉素的TetActivator线组合产生渗漏表达预期导致发育缺陷或毒性行。 注:春节应答线是在胚胎鲁棒诱导可以部分或完全沉默的后期幼体或成鱼。因此，如果线是指在成鱼中使用，几个独立的线路应被表征为诱导成人。如果诱导感兴趣的基因的是进一步发展兼容，它已被证明是有益的，以创造双转基因胚胎（TetActivator x TetResponder），以诱导GOI表达在胚胎和选择并提高仅个人表示最健壮和至少镶嵌感应。 </li><li>为了保持并按照2.5节所述传播建立TetResponder线，型成鱼）。 </li></ol></li><li>胚胎多西环素治疗<ol><li>准备2000倍多西环素（DOX）的股票。解决DOX在50％EtOH中以达到50毫克/毫升（97毫摩尔）的浓度。商店DOX股避光在-20℃。 </li><li>从胚胎中倒出了广大E3媒体和其替换含有DOX以25微克/毫升阿霉素的最终浓度E3胚胎培养基（2.5微升2000倍的DOX股票每50毫升E3）。 Dechorionation不是必需的。 </li><li>通过评估外观YPet荧光感兴趣的标记基因的表达返回前的胚胎28.5℃，最低6小时的。 </li><li>通过把胚胎只有乙醇溶剂评估TetActivator / TetResponder组合的泄漏。 注: 在原位杂交（ISH）或RT-PCR，以检测TetResponder的mRNA可以用作泄漏比感兴趣的标记基因的荧光的更敏感的措施。泄漏的程度将取决于特定TetActivator / TetResponder线组合，它也可以组织和发育阶段之间变化。 </li></ol></li><li>成年斑马鱼麻醉<ol><li>准备好10厘米直径的陪替氏培养皿或小烧杯充满含有鱼系统水1mg / ml的三卡因（3-氨基苯甲酸乙酯methanesulfonate，MS-222）。 </li><li>鱼转移到含有麻醉剂的容器，并等待，直到它达到麻醉4水平的均衡完全丧失（鱼就在于在其一侧），无运动和触摸11没有回应表示。 </li><li>仔细用镊子或勺子载玻片上，盖的塑料培养皿中，或琼脂糖涂层培养皿转移鱼类和执行鳍截肢或成像（参见3.2节）。 </li><li>返回鱼含有大量的新鲜鱼系统水的容器中，并进行监控。如果刺的动作不会在3分钟内恢复，帮助超过使用塑料移液管鳃吹水。 </li></ol></li><li>的TetResponder鱼基于PCR的基因分型<ol><li>执行尾鳍12的部分截肢。将鱼放入单独的，编号为坦克（养殖箱;见材料表）。这允许对转基因鱼以下成功基因型分型的恢复。 </li><li> TRAnsfer鳍组织到一个96孔PCR板预先填充与20μl无DNase的水为基因组DNA 13隔离各个孔。 <ol><li>添加120微升的1M氢氧化钠至每孔，并使用热循环仪在95℃孵育样品20分钟。 </li><li>寒意样品4℃，加入14微升的1M的Tris-HCl（pH值8.0），每孔，并简要涡样品。在16000×g离心离心机样品5分钟以沉淀细胞碎片。直到需要存储的基因组DNA在4℃下。 </li></ol></li><li>设计引物以特异性地扩增TetResponder转基因的片段。使用2微升分离的DNA在标准的PCR反应，分析PCR产物在琼脂糖凝胶上。 </li></ol></li></ol>转基因表达在成人再生斑马鱼尾鳍3组织特异性的诱导<ol><li>建立TetActivator的; TetResponder双转基因鱼<ol><li>队友TetActivator携带者TetResponder CARRIERS。 </li><li>选择胚胎携带TetActivator转基因在发育阶段时，调节元件驱动TetActivator盒活跃。通过外观AmCyan荧光使用立体声荧光显微镜转基因胚胎很容易被识别。 注:TetActivator转基因阳性的鱼可替代地在成年期由成像的麻醉鱼，例如鉴定。以下尾鳍的截肢和在再生AmCyan荧光的出现。 </li><li>提高选胚胎到成年。 </li><li>通过其诱导在感兴趣的组织的TetResponder的表达能力通过基于PCR的基因分型或（优选）确定双转基因成鱼（鱼携带TetResponder除了TetActivator）。一种协议，用于TetResponder诱导和检测再生尾鳍在2.3节和第4节进行说明。 注:如果瞬时表达TetResponder兼容进一步胚胎或幼虫发育，上面所描述的胚胎发育过程中，以确定下治疗DOX携带TetActivator和TetResponder转基因双转基因胚胎是非常有用的（2.3节， 图1H）。双阳性的胚胎会显示AmCyan和YPet荧光。积极提高胚胎到成年。 </li></ol></li><li>斑马鱼尾鳍截肢和诱导转基因表达在再生鳍<ol><li>执行尾鳍上麻醉鱼的部分截肢（参见2.4节）12。与DOX或者立即把鱼或退回给鱼住房制度。 </li><li>准备繁殖箱（ 图2A;见材料表）充满了1升含25微克/毫升DOX鱼制水（加入500μl的2000倍50毫克/毫升股票1升的鱼制水）。 