Name: in vivo modeling of the morbid human genome using danio rerio
Uploaded: 2013-08-24T14:00:00.000+00:00
Duration: 12 min 31 s
Description: Watch this Scientific Journal Video about In Vivo Modeling of the Morbid Human Genome using Danio rerio at JoVE.com

我们从一个杜克大学大学学院院长的暑期研究奖学金（AN），美国心脏协会（AHA）奖学金11POST7160006（CG），美国国立卫生研究院（NIH）拨款R01-EY021872从国家眼科研究所（EED），R01HD04260从承认支持国立儿童健康和发展研究所（NK），R01DK072301 R01DK075972从国家糖尿病消化和肾脏疾病研究所（NK），欧洲联盟（欧盟​​7 日 FP资助下GA NR。241955，项目SYSCILIA; EED NK）NK是一个杰出的吉恩和乔治·W·布伦利教授。

1。生物信息学分析<ol><li>确定人类基因的斑马鱼的同源基因，如果是这样，多少份。我们建议互惠的的BLAST（ <a href="http://blast.ncbi.nlm.nih.gov/" target="_blank">http://blast.ncbi.nlm.nih.gov/</a> ）人的蛋白质对斑马鱼的基因组中，最好的斑马鱼撞到了人类基因组和随后爆炸。真同源将是最好的打在每个实例。 </li><li>确定人类基因的开放阅读框（ORF）的大小。如果超过6 KB，这种模式是目前棘手的，因为长的模板体外转录的高品质的局限性。 </li><li>获取或生成一个结构，其中包含人类ORF PCS2 +载体骨架（或等效，5&#39;有SP6转录位点和3&#39;多聚腺苷酸信号的向量）。 </li><li>设计一个MO阻止有针对性的斑马鱼基因的剪接或翻译。如果存在多个副本在斑马鱼的基因组中，有E是两个选项:a）额外MOS设计或b）完全保守的副本之间的剪接位点的识别对单一MO可以有效。一些出版MO序列提供<a href="http://www.zfin.org" target="_blank">www.zfin.org</a> 。 </li></ol> 2。发展的斑马鱼胚胎中的表达分析<ol><li>确定是否在斑马鱼的直向同源物在时空上下文相关的表型读出表示。如果没有感兴趣的基因表达数据，进行逆转录（RT）-PCR方法从整个斑马鱼胚胎或原位杂交基因。 （见图2,3）。 </li></ol> 3。定点突变<ol><li>设计诱变引物，25〜45个碱基的长度，在中心与所需的突变。的引物的熔融温度应大于或等于78℃。设计一个正向和反向的诱变引物退火到相反链的质粒。 </li><li>得到的引物序列的ORF诱变后确认，这些跨〜300个碱基对的部分，以覆盖整个ORF的瓷砖。 </li><li>组装诱变反应与一个高保真聚合酶和周期如下:（1:95℃30秒，2:95℃30秒，3:55℃1分钟，4:68℃，6分钟，5: 2 18X，6:4°C天长地久，7月底）。 </li><li>加入1微升DpnI限制性内切酶每反应除去坝甲基化模板;在37°C孵育2小时。 </li><li>根据标准的协议变换的2微升到20微升的感受态细胞的诱变反应。 </li><li>挑选3-4个菌落，接种5毫升含有适当抗生素的LB培养基。在225转摇在37℃过夜。 </li><li>少量的DNA，并确定浓度。 </li><li>序列的突变位点，确定感兴趣的突变的存在。 </li><li>整个ORF序列确认序列的完整性。 </li></ol> （4） 在体外的mRNA转录<ol><li>使用一个线性PCS2的+模板，生成皑皑的mRNA使用mMessage mMachine SP6试剂盒（Ambion公司）。我们建议使用反应元件数量的一半。 </li><li>与氯化锂沉淀或试剂盒说明书中所描述的酚 - 氯仿纯化的RNA样品。 </li><li>确定使用的吸光度的mRNA的浓度，确保使用凝胶电泳的mRNA的完整性，并在三个或更多的等分试样在-80℃下储存样品，直到准备使用。我们不建议多个冻融循环的mRNA等分。 </li></ol> 5， 在体内含量的变异功能丧失<ol><li>从天然斑马鱼交配获得斑马鱼胚胎，并保持在28°C在胚胎水在6或10厘米的菜。 </li><li>进行吗啉的剂量反应曲线来评估型特异性，MO效率和MO毒性的。注入剂量曲线至少有三种不同的浓度成50-100（1-4细胞阶段）斑马鱼胚胎/批1-10纳克MO之间。高效的MO产生剂量依赖性的增加的比例在一个批处理中受影响的胚胎。 </li><li>在适当的表型胚胎发育阶段的基础上针对性的斑马鱼基因表达的兴趣和在哪个阶段，将观察相关表型。这可以是定量的（如解剖结构之间的测量）或质量（基于标准化的表型标准）。所有注射得分&gt; 24 HPF:胚胎应被视为与PTU（0.003％1 - 苯基-2 - 硫脲在胚胎媒体）在24 HPF最大限度地减少黑色素细胞的形成。 </li><li>对于拼接 - 阻塞的MO中，通过提取总RNA的全胚胎裂解物试验MO效率的表型的得分在时间点，产生的cDNA的靶基因侧翼MO目标站点的使用引物进行RT-PCR检测。 </li><li>要验证压制EFFICiency的TB-MO，收获全胚胎蛋白裂解，并进行免疫比较有针对性的蛋白质与控制水平。然而，这种方法不适合于所有的靶基因，因为是有限的为许多商业斑马鱼的蛋白质的抗体的交叉反应性。表示MO特异性的间接方法，包括:一）证明，有一个对表型的剂量依赖效应;和b）示出野生型的mRNA与tb的-MO，共注射有效抢救的表型。出于这些原因，我们建议拼接阻滞剂时可能的，因为可以直接监控效率。 </li><li>如果观察到的表型进行到步骤5.7，如果没有观察到的表型进行到步骤6.1。 </li><li>对于定性的表型，选择一个MO剂量50-75％的胚胎受到影响;定量表型，选择一个MO剂量表型的措施是显着不同的野生型（P &lt;0.001）。注入新的批次斑马鱼EMBryos（1-4细胞; N = 50-100/batch）位置与MO的"试验"剂量和剂量曲线的人体重量的mRNA（范围从10-200皮克表达的鸡尾酒;这些剂量，以确保大幅度过度表达上述任何单一的成绩单的基线，占多聚腺苷酸总mRNA在斑马鱼胚胎的0.25-0.5％）。 </li><li>注射批次进行屏蔽的得分;选择WT mRNA的剂量在比较MO单独的最显着的救援，这是"试验"剂量的mRNA。 </li><li> MO和人类突变基因检测剂量测定剂量注入新的批次（1-4细胞阶段; N = 50-100/batch时）。表型的胚胎在适当阶段和比较结果WT的人类mRNA的救援用适当的统计检验（t检验或卡方）。参见图1，结果及进行第7步。注入试验剂量WT和突变基因控制基因毒性效应。 </li></ol> 6。 在体内含量的变异DOMinant负增益功能的影响<ol><li>如果没有损失的功能表型观察（步骤5.5），或者如果突变基因产生的表型没有显着不同的从MO单独（步骤5.7），注入剂量曲线从10-200皮克WT人类基因（我们建议25，50作为初始试验，100皮克）成胚胎批次（1-4细胞期50-100胚胎/批次）。 </li><li>在适当的阶段（见5.3段），进行表型得分，并确定最高剂量是没有注射的对照组比较有统计学显着数量的死亡和/或受影响的胚胎。这是"试验"剂量。 </li><li>注入检测人类基因突变剂量（1-4细胞期50-100胚胎/批次）。表型胚胎和比较WT人类基因注射或检测MO浓度的测定剂量得分。参见图1的结果。 </li><li>如果结果是无法区分从MO，滴定人类的突变mRNA与WT表达比较人类WT和突变的mRNA注射。表型与突变加上WT的mRNA注入批次的改进表示了显性负。没有改善表示增益功能。 </li></ol> 7。 体内试验结果重现<ol><li> 在体内试验中重复一个最低的3倍。 </li></ol> 8。与其他行的证据，斑马鱼体内的致病性数据整合<ol><li>致病性比较从斑马鱼实验获得的数据:遗传谱系内的数据（如适用），控制人口的频率数据， 体外研究（基于细胞的检测蛋白质的稳定性，细胞定位，信号输出，或酶活性）。 </li></ol>

作者宣称，他们有没有竞争经济利益。

