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?...

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 border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<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&#8217;, 6- diamidino-2-phenylinodole (Dapi)</td>
			<td>Sigma-Aldrich</td>
			<td>D9542</td>
			<td>use 1/5000 in PBS 
			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) 
			for TetResponder induction</td>
		</tr>
		<tr>
			<td>E3 embryo medium&#160;</td>
			<td></td>
			<td></td>
			<td>5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2&#903; 2 H2O, 0.33 mM MgSO4&#903;7 H2O, 0.2 &#8240; (w/v) methylene blue, pH 6.5 
			for embryo/larvae husbandry</td>
		</tr>
		<tr>
			<td>Paraformaldehyde (PFA)</td>
			<td>Sigma-Aldrich</td>
			<td>P6148</td>
			<td>4 % (w/v) paraformaldehyde in PBS, pH 7.5 
			for fixation</td>
		</tr>
		<tr>
			<td>1x Phosphat-buffer saline (PBS)</td>
			<td></td>
			<td></td>
			<td>1.7 mM KH2PO4, 5.2 mM Na2HPO4, 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&#160;</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 
			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

Hepatocyte-specific Ablation in Zebrafish to Study Biliary-driven Liver Regeneration

The liver has a great capacity to regenerate. Hepatocytes, the parenchymal cells of the liver, can regenerate in one of two ways: hepatocyte- or biliary-driven liver regeneration. In hepatocyte-driven liver regeneration, regenerating hepatocytes are derived from preexisting hepatocytes, whereas, in biliary-driven regeneration, regenerating hepatocytes are derived from biliary epithelial cells (BECs). For hepatocyte-driven liver regeneration, there are excellent rodent models that have significantly contributed to the current understanding of liver regeneration. However, no such rodent model exists for biliary-driven liver regeneration. We recently reported on a zebrafish liver injury model in which BECs extensively give rise to hepatocytes upon severe hepatocyte loss. In this model, hepatocytes are specifically ablated by a pharmacogenetic means. Here we present in detail the methods to ablate hepatocytes and to analyze the BEC-driven liver regeneration process. This hepatocyte-specific ablation model can be further used to discover the underlying molecular and cellular mechanisms of biliary-driven liver regeneration. Moreover, these methods can be applied to chemical screens to identify small molecules that augment or suppress liver regeneration.

To help assess the molecular mechanisms underlying zebrafish biliary-driven liver regeneration, we established a liver injury model in which the nitroreductase-expressing hepatocytes are genetically ablated upon metronidazole treatment. In this protocol, we describe how to adeptly manipulate, monitor and analyze hepatocyte ablation and biliary-driven liver regeneration.

肝细胞特异性消融在斑马鱼研究胆驱动肝再生

The liver has a great capacity to regenerate. Hepatocytes, the parenchymal cells of the liver, can regenerate in one of two ways: hepatocyte- or ...

科研

发育生物学

Tracking Cells in GFP-transgenic Zebrafish Using the Photoconvertible PSmOrange System

The rapid development of transparent zebrafish embryos (Danio rerio) in combination with fluorescent labelings of cells and tissues allows visualizing developmental processes as they happen in the living animal. Cells of interest can be labeled by using a tissue specific promoter to drive the expression of a fluorescent protein (FP) for the generation of transgenic lines. Using fluorescent photoconvertible proteins for this purpose additionally allows to precisely follow defined structures within the expression domain. Illuminating the protein in the region of interest, changes its emission spectrum and highlights a particular cell or cell cluster leaving other transgenic cells in their original color. A major limitation is the lack of known promoters for a large number of tissues in the zebrafish. Conversely, gene- and enhancer trap screens have generated enormous transgenic resources discretely labeling literally all embryonic structures mostly with GFP or to a lesser extend red or yellow FPs. An approach to follow defined structures in such transgenic backgrounds would be to additionally introduce a ubiquitous photoconvertible protein, which could be converted in the cell(s) of interest. However, the photoconvertible proteins available involve a green and/or less frequently a red emission state1 and can therefore often not be used to track cells in the FP-background of existing transgenic lines. To circumvent this problem, we have established the PSmOrange system for the zebrafish2,3. Simple microinjection of synthetic mRNA encoding a nuclear form of this protein labels all cell nuclei with orange/red fluorescence. Upon targeted photoconversion of the protein, it switches its emission spectrum to far red. The quantum efficiency and stability of the protein makes PSmOrange a superb cell-tracking tool for zebrafish and possibly other teleost species.