注:转基因诱导表达后，立即截肢允许作为sessment对再生过程中的早期事件的影响，而感应在后3天截肢（3 DPA），应使用在再生生长来评估转基因表达或功能。 </li><li>传输高达10以前截肢，双转基因鱼的养殖箱，接近盒的盖子，这样的鱼无法逃脱（ 图2A）。 </li><li>将养殖箱到黑暗减轻压力。我们通常把箱子到28.5℃的空气孵化器，以确保持续的黑暗和温度。 </li><li>与乙醇只（250元1L鱼制水微升）治疗阴性对照双转基因鱼。 注:单转基因或用阿霉素处理的野生型鱼可以用作附加的阴性对照，尽管我们从未观察到这里使用的鳍再生的DOX剂量的不利影响。 </li><li>如在2.4节和使用镊子或勺子鱼转移到描述湿润的琼脂糖涂层的培养皿麻醉鱼<html>菜。仔细用镊子传播尾鳍。 </li><li>屏幕鱼用荧光立体显微镜（ 图2B）的再生内外观YPet的。 注:通常情况下，TetResponder驱动的荧光可以观察到稳健以下6个小时DOX治疗。但是，我们建议TetResponder表达的动态表征为生成的每个行。 </li><li>回到鱼鱼住房制度终止TetResponder转基因诱导或继续治疗。 注:对于治疗&gt; 24小时（长期治疗）有必要交换水和每日彻底清洗育种框。 </li><li>在长期的治疗，饲料鱼每隔2-3天喂养后，他们返回到养殖箱前转移到鱼干净的容器中，新鲜的鱼水系统。 </li></ol></li></ol>在鳍再生TetResponder表达4.表征吨"&gt;注意:我们通常验证组织特异性基因表达中使用的冷冻切片的荧光成像在3 DPA再生尾鳍在这个时间点一个再生的不同组织区室已经形成，并且可以清楚地识别在组织切片，和cryosectioning比在以后再生阶段简单。 图2C示出了可以在再生区分开的组织结构域，并列出了几个分子标记对这些域。以下方案描述了制备的纵向或横向的冰冻切片进行直接成像或免疫染色。 <ol><li>成人尾翼Cryosectioning <ol><li>治疗鱼的尾鳍从2 DPA如第3.2节截肢，直到3政治部与DOX </li><li>如在2.4节所述麻醉鱼</li><li>用勺子或镊子轻轻地转移到鱼的塑料培养皿的盖子。 </li><li>再截肢的鳍再生约4-5段骨s近端到初始截肢平面使用手术刀和鱼返回到新鲜的鱼水系统。 </li><li>小心转移再生的小皮氏培养皿（ 例如，35毫米）含有4％（重量/体积）低聚甲醛（PFA）在1×磷酸盐缓冲盐水（PBS;见材料表），使用细镊子。请勿触摸再生，但组织只有残端的一部分。 </li><li>将组织平放在培养皿底部和修复O / N在4℃。 </li><li>洗涤再生两次在1×PBS中含有0.1％吐温（PBT）以除去煤灰和孵化再生在o / n 4℃在0.5M的EDTA-PBS（pH7.5）中脱钙骨基质。 </li><li>在10％的蔗糖的PBS，20％蔗糖的PBS，30％蔗糖的PBS和30％的孵育回收物在RT蔗糖的PBS:组织冷冻介质以1:1的比例（TFM;见材料表）为各30分钟。随后，孵化鳍在4℃&gt; 2小时组织冷冻介质。 </li><li>将cryomolds到显微镜载玻片，并填写米，组织冷冻介质。避免引入气泡。 </li><li>转印再生成cryomolds和定向组织适当地获得鳍回收物（ 图2D）的纵向或横向的部分。确保再生是直的，这有利于切片。 </li><li>转印与显微镜载玻片到定位在一个床干冰启动冷冻过程（ 图2D）的金属架一起cryomolds。 </li><li>一旦组织冷冻介质已成为固体，地点cryomold到工作台并让坐在RT几分钟，使得组织冷冻培养基解冻在塑料介质界面。 </li><li>使用钢笔或类似推冻堵出cryomold的。商店cryoblocks在一个盒子在-80℃直至组织切片。 </li><li>用冷冻切片机准备10-14微米厚的冰冻切片和收集粘连显微镜载玻片部分（见材料表）。商店载玻片在-20℃直至ねEDED。 </li></ol></li><li> TetResponder表达对基于YPet荧光翅片区段表征<ol><li>治疗翅片区段30分钟用100％MeOH中冷却至-20℃，以提高粘附到显微镜载片随后在PBT中洗涤两次，在室温和1次洗涤在PBS，每次5分钟。 </li><li>可视化晶核孵育切片10分钟，4&#39;，6-二脒基-2- phenylinodole（DAPI）在PBS中（1:5000），随后洗涤两次，每次10分钟的PBS。 </li><li>山段75％甘油PBS或市售的抗淬灭坐骑，并盖上盖玻片。 </li><li>图像YPet和共焦或宽视场荧光显微镜下AmCyan荧光（ 图1E）。 注意:为了便于明确识别表达YPet，免疫荧光或免疫组织化学与抗细胞类型特异性抗原连同抗GFP抗体的细胞类型（其与YPet反应，但不AmCyan）可以执行。或者，为了便于检测弱YPet表达，RNA 原位杂交对YPet RNA可被执行或者在整个安装翅片或翼片的石蜡切片。 </li></ol></li></ol>