这里描述的方法表示的一般协议，适用于非同义变化与人类遗传病的表型（ 表2，图3）的各种测定。我们的方法已被证明可用于评价疾病表型变异的潜在影响，并帮助剖析疾病的机制（如显性负突变Bardet-Biedl综合征的贡献，主要是常染色体隐性遗传疾病，17）。迄今为止，通过所提出的决策树的发展，我们已以合理的成本和时间超过200个基因与遗传性疾病的因果关系模型，过量的1,000个等位基因。 虽然没有在这里详细讨论，我们也表明这些方法是适当的其他类型的遗传病变，如拷贝数变异（CNVs），以及和遗传相互作用模型。这类事件的分析范围之外本方法的描述，虽然他们从根本上依赖于系统的测试原理相同的候选基因（包括对基因同时注入），以确定适合临床表型诱导或加重。例如，要澄清的29个基因在16p11.2 CNV可能会对相关头畸形所观察到的在重复的660 kb的基因组分部患者中观察到，每个段内的29个基因的mRNA的相对应的注入和头部2 DPF和DPF进行尺寸测量，一个单一的成绩单，KCTD13揭示了重大贡献。21此外，我们已经使用这个模型来检测基因病变的遗传相互作用Bardet Biedl综合征和先天性巨结肠症的患者22 MO抑制的因果基因的两个临床身份分别和同时通过比较，我们能够识别所得到的表型为贝纳克的协同互动，而不是仅仅添加剂的严重性。 尽管建立了高灵敏度（98％）和特异性（&gt; 82％）有助于ciliopathies 17的变种，我们尚未有足够的数据，以确定是否这些都是推广到在斑马鱼模型中的所有表型的读数。要做到这一点，有大量的等位基因，基因预测是良性的或致病的，必须进行测试，在每个表型类别之内。这种检测在临床上实施，这将是尤为重要，其中功能解释VUSs可以告知只有充分理解这样的结果给医生和患者的假阳性和假阴性，可以陪交付的诊断和管理。然而，这些方法可以显着贡献，对更好地了解人类遗传疾病的景观。我们预计，这些模型不仅可以作为一个FOUNdation改善临床遗传信息的解释，但也将被作为有用的模型进行治疗屏幕在体内的数据可能也被比较到硅片计算预测从如PolyPhen 23来源，SIFT 24，单核苷酸多态性与GO 25，或MutPred 26表现出一致性。需要注意的是，在先前的研究中，预测数据库的单核苷酸多态性＆GO和MutPred的被认为是最准确的，精度达到0.82和0.81，分别27 尽管我们已经简要介绍了这些方法的鲁棒性小儿解剖缺陷（ 表2，图3）的一个子集，通过这些方法，某些表型不听话。尽管有些例外，主要有三个类的疾病不适合我们的通讯协议。成人发病的疾病（如帕金森氏症）是指胚胎系统模型，是一个挑战。慢PROG退行性ression的表型（如额颞叶痴呆），可能需要更多的时间比MO活动产生的表型的7个单丝旦窗口。其他的基因敲除技术，例如RNA干扰的siRNA可干扰或降解的靶标基因，但它已经表明，没有具体的，稳定的，无毒的，或长期持久的MO 28，从而也限制了时间范围表型。第三，一些脊椎动物的结构，如哺乳动物肺，不具备足够的斑马鱼胚胎中的直向同源结构。我们还提供了建议的应急计划，以调查这些案件中，WT人类基因注射导致的表型，但我们提醒这是一个不寻常的和不良的情况。 某些疾病表型可能需要更大程度的抽象和代孕。这是可能的基因的功能已充分发散减弱模型的表型之​​间的相似性和trUE的表型，或斑马鱼生理固有的复杂性引起的疾病的效果。在这种情况下，我们建议进一步解剖解雇前的生产型。我们已经产生了一些成功的例子，其中有问题的表型这个实验已在斑马鱼胚胎模型。例如，突变TCF8，相关的基因用Fuchs的角膜营养不良（FCD），分别测定使用我们的协议，通过使用原肠胚缺陷作为一个替代的表型的基础上读出该转录本在早期发展中已知的角色29在其他情况下，例如成人发病型肌营养不良症的突变引起的DNAJB6，我们能够产生肌纤维表型在5dpf胚胎尽管事实上，人类是他们的第一个三，四十年的生活失去了明显的肌肉病理19 在这里提出的的瞬时突变体模型中，其他人也采取了优势的GE这个短暂的系统来模拟人类疾病的各种身体系统。在一个实施例中，视网膜色素变性建模在斑马鱼RP2基因的敲除，导致视网膜细胞死亡和减少视网膜层叠。救援与人野生型的mRNA的发展，导致视网膜层压所有三个层，而四的5个突变体的mRNA没有抢救30尽管这种模式的一个人的感觉障碍的形态表型的基础上，也有可能检测响应声惊吓或脉冲抑制刺激如47 近日，斑马鱼模型被用来研究阿尔茨海默病的发病机制，通过淀粉样前体蛋白31研究人员发现，基因敲除引起轴突生长受损运动神经元，这可能与人类的基因被救出。这种模式已经被证明是特别翔实，小鼠模型显示只有细微的phenotYPES（单击倒）或产后杀伤力（双击倒）。评估在体内的斑马鱼胚胎的能力，在整个开发过程帮助辨别降低的淀粉样前体蛋白（APP），以及提供了直接证据，该蛋白既需要一个正常功能的细胞外和细胞内结构域的致病效果。其他著名的车型包括额外的肌营养不良症32，钻石Blackfan贫血症33，AXENFELD里格综合征（眼和颅面部发育）34，炎症性肠病35（抗菌活性），帕金森病36（神经元和运动损失），并扣押37 （脑积水和多动症）。 更常见的是已被证明也复述人类疾病表型突变的斑马鱼线。 1,38，型号包括白血病，黑色素瘤，扩张性心肌病，杜氏肌营养不良症，和其他许多人。