We established the photoconvertible PSmOrange system as a powerful, straight-forward and cost inexpensive tool for in vivo cell tracking in GFP transgenic backgrounds. This protocol describes its application in the zebrafish model system.

在GFP-转基因斑马鱼使用Photoconvertible PSmOrange系统追踪单元

The rapid development of transparent zebrafish embryos (Danio rerio) in combination with fluorescent labelings of cells and tissues allows visualizing ...

Development of an Ethanol-induced Fibrotic Liver Model in Zebrafish to Study Progenitor Cell-mediated Hepatocyte Regeneration

Sustained liver fibrosis with continuation of extracellular matrix (ECM) protein build-up results in the loss of cellular competency of the liver, leading to cirrhosis with hepatocellular dysfunction. Among multiple hepatic insults, alcohol abuse can lead to significant health problems including liver failure and hepatocellular carcinoma. Nonetheless, the identity of endogenous cellular sources that regenerate hepatocytes in response to alcohol has not been properly investigated. Moreover, few studies have effectively modeled hepatocyte regeneration upon alcohol-induced injury. We recently reported on establishing an ethanol (EtOH)-induced fibrotic liver model in zebrafish in which hepatic progenitor cells (HPCs) gave rise to hepatocytes upon near-complete hepatocyte loss in the presence of fibrogenic stimulus. Furthermore, through chemical screens using this model, we identified multiple small molecules that enhance hepatocyte regeneration. Here we describe in detail the procedures to develop an EtOH-induced fibrotic liver model and to perform chemical screens using this model in zebrafish. This protocol will be a critical tool to delineate the molecular and cellular mechanisms of how hepatocyte regenerates in the fibrotic liver. Furthermore, these methods will facilitate potential discovery of novel therapeutic strategies for chronic liver disease in vivo.

Sustained fibrosis with deposition of excessive extracellular matrix proteins leads to cirrhosis. Alcohol abuse is one of the main causes of severe liver disease. We established an ethanol-induced zebrafish fibrotic liver model to study the mechanisms and strategies of promoting hepatocyte regeneration upon alcohol-induced injury.

乙醇引起的肝纤维化模型的斑马鱼发展研究祖细胞介导的​​肝细胞的再生

Sustained liver fibrosis with continuation of extracellular matrix (ECM) protein build-up results in the loss of cellular competency of the liver, leading ...

Use of Immunolabeling to Analyze Stable, Dynamic, and Nascent Microtubules in the Zebrafish Embryo

Microtubules (MTs) are dynamic and fragile structures that are challenging to image in vivo, particularly in vertebrate embryos. Immunolabeling methods are described here to analyze distinct populations of MTs in the developing neural tube of the zebrafish embryo. While the focus is on neural tissue, this methodology is broadly applicable to other tissues. The procedures are optimized for early to mid-somitogenesis-stage embryos (1 somite to 12 somites), however they can be adapted to a range of other stages with relatively minor adjustments. The first protocol provides a method to assess the spatial distribution of stable and dynamic MTs and perform a quantitative analysis of these populations with image-processing software. This approach complements existing tools to image microtubule dynamics and distribution in real-time, using transgenic lines or transient expression of tagged constructs. Indeed, such tools are very useful, however they do not readily distinguish between dynamic and stable MTs. The ability to image and analyze these distinct microtubule populations has important implications for understanding mechanisms underlying cell polarization and morphogenesis. The second protocol outlines a technique to analyze nascent MTs specifically. This is accomplished by capturing the de novo growth properties of MTs over time, following microtubule depolymerization with the drug nocodazole and a recovery period after drug washout. This technique has not yet been applied to the study of MTs in zebrafish embryos, but is a valuable assay for investigating the in vivo function of proteins implicated in microtubule assembly.

Immunolabeling methods to analyze distinct populations of microtubules in the developing zebrafish brain are described here, which are broadly applicable to other tissues. The first protocol outlines an optimized method for immunolabeling stable and dynamic microtubules. The second protocol provides a method to image and quantify nascent microtubules specifically.

利用 Immunolabeling 分析斑马鱼胚胎中的稳定、动态和初生的微管

Microtubules (MTs) are dynamic and fragile structures that are challenging to image in vivo, particularly in vertebrate embryos. Immunolabeling methods ...