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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditional+expression+of+a+myocardium-specific+transgene+in+zebrafish+transgenic+lines.">Conditional expression of a myocardium-specific transgene in zebrafish transgenic lines.</a> Dev Dyn. 233 (4), 1294-1303 (2005).</li><li>Knopf, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dually+inducible+TetON+systems+for+tissue-specific+conditional+gene+expression+in+zebrafish.">Dually inducible TetON systems for tissue-specific conditional gene expression in zebrafish.</a> Proceedings of the National Academy of Sciences of the United States of America. 107 (46), 19933-19938 (2010).</li><li>Provost, E., Rhee, J., Leach, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+2A+peptides+allow+expression+of+multiple+proteins+from+a+single+ORF+in+transgenic+zebrafish+embryos.">Viral 2A peptides allow expression of multiple proteins from a single ORF in transgenic zebrafish embryos.</a> Genesis. 45 (10), 625-629 (2007).</li><li>Suster, M. L., Abe, G., Schouw, A., Kawakami, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transposon-mediated+BAC+transgenesis+in+zebrafish.">Transposon-mediated BAC transgenesis in zebrafish.</a> Nat Protoc. 6 (12), 1998-2021 (2011).</li><li>Bussmann, J., Schulte-Merker, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+BAC+selection+for+tol2-mediated+transgenesis+in+zebrafish.">Rapid BAC selection for tol2-mediated transgenesis in zebrafish.</a> Development. 138 (19), 4327-4332 (2011).</li><li>Yuan, S., Sun, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+of+mRNA+and+morpholino+antisense+oligonucleotides+in+zebrafish+embryos.">Microinjection of mRNA and morpholino antisense oligonucleotides in zebrafish embryos.</a> J Vis Exp. (27), (2009).</li><li>Kim, J. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+cleavage+efficiency+of+a+2A+peptide+derived+from+porcine+teschovirus-1+in+human+cell+lines,+zebrafish+and+mice.">High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice.</a> PloS one. 6 (4), e18556 (2011).</li><li>Matthews, M., Varga, Z. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anesthesia+and+euthanasia+in+zebrafish.">Anesthesia and euthanasia in zebrafish.</a> ILAR journal / National Research Council, Institute of Laboratory Animal Resources. 53 (2), 192-204 (2012).</li><li>Hyde, D. R., Godwin, A. R., Thummel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+electroporation+of+morpholinos+into+the+regenerating+adult+zebrafish+tail+fin.">In vivo electroporation of morpholinos into the regenerating adult zebrafish tail fin.</a> J Vis Exp. (61), (2012).</li><li>Meeker, N. D., Hutchinson, S. A., Ho, L., Trede, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+for+isolation+of+PCR-ready+genomic+DNA+from+zebrafish+tissues.">Method for isolation of PCR-ready genomic DNA from zebrafish tissues.</a> Biotechniques. 43 (5), 610 (2007).</li><li>Thermes, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=I-SceI+meganuclease+mediates+highly+efficient+transgenesis+in+fish.">I-SceI meganuclease mediates highly efficient transgenesis in fish.</a> Mech Dev. 118 (1-2), 91-98 (2002).</li><li>Suster, M. L., Kikuta, H., Urasaki, A., Asakawa, K., Kawakami, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenesis+in+zebrafish+with+the+tol2+transposon+system.">Transgenesis in zebrafish with the tol2 transposon system.</a> Methods Mol Biol. , 56141-56163 (2009).</li><li>Grabher, C., Wittbrodt, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Meganuclease+and+transposon+mediated+transgenesis+in+medaka.">Meganuclease and transposon mediated transgenesis in medaka.</a> Genome Biol. 8, (2007).</li></ol>