功能解释遗传信息和基因的基因型预测的临床应用价值的分配是一个重大的问题，在医学遗传学，并日益尖锐的全基因组测序的加速技术和经济可行性。因此，有必要制定和实施新的范例测试意义不明的变体（VUS）检测患者的致病性。这些检测必须是​​准确的，时间和成本效率，和海港的潜在催化过渡到临床实用。 当鼠标历来在该领域的首选工具的人类疾病模型，斑马鱼是新兴科学和经济有利的替代品。斑马鱼的生物学与鼠标，可以方便及时地获取所有发育阶段，借助于光学清晰度的胚胎，它允许实时成像发展中的病症。 <sup&gt; 1突变的斑马鱼线比较近的一代提供了额外的测试和建模选项，雇用了许多功能的研究，但这项技术仍然有限（1,38审查）。不仅是敲插件费力达到的特定的突变的遗传突变体，它们也适合于中等或高通量分析的范围内，在一个单一的基因突变的测试。重要的是，可以提供一个单一的测试套件不仅对致病潜力的等位基因的信息，而且在细胞水平上（ 如失去功能与增益的关系的函数），这是用于通知的遗传方式的关键效果的方向在家庭，尤其是小的时候的人类血统怀有有限的信息遗传传输模式。可用鼠标和斑马鱼模型的用途作进一步比较， 见表1。 我们也注意到，重的斑马鱼模型系统固有的局限性。尽管D.斑马鱼的器官系统有初步发展迅速，性成熟需要约3个月。正因为如此，产前和婴幼儿发性疾病是最适合于这种瞬时表达模式。虽然适合进行大型化合物屏幕，使用遗传突变是不可行的成千上万的非同义变异，有助于系统测试，并继续在儿科疾病中被检测到。 互补这里描述的测试利用这个实验的可追踪性，高度的同源性，保护人类和斑马鱼的蛋白质之间的功能，特别是保守的发育过程中所必需的分子。 图1列出了各种等位基因效果的试验和鉴定的策略。可以执行这两个功能丧失（LOF）和主检测。对于LOF，在实验开始与感兴趣的基因吗啉击倒，抑制和表型，可能是根据调查的临床表型相关的化验。抑制可达到通过目标MO的斑马鱼（翻译阻断剂吗啉; tbMO）的轨迹的翻译起始位点处或附近，或者通过干扰剪接通过放置一个拼接位点上的MO，通常诱导任列入一个阻挡翻译内含子或异常外显子跳跃（拼接阻塞吗啉; SBMO）的。 随后，介绍了从人类同源成绩单皑皑的mRNA和表型的量化救援测量。一旦建立后，测定在人的消息中的候选基因突变可以引入并测定他们的能力抢救的MO-诱导的表型在同一作为WT​​人类mRNA的效率。相反，对于候选优势等位基因，人类基因（但不是MO）是引入与，WT人类基因不会严重影响斑马鱼的解剖学和生理学，而引进测试突变，有一个主导的作用将类似于在人体临床条件下观察到的表型诱导的期望。这个实验可以是细粒度的进一步解剖是否发生的显着效果由增益函数（GOF）或显性负性的野生型和突变型人mRNA的混合机制; GOF事件，除了野生型人类mRNA的预期为不相关的，而显性负等位基因的野生型和突变型mRNA的混合应该改变的表型诱导的突变体消息的严重程度。在所有情况下，我们建议注射的所有组合（MO吗啉代的WT人类基因与突变的人类基因等，最好是在同一个离合器的胚胎（ 见图1）。释义如下: 对于LOF试验: <ul><li>如果击倒产生的表型突变体和野生型的表达可以等价获救，等位基因可能是良性的。 </li><li>如果抢救击倒型的突变从击倒表型本身没有什么区别，等位基因可能是一个功能性的空。实验无法区分真正的空值（无功能的蛋白质）和超低的蛋白质的活性水平，没有救援能力。 </li><li>如果击倒表型突变救援显优于MO，但比野生差，等位基因可能是一个hypomorph这一结果表明部分功能丧失。 </li></ul> 对于显性的测试: <ul><li>如果没有击倒型，但注射WT基因产生的表型，必须使用一个应急计划，如果实验继续进行（见下文）。 </li><li>如果没有拦截表型和野生型的mRNA注射不产生表型，像往常一样进行实验。 </li><li>如果注射突变的mRNA是相当于野生型的mRNA的等位基因可能是良性或丧失功能，或可能已发生故障的检测。这需要进一步的实验来区分这些选项。 </li><li>如果突变的mRNA注射的基因产物的功能区分MO拦截，有可能以某种方式改变。为了区分在功能上的变化，应进行的突变体与野生型的mRNA的mRNA的滴定。 </li><li>如果滴定的结果是无法区分野生型的mRNA的单独的，它已被证明，该突变体蛋白产品使用的野生型蛋白作为底物，其效果是不同的量滴定。这表明一个显性阴性表型。 </li><li>如果滴定的结果是无法区分的单突变的mRNA，它已被显示，该突变体的蛋白质产物不再具有与野生型相同的功能，并因而是不会受到量的野生型蛋白质产物预发送。这表明，等位基因是可能的增益功能。 </li></ul> 应急预案: <ul><li>如果没有表型呈现从MO击倒，但不存在与野生型基因，进一步的实验可以发生，但我们强调的是，这种情况是不理想。 WT人类mRNA的滴定，以尽量减少表型，也可以用来作为一个新的设定点。进一步共注射野生型和突变型人mRNA的可评估的基础上的救援能力的突变。 </li></ul>

<table><tbody><tr><td colspan="4">&lt;strong&gt;Reagent&lt;/strong&gt;</td></tr><tr><td>Phusion High-Fidelity DNA Polymerase</td><td>NEB</td><td>M0530S, M0530L</td><td></td></tr><tr><td>DpnI restriction endonuclease</td><td>NEB</td><td>R0176L, R0176S</td><td></td></tr><tr><td>Max Efficiency DH5&amp;alpha; competent cells</td><td>Invitrogen</td><td>18258-012</td><td></td></tr><tr><td>Big Dye Terminator</td><td>Applied Biosystems</td><td>4337455</td><td></td></tr><tr><td>mMESSAGE mMACHINE Kit </td><td>Invitrogen</td><td>AM1340, AM1344, AM1348</td><td></td></tr><tr><td>Morpholino</td><td>Gene-Tools</td><td>n/a</td><td></td></tr><tr><td>1-phenyl-2-thiourea (PTU) </td><td>Sigma Aldrich</td><td>P7629</td><td>Prepare as 0.003% PTU in embryo media </td></tr><tr><td>Paraformaldehyde (PFA)</td><td>Sigma Aldrich</td><td>P6148</td><td>For embryos that must be fixed prior to phenotyping, prepare as 4%</td></tr><tr><td>Tricaine methane sulfonate</td><td>Western Chemical</td><td>N/A</td><td>For anesthetization and euthanasia </td></tr><tr><td colspan="4">&lt;strong&gt;Equipment&lt;/strong&gt;</td></tr><tr><td>PTC-225 Tetrad Thermal Cycler</td><td>BioRad</td><td>Any equivalent thermal cycler</td><td></td></tr><tr><td>Nano Drop 2000 spectrophotometer</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>SMZ 745T Stereomicroscope</td><td>Nikon</td><td></td><td></td></tr><tr><td>AZ100 Stereomicroscope</td><td>Nikon</td><td></td><td></td></tr><tr><td>DS Fi1 Digital Camera</td><td>Nikon</td><td>For color/fluorescent imaging</td><td></td></tr><tr><td>DS QiMC Digital Camera</td><td>Nikon</td><td>For black/white imaging</td><td></td></tr><tr><td>Advanced Resarch 3.2 Imaging Software</td><td>NIS- Elements</td><td></td><td></td></tr></tbody></table>