Methods for the Study of Regeneration in Stentor

Cells need to be able to regenerate their parts to recover from external perturbations. The unicellular ciliate Stentor coeruleus is an excellent model organism to study wound healing and subsequent cell regeneration. The Stentor genome became available recently, along with modern molecular biology methods, such as RNAi. These tools make it possible to study single-cell regeneration at the molecular level. The first section of the protocol covers establishing Stentor cell cultures from single cells or cell fragments, along with general guidelines for maintaining Stentor cultures. Culturing Stentor in large quantities allows for the use of valuable tools like biochemistry, sequencing, and mass spectrometry. Subsequent sections of the protocol cover different approaches to inducing regeneration in Stentor. Manually cutting cells with a glass needle allows studying the regeneration of large cell parts, while treating cells with either sucrose or urea allows studying the regeneration of specific structures located at the anterior end of the cell. A method for imaging individual regenerating cells is provided, along with a rubric for staging and analyzing the dynamics of regeneration. The entire process of regeneration is divided in three stages. By visualizing the dynamics of the progression of a population of cells through the stages, the heterogeneity in regeneration timing is demonstrated.

Methods for the Study of Regeneration in Stentor

The giant ciliate, Stentor coeruleus, is an excellent system to study regeneration and wound healing. We present procedures for establishing Stentor cell cultures from single cells or cell fragments, inducing regeneration by cutting cells, chemically inducing the regeneration of membranellar band and oral apparatus, imaging, and analysis of cell regeneration.

Stentor再生的研究方法

Cells need to be able to regenerate their parts to recover from external perturbations. The unicellular ciliate Stentor coeruleus is an excellent model ...

Whole-Mount In Situ Hybridization in Zebrafish Embryos and Tube Formation Assay in iPSC-ECs to Study the Role of Endoglin in Vascular Development

Vascular development is determined by the sequential expression of specific genes, which can be studied by performing in situ hybridization assays in zebrafish during different developmental stages. To investigate the role of endoglin(eng) in vessel formation during the development of hereditary hemorrhagic telangiectasia&#160;(HHT), morpholino-mediated targeted knockdown of eng in zebrafish are used to study its temporal expression and associated functions. Here, whole-mount in situ RNA hybridization (WISH) is employed for the analysis of eng and its downstream genes in zebrafish embryos. Also, tube formation assays are performed in HHT patient-derived induced pluripotent stem cell-differentiated&#160;endothelial cells (iPSC-ECs; with eng mutations). A specific signal amplifying system using the whole amount In Situ Hybridization &#8211; WISH provides higher resolution and lower background results compared to traditional methods. To obtain a better signal, the post-fixation time is adjusted to 30 min after probe hybridization. Because fluorescence staining is not sensitive in zebrafish embryos, it is replaced with diaminobezidine (DAB) staining here. In this protocol, HHT&#160;patient-derived iPSC lines containing an eng mutation are differentiated into endothelial cells. After coating a plate with basement membrane matrix for 30 min at 37 &#176;C, iPSC-ECs are seeded as a monolayer into wells and kept at 37 &#176;C for 3 h. Then, the tube length and number of branches are calculated using microscopic images. Thus, with this improved WISH protocol, it is shown that reduced eng expression affects endothelial progenitor formation in zebrafish embryos. This is further confirmed by tube formation assays using iPSC-ECs derived from a patient with HHT. These assays confirm the role for eng in early vascular development.

Presented here is a protocol for whole-mount in situ RNA hybridization analysis in zebrafish and tube formation assay in patient-derived induced pluripotent stem cell-derived endothelial cells to study the role of endoglin in vascular formation.

斑马鱼胚胎全山原位杂交和管状测定在iPSC-ECs中研究恩多林在血管发育中的作用

Vascular development is determined by the sequential expression of specific genes, which can be studied by performing in situ hybridization assays in ...

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, ...

Light-Induced GFP Expression in Zebrafish Embryos using the Optogenetic TAEL/C120 System

Inducible gene expression systems are an invaluable tool for studying biological processes. Optogenetic expression systems can provide precise control over gene expression timing, location, and amplitude using light as the inducing agent. In this protocol, an optogenetic expression system is used to achieve light-inducible gene expression in zebrafish embryos. This system relies on an engineered transcription factor called TAEL based on a naturally occurring light-activated transcription factor from the bacterium E. litoralis. When illuminated with blue light, TAEL dimerizes, binds to its cognate regulatory element called C120, and activates transcription. This protocol uses transgenic zebrafish embryos that express the TAEL transcription factor under the control of the ubiquitous ubb promoter. At the same time, the C120 regulatory element drives the expression of a fluorescent reporter gene (GFP). Using a simple LED panel to deliver activating blue light, induction of GFP expression can first be detected after 30 min of illumination and reaches a peak of more than 130-fold induction after 3 h of light treatment. Expression induction can be assessed by quantitative real-time PCR (qRT-PCR) and by fluorescence microscopy. This method is a versatile and easy-to-use approach for optogenetic gene expression.