作者什么都没有透露。

成年斑马鱼有一个惊人的能力，成功地再生许多内部器官和附属物。深入了解涉及的分子和细胞机制的需要的基因功能和信号通路组织特异性分析。朝此，特顿系统提供了在胚胎和成年斑马鱼spatiotemporally控制基因表达的有效工具。特顿体系结构和方法论在这个手稿中描述已成功应用于最近我们实验室3的研究。建立系统顿时下列几个问题应考虑: TetActivator转基因设?...

斑马鱼是一种行之有效的脊椎动物模式生物研究的发展，生理学，疾病和再生的许多方面。随着越来越多地采用斑马鱼作为模型用于后胚胎的生物过程，实验工具为诱导型，组织特异性操纵基因表达或信号转导途径已经变得越来越重要。具体地讲，研究成器官和附属物再生在成年斑马鱼都遭受缺乏工具的过程中，这些再生过程信号通路的时空要求清扫。 目前，三种不同的系统已被用于实现在再生成年斑马鱼的内脏有条件的，组织特异性的基因表达:在的Cre-lox系统中，使用转座子介导的体细胞的转基因热休克诱导转基因拼接表达，并且顿系统1 -3。堤是指四环素续的变体轧制转录激活系统，其中表达在抗生素四环素或其衍生物， 例如多西环素存在下激活。在的Cre-lox系统，因为它迄今已在成年鱼使用的，依赖于他莫昔芬控制Cre重组酶（CreERT2），其表达在空间上受限于组织特异性调控元件。 Cre的驱动除去一个STOP盒的促进的兴趣的启动子应是活性在所有细胞类型1驱动的基因的表达。转座子介导的创作mosaically表达的转基因体细胞提供了在单个细胞系诱导转基因表达系统。注射斑马鱼的胚胎与TOL2座子携带目的基因下的热休克启动子的结果在嵌合个体通常携带外源基因仅在再生器官2的离散细胞谱系的转录控制。虽然这两个系统允许有条件的组织SPEcific基因表达方法，将Cre-lox系统是不可逆的，并且使用转座子为基础的克隆标记策略从它的随机性质受到影响。因此，我们和其他人已经最近适于转基因TETON系统用于在斑马鱼中，这有利于在时间上和空间上控制基因的表达是在附加的可调和可逆3-5。 此处所用的提顿系统包括转基因驱动线（TetActivator），其中多西环素（DOX）诱导性转录激活（改进反向四环素反式激活，irtTA，短TETA）是根据组织特异性基因调控序列的控制。其次，它要求的转基因应答线（TetResponder），该港口的在转录控制下的四环素操作员感兴趣的基因（四环素响应元件; TetRE）（ 图1A）。因此，使用的TetActivator和TetResponder线路特定组合允许有条件组织特定基因表达的FIC操纵。 最近，我们利用了系统顿探测的Wnt /β-catenin信号通路的成人斑马鱼再生尾鳍3组织特异性的功能。在这里介绍的协议中，我们描述了用于在斑马鱼中的建立的顿系统和使用，特别是用于鳍再生的研究工作流。这包括如何生成稳定的转基因TetActivator和TetResponder线的详细说明和协议，用于在胚胎和成年斑马鱼转基因诱导。此外，我们描述了在鳍再生技术验证组织特异性基因表达，包括一个协议的成年斑马鱼翅片冷冻切片的制备方法。此外，我们将讨论考虑的TetActivator转基因的表达TetResponder的设计，的转基因方法的选择和检测。因此，该协议的总体目标是作为一个蓝图设置式的官能顿系统在斑马鱼实现有条件组织特异性基因的表达，这可以适用于任何感兴趣的组织。 我们已经创建了一个TetActivator向量允许I-SceI的或TOL2介导的一代使用短基因的调控序列稳定TetActivator线（增强片段; Weidinger实验室质粒数据库中没有1247; 图1B）。此构建体含有一个TetActivator盒，其组成稠合与单纯疱疹病毒VP16的激活结构域衍生3F [irtTAM2（3F）]反向的Tet阻抑物结构域的M2突变体变体。所述TetActivator（TETA）的表达可以容易地监测，因为它是共表达与来自相同的开放阅读框架的荧光团AmCyan;一个P2A肽介导核糖体跳跃，这将导致生产TETA和AmCyan截至1独立的蛋白质:1的比例5,6。该构建体也包含一个poylinker 5&#39;T形etActivator盒，以便于插入的兴趣使用常规的克隆方法的基因组调节序列。 此外，我们已经创建了一个构建体由上述TetActivator盒外加一个卡那霉素选择盒（Weidinger实验室质粒数据库没有1180; 图1C）含有大量的基因组区域，可以重组到细菌人工染色体（BAC）（通常成基因的表达模式是由转基因来模拟）的起始密码子。这两种结构都可以从应要求Weidinger实验室。

<table><tbody><tr><td>Breeding boxes</td><td>Aqua Schwarz</td><td>AquaBox 1</td><td></td></tr><tr><td>Compound fluorescent microscope</td><td>e.g., Leica, Zeiss</td><td>varies with the manufacturer</td><td>to image fluorescent tissue sections</td></tr><tr><td>Confocal microscope</td><td>e.g., Leica, Zeiss</td><td>varies with the manufacturer</td><td>to image fluorescent tissue sections</td></tr><tr><td>Cryostat</td><td>e.g., Leica, Thermo-Scientific</td><td>varies with the manufacturer</td><td>for cryosectioning</td></tr><tr><td>4&amp;rsquo;, 6- diamidino-2-phenylinodole (Dapi)</td><td>Sigma-Aldrich</td><td>D9542</td><td>use 1/5,000 in PBS&lt;br/&gt; for visualization of nuclei</td></tr><tr><td>Doxycycline</td><td>Sigma-Aldrich</td><td>D9891</td><td>prepare stocks in 50% EtOH at 50 mg/ml (97 mM)&lt;br/&gt; for TetResponder induction</td></tr><tr><td>Paraformaldehyde (PFA)</td><td>Sigma-Aldrich</td><td>P6148</td><td>4% (w/v) paraformaldehyde in PBS, pH 7.5&lt;br/&gt; for fixation</td></tr><tr><td>1x Phosphat-buffer saline (PBS)</td><td></td><td></td><td>1.7 mM KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;, 5.2 mM Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;, 150 mM NaCl, pH 7.5</td></tr><tr><td>1x Phosphat-buffer saline + Tween 20 (PBT)</td><td></td><td></td><td>1x PBS with 0.1% Tween 20</td></tr><tr><td>Superfrost Ultra Plus adhesion microscope slides</td><td>Thermo Scientific&amp;nbsp;</td><td>1014356190</td><td>for collection of tissue sections</td></tr><tr><td>Stereo fluorescent microscope</td><td>e.g., Leica, Zeiss</td><td>varies with the manufacturer</td><td>for fluorescence-based genotyping</td></tr><tr><td>Thermocycler</td><td>e.g., Biorad, Applied Biosystems</td><td>varies with the manufacturer</td><td>for PCR-based genotyping</td></tr><tr><td>Tissue freezing medium (TFM)</td><td>Triangel Biomedical Sciences</td><td>TFM-C</td><td>for embedding of tissue samples</td></tr><tr><td>Tricaine (L-Ethyl-m-amino-benzoate-methane sulfonate/MS-222)</td><td>Sigma-Aldrich</td><td>E10521</td><td>for anesthesia&lt;br/&gt; use at 1 mg/ml in E3 embryo medium</td></tr></tbody></table>

use of the teton system to study molecular mechanisms of zebrafish regeneration

The zebrafish has become a very important model organism for studying vertebrate development, physiology, disease, and tissue regeneration. A thorough understanding of the molecular and cellular mechanisms involved requires experimental tools that allow for inducible, tissue-specific manipulation of gene expression or signaling pathways. Therefore, we and others have recently adapted the TetON system for use in zebrafish. The TetON system facilitates temporally and spatially-controlled gene expression and we have recently used this tool to probe for tissue-specific functions of Wnt/beta&#8211;catenin signaling during zebrafish tail fin regeneration. Here we describe the workflow for using the TetON system to achieve inducible, tissue-specific gene expression in the adult regenerating zebrafish tail fin. This includes the generation of stable transgenic TetActivator and TetResponder lines, transgene induction and techniques for verification of tissue-specific gene expression in the fin regenerate. Thus, this protocol serves as blueprint for setting up a functional TetON system in zebrafish and its subsequent use, in particular for studying fin regeneration.

建立用于组织特异性诱导的基因表达时，转基因TetActivator和TetResponder线的官能TETON系统需要生成（ 图1A）。这是通过显微注射TetActivator（ 图1B - C）的完成或TetResponder（ 图1E）构造成早期斑马鱼的胚胎和随后的生殖系融合。官能TetActivator构建体既可以由短的调节序列（增强子元件）的TetActivator盒（ 图1B）的上游克隆产生，或者由重组的...

Watch this Scientific Journal Video about Use of the TetON System to Study Molecular Mechanisms of Zebrafish Regeneration at JoVE.com

Use of the TetON System to Study Molecular Mechanisms of Zebrafish Regeneration

Here we outline the workflow for using the TetON system to achieve tissue-specific gene expression in the adult regenerating zebrafish tail fin.