Optogenetics is a powerful tool with wide-ranging applications. This protocol demonstrates how to achieve light-inducible gene expression in zebrafish embryos using the blue light-responsive TAEL/C120 system.

使用光遗传TAEL/C120系统在斑马鱼胚胎中的光诱导GFP表达

Inducible gene expression systems are an invaluable tool for studying biological processes. Optogenetic expression systems can provide precise control ...

Eye Removal in Living Zebrafish Larvae to Examine Innervation-dependent Growth and Development of the Visual System

Zebrafish exhibit remarkable life-long growth and regenerative abilities. For example, specialized stem cell niches established during embryogenesis support continuous growth of the entire visual system, both in the eye and the brain. Coordinated growth between the retinae and the optic tectum ensures accurate retinotopic mapping as new neurons are added in the eyes and brain. To address whether retinal axons provide crucial information for regulating tectal stem and progenitor cell behaviors such as survival, proliferation, and/or differentiation, it is necessary to be able to compare innervated and denervated tectal lobes within the same animal and across animals. 
Surgical removal of one eye from living larval zebrafish followed by observation of the optic tectum achieves this goal. The accompanying video demonstrates how to anesthetize larvae, electrolytically sharpen tungsten needles, and use them to remove one eye. It next shows how to dissect brains from fixed zebrafish larvae. Finally, the video provides an overview of the protocol for immunohistochemistry and a demonstration of how to mount stained embryos in low-melting-point agarose for microscopy.

The article explains how to surgically remove eyes from living zebrafish larvae as the first step toward investigating how retinal input influences optic tectum growth and development. In addition, the article provides information about larval anesthetization, fixation, and brain dissection, followed by immunohistochemistry and confocal imaging.

活斑马鱼幼虫的眼睛去除以检查视觉系统的神经支配依赖性生长和发育

Zebrafish exhibit remarkable life-long growth and regenerative abilities. For example, specialized stem cell niches established during embryogenesis ...

Primary Cell Cultures to Study the Regeneration Potential of Murine M&#252;ller Glia after MicroRNA Treatment

M&#252;ller glia (MG) are the predominant glia in the neural retina and can function as a regenerative source for retinal neurons. In lower vertebrates such as fish, MG-driven regeneration occurs naturally; in mammals, however, stimulation with certain factors or genetic/epigenetic manipulation is required. Since MG comprise only 5% of the retinal cell population, there is a need for model systems that allow the study of this cell population exclusively. One of these model systems is primary MG cultures that are reproducible and can be used for a variety of applications, including molecule/factor screening and identification, testing of compounds or factors, cell monitoring, and/or functional tests. This model is used to study the potential of murine MG to convert into retinal neurons after supplementation or inhibition of microRNAs (miRNAs) via transfection of artificial miRNAs or their inhibitors. The use of MG-specific reporter mice in combination with immunofluorescent labeling and single-cell RNA sequencing (scRNA-seq) confirmed that 80%-90% of the cells found in these cultures are MG. Using this model, it was discovered that miRNAs can reprogram MG into retinal progenitor cells (RPCs), which subsequently differentiate into neuronal-like cells. The advantages of this technique are that miRNA candidates can be tested for their efficiency and outcome before their usage in in vivo applications.

Primary Cell Cultures to Study the Regeneration Potential of Murine M&amp;#252;ller Glia after MicroRNA Treatment

M&#252;ller glia primary cultures obtained from mouse retinas represent a very robust and reliable tool to study the glial conversion into retinal progenitor cells after microRNA treatment. Single molecules or combinations can be tested before their subsequent application of in vivo approaches.

原代细胞培养物研究小鼠穆勒胶质细胞在微RNA处理后的再生潜力

M&#252;ller glia (MG) are the predominant glia in the neural retina and can function as a regenerative source for retinal neurons. In lower vertebrates ...