使用提顿体系的研究斑马鱼再生的分子机制

The zebrafish has become a very important model organism for studying vertebrate development, physiology, disease, and tissue regeneration. A thorough ...

use-teton-system-to-study-molecular-mechanisms-zebrafish

Research

JoVE Journal

Developmental Biology

10.6K 次观看.  Ulm University. Here we outline the workflow for using the TetON system to achieve tissue-specific gene expression in the adult regenerating zebrafish tail fin.

视频: Use of the TetON System to Study Molecular Mechanisms of Zebrafish Regeneration

Kidney Regeneration in Adult Zebrafish by Gentamicin Induced Injury

The kidney is essential for fluid homeostasis, blood pressure regulation and filtration of waste from the body. The fundamental unit of kidney function is the nephron. Mammals are able to repair existing nephrons after injury, but lose the ability to form new nephrons soon after birth. In contrast to mammals, adult fish produce new nephrons (neonephrogenesis) throughout their lives in response to growth requirements or injury. Recently, lhx1a has been shown to mark nephron progenitor cells in the adult zebrafish kidney, however mechanisms controlling the formation of new nephrons after injury remain unknown. Here we show our method for robust and reproducible injury in the adult zebrafish kidney by intraperitoneal (i.p.) injection of gentamicin, which uses a noninvasive visual screening process to select for fish with strong but nonlethal injury. Using this method, we can determine optimal gentamicin dosages for injury and go on to demonstrate the effect of higher temperatures on kidney regeneration in zebrafish.

Here we present a reliable method to study adult kidney regeneration by inducing acute kidney injury by gentamicin injection. We show that injury is dependent on gentamicin dosage and environmental temperature using in situ hybridization to label lhx1a+ developing new nephrons.

肾再生的成年斑马鱼庆大霉素性损伤

The kidney is essential for fluid homeostasis, blood pressure regulation and filtration of waste from the body. The fundamental unit of kidney function is ...

科研

发育生物学

Capturing Tissue Repair in Zebrafish Larvae with Time-lapse Brightfield Stereomicroscopy

The zebrafish larval tail fin is ideal for studying tissue regeneration due to the simple architecture of the larval fin-fold, which comprises of two layers of skin that enclose undifferentiated mesenchyme, and because the larval tail fin regenerates rapidly within 2-3 days. Using this system, we demonstrate a method for capturing the repair dynamics of the amputated tail fin with time-lapse video brightfield stereomicroscopy. We demonstrate that fin amputation triggers a contraction of the amputation wound and extrusion of cells around the wound margin, leading to their subsequent clearance. Fin regeneration proceeds from proximal to distal direction after a short delay. In addition, developmental growth of the larva can be observed during all stages. The presented method provides an opportunity for observing and analyzing whole tissue-scale behaviors such as fin development and growth in a simple microscope setting, which is easily adaptable to any stereomicroscope with time-lapse capabilities.

We present a protocol for capturing the dynamics of zebrafish larval tail fin regeneration on a whole-tissue scale using brightfield-based stereomicroscopy. This technique enables capturing the regeneration dynamics with single cell resolution. This methodology can be adapted to any stereomicroscope equipped with a CCD camera and time-lapse software.

捕获组织修复的斑马鱼幼虫与时间推移明视立体显微镜

The zebrafish larval tail fin is ideal for studying tissue regeneration due to the simple architecture of the larval fin-fold, which comprises of two ...

Nanoparticle-mediated siRNA Gene-silencing in Adult Zebrafish Heart

Mammals have a very limited capacity to regenerate the heart after myocardial infarction. On the other hand, the adult zebrafish regenerates its heart after apex resection or cryoinjury, making it an important model organism for heart regeneration study. However, the lack of loss-of-function methods for adult organs has restricted insights into the mechanisms underlying heart regeneration. RNA interference via different delivery systems is a powerful tool for silencing genes in mammalian cells and model organisms. We have previously reported that siRNA-encapsulated nanoparticles successfully enter cells and result in a remarkable gene-specific knockdown in the regenerating adult zebrafish heart. Here, we present a simple, rapid, and efficient protocol for the dendrimer-mediated siRNA delivery and gene-silencing in the regenerating adult zebrafish heart. This method provides an alternative approach for determining gene functions in adult organs in zebrafish and can be extended to other model organisms as well.

It remains a major challenge to develop conditional gene-knockout or effective gene-knockdown in adult zebrafish organs. Here we report a protocol for performing nanoparticle-mediated siRNA gene-silencing in adult zebrafish heart, thus providing a new loss-of-function method for studying adult organs in zebrafish and other model organisms.

纳米粒子介导的 siRNA 基因沉默在斑马鱼心脏中的表达

Mammals have a very limited capacity to regenerate the heart after myocardial infarction. On the other hand, the adult zebrafish regenerates its heart ...

Spinal Cord Transection in the Larval Zebrafish

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability to reinitiate spinal neurogenesis. However, some anamniotes including the zebrafish Danio rerio exhibit both sensory and functional recovery even after complete transection of the spinal cord. The adult zebrafish is an established model organism for studying regeneration following spinal cord injury, with sensory and motor recovery by 6&#160;weeks post-injury. To take advantage of in vivo analysis of the regenerative process available in the transparent larval zebrafish as well as genetic tools not accessible in the adult, we use the larval zebrafish to study regeneration after spinal cord transection. Here we demonstrate a method for reproducibly and verifiably transecting the larval spinal cord. After transection, our data shows sensory recovery beginning at 2&#160;days post-injury (dpi), with the C-bend movement detectable by 3 dpi and resumption of free swimming by 5 dpi. Thus we propose the larval zebrafish as a companion tool to the adult zebrafish for the study of recovery after spinal cord injury.

After spinal transection, adult zebrafish have functional recovery by six weeks post-injury. To take advantage of larval transparency and faster recovery, we present a method for transecting the larval spinal cord. After transection, we observe sensory recovery beginning at 2&#160;days post-injury, and C-bend movement by 3 days post-injury.

在斑马鱼幼虫脊髓横断

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability ...

神经科学

Stab Wound Injury of the Zebrafish Adult Telencephalon: A Method to Investigate Vertebrate Brain Neurogenesis and Regeneration

Adult zebrafish have an amazing capacity to regenerate their central nervous system after injury. To investigate the cellular response and the molecular mechanisms involved in zebrafish adult central nervous system (CNS) regeneration and repair, we developed a zebrafish model of adult telencephalic injury.
In this approach, we manually generate an injury by pushing an insulin syringe needle into the zebrafish adult telencephalon. At different post injury days, fish are sacrificed, their brains are dissected out and stained by immunohistochemistry and/or in situ hybridization (ISH) with appropriate markers to observe cell proliferation, gliogenesis, and neurogenesis. The contralateral unlesioned hemisphere serves as an internal control. This method combined for example with RNA deep sequencing can help to screen for new genes with a role in zebrafish adult telencephalon neurogenesis, regeneration, and repair.

To shed light on the cellular and molecular mechanisms of zebrafish adult neurogenesis and regeneration, we developed a protocol for invasive surgery causing mechanical injuries in the zebrafish adult telencephalon and subsequent monitoring of changes in the stabbed hemisphere by immunohistochemistry or in situ hybridization.

斑马鱼成人端脑的一刀，受伤:一个方法调查脊椎动物脑部神经发​​生与再生

Adult zebrafish have an amazing capacity to regenerate their central nervous system after injury. To investigate the cellular response and the molecular ...

Examining Muscle Regeneration in Zebrafish Models of Muscle Disease

Skeletal muscle has a remarkable ability to regenerate following injury, which is driven by obligate tissue resident muscle stem cells. Following injury, the muscle stem cell is activated and undergoes cell proliferation to generate a pool of myoblasts, which subsequently differentiate to form new muscle fibers. In many muscle wasting conditions, including muscular dystrophy and ageing, this process is impaired resulting in the inability of muscle to regenerate. The process of muscle regeneration in zebrafish is highly conserved with mammalian systems providing an excellent system to study muscle stem cell function and regeneration, in muscle wasting conditions such as muscular dystrophy. Here, we present a method to examine muscle regeneration in zebrafish models of muscle disease. The first step involves the use of a genotyping platform that allows the determination of the genotype of the larvae prior to eliciting an injury. Having determined the genotype, the muscle is injured using a needle stab, following which polarizing light microscopy is used to determine the extent of muscle regeneration. We therefore provide a high throughput pipeline which allows the examination of muscle regeneration in zebrafish models of muscle disease.

Skeletal muscle regeneration is driven by tissue resident muscle stem cells, which are impaired in many muscle diseases such as muscular dystrophy, and this results in the inability of muscle to regenerate. Here, we describe a protocol that allows the examination of muscle regeneration in zebrafish models of muscle disease.

检查斑马鱼肌肉疾病模型中的肌肉再生

Skeletal muscle has a remarkable ability to regenerate following injury, which is driven by obligate tissue resident muscle stem cells. Following injury, ...

Stab Wound Injury Model of the Adult Optic Tectum Using Zebrafish and Medaka for the Comparative Analysis of Regenerative Capacity

While zebrafish have a superior capacity to regenerate their central nervous system (CNS), medaka has a lower CNS regenerative capacity. A brain injury model was developed in the adult optic tectum of zebrafish and medaka and comparative histological and molecular analyses were performed to elucidate the molecular mechanisms regulating the high regenerative capacity of this tissue across these fish species. Here a stab wound injury model is presented for the adult optic tectum using a needle and histological analyses for proliferation and differentiation of the neural stem cells (NSCs). A needle was manually inserted into the central region of the optic tectum, and then the fish were intracardially perfused, and their brains were dissected. These tissues were then cryosectioned and evaluated using immunostaining against the appropriate NSC proliferation and differentiation markers. This tectum injury model provides robust and reproducible results in both zebrafish and medaka, allowing for comparing NSC responses after injury. This method is available for small teleosts, including zebrafish, medaka, and African killifish, and enables us to compare their regenerative capacity and investigate unique molecular mechanisms.

A mechanical brain injury model in the adult zebrafish is described to investigate the molecular mechanisms regulating their high regenerative capacity. The method explains to create a stab wound injury in the optic tectum of multiple species of small fish to evaluate the regenerative responses using fluorescent immunostaining.

基于斑马鱼和青鳉鱼的成年视锥刺伤损伤模型的再生能力比较分析

While zebrafish have a superior capacity to regenerate their central nervous system (CNS), medaka has a lower CNS regenerative capacity. A brain injury ...

<meta charset="utf8"></head><body>用于再生和发育研究的稳健斑马鱼细胞移植

High-resolution Cell Transplantation in Embryonic and Larval Zebrafish

Development and regeneration occur by a process of genetically encoded spatiotemporally dynamic cellular interactions. The use of cell transplantation between animals to track cell fate and to induce mismatches in the genetic, spatial, or temporal properties of donor and host cells is a powerful means of examining the nature of these interactions. Organisms such as chick and amphibians have made crucial contributions to our understanding of development and regeneration, respectively, in large part because of their amenability to transplantation. The power of these models, however, has been limited by low genetic tractability. Likewise, the major genetic model organisms have lower amenability to transplantation. 
The zebrafish is a major genetic model for development and regeneration, and while cell transplantation is common in zebrafish, it is generally limited to the transfer of undifferentiated cells at the early blastula and gastrula stages of development. In this article, we present a simple and robust method that extends the zebrafish transplantation window to any embryonic or larval stage between at least 1 and 7 days post fertilization. The precision of this approach allows for the transplantation of as little as one cell with near-perfect spatial and temporal resolution in both donor and host animals. While we highlight here the transplantation of embryonic and larval neurons for the study of nerve development and regeneration, respectively, this approach is applicable to a wide range of progenitor and differentiated cell types and research questions.

<meta charset="utf8"></head><body>作者聚焦:绘制发育和再生过程中斑马鱼神经系统中的细胞连接图

Here we present a protocol to transplant cells with high spatial and temporal resolution in zebrafish embryos and larvae at any stage between at least 1 and 7 days post fertilization.

胚胎和幼体斑马鱼的高分辨率细胞移植

Development and regeneration occur by a process of genetically encoded spatiotemporally dynamic cellular interactions. The use of cell transplantation ...

使用提顿体系的研究斑马鱼再生的分子机制

摘要

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此视频中的章节

肾再生的成年斑马鱼庆大霉素性损伤

捕获组织修复的斑马鱼幼虫与时间推移明视立体显微镜

纳米粒子介导的 siRNA 基因沉默在斑马鱼心脏中的表达

在斑马鱼幼虫脊髓横断

斑马鱼成人端脑的一刀，受伤:一个方法调查脊椎动物脑部神经发生与再生

检查斑马鱼肌肉疾病模型中的肌肉再生

基于斑马鱼和青鳉鱼的成年视锥刺伤损伤模型的再生能力比较分析

胚胎和幼体斑马鱼的高分辨率细胞移植

胚胎和幼体斑马鱼的高分辨率细胞移植

胚胎和幼体斑马鱼的高分辨率细胞移植

使用提顿体系的研究斑马鱼再生的分子机制

摘要

探索更多视频

此视频中的章节

肾再生的成年斑马鱼庆大霉素性损伤

捕获组织修复的斑马鱼幼虫与时间推移明视立体显微镜

纳米粒子介导的 siRNA 基因沉默在斑马鱼心脏中的表达

在斑马鱼幼虫脊髓横断

斑马鱼成人端脑的一刀，受伤:一个方法调查脊椎动物脑部神经发​​生与再生

检查斑马鱼肌肉疾病模型中的肌肉再生

基于斑马鱼和青鳉鱼的成年视锥刺伤损伤模型的再生能力比较分析

胚胎和幼体斑马鱼的高分辨率细胞移植

胚胎和幼体斑马鱼的高分辨率细胞移植

胚胎和幼体斑马鱼的高分辨率细胞移植

斑马鱼成人端脑的一刀，受伤:一个方法调查脊椎动物脑部神经发生与再生