in vivo modeling of the morbid human genome using danio rerio

在这里，我们介绍的方法分析查询使用在斑马鱼体内互补的潜在临床意义的同义变化的发展。斑马鱼 （Danio rerio）是有用的动物系统中，由于他们的实验的易处理性，胚胎是透明的，使轻便观看，经过快速发展体外，可以操纵基因。1。在这些方面的允许分析胚胎发育的显着的进步，分子过程，和骨形态发生信号。脊椎动物模型的优点，这两者合计，使斑马鱼的高度适合建模儿科疾病的发育缺陷，和在某些情况下，成人发病的疾病。因为斑马鱼的基因组与人类（〜70％的同源）是高度保守的，它是可以概括在斑马鱼的人类疾病状态。这是通过或者通过注射突变型人米RNA诱导功能等位基因显性负或收益，或利用吗啉（MO）的反义寡核苷酸抑制基因模仿丧失功能的变种。通过MO-诱导皑皑的人类基因表型的互补，我们的方法能够解释的基础上的人类蛋白质序列突变基因的能力拯救一个可衡量的，有关生理表型突变的有害影响。人类疾病的等位基因建模通过注射MO和/或在1-4细胞阶段的人类基因，7天受精后（DPF）和表型的斑马鱼胚胎发生。这是一般的策略可以扩展到很宽范围的疾病表型，这表现在下面的协议。我们提出我们建立的模型形态信号，颅面，心脏，血管的完整性，肾功能，骨骼肌肉疾病的表型，以及其他。

隐性和Pseudorecessive失调初级纤毛近乎无处不在的结构在脊椎动物体内计划，发挥细胞信号传导的角色，在多种细胞的命运，包括增殖，极性，分化和组织维护这些细胞器的功能障碍导致了广泛的人类遗传性疾病统称为称为ciliopathies 6,7这样一个临床实体Bardet Biedl综合征（BBS），多系统的儿科疾病，视网膜变性，肥胖，性腺功能低下，多指（趾），肾功能不全特征。 在体内检测致病等位基因的发展是必要的论坛因为）它是一种遗传异质性疾病引起的主要是私人的同义变化发生在至少17个基因7-10;和b）寡继承&gt; BBS家庭的25％，其中杂合子的变化在第二论坛除了隐性主要的致病突变基因（）可以调节临床外显率和表现。通常情况下，这样的第三等位基因杂合子的同义变化，从遗传学的角度来看，还不清楚致病潜力，因此需要准确的解释，他们对蛋白质功能的生物效应。11-13 探讨潜在的致病突变论坛突变负荷做出贡献，我们初步确定BBS1 - BBS14测试了所有的错义变化。我们和其他人已经表明，基体蛋白损失产生失调的平面细胞极性（PCP非经典Wnt信号）表现为收敛扩展的缺陷，在斑马鱼胚胎中体节14使用此有关生理表型读出，已经发现，抑制BBS基因体轴缩短，更广泛和更薄的体节，而宽，扭结notochords的15 &lt;/线上（图2）引起的MO抑制产生的原肠胚形成的缺陷，和共注射与人类mRNA的显着获救（再现性）这些表型的得分由三种不同的体内方法。首先，胚胎被现场拿下根据定性的表型标准（正常，I类和II类的表型类的详细定义见15）。接下来，我们在外包过程中定量细胞迁移（早期发育阶段的特点是在卵黄细胞16）通过采用荧光示踪可视化迁移细胞的细胞层变薄和传播。最后，我们测量体节的躯干长度在9体节胚胎原位杂交的鸡尾酒krox20，PAX2和myoD的的的核糖探针，持平安装形态分析。 这种方法已经被用来测试500等位基因在睫状肌突变空间超出。在一个体内检测&gt; 130等位基因独自学习，产生了一系列的表型的分数，我们的协议表示（图1）完成救援被列为良性（不显着不同的从WT救援），部分抢救被列为hypomorphs的（显着改善来自密苏里州，但更严重的比WT救援），故障救援功能空（没有显着不同MO）和表型诱导突变基因被列为主导底片相比，MO被列为。 我们也评估，灵敏度和特异性的斑马鱼体内互补实验。特异性证实了合作注入常见SNPs（&gt; 5％的次要等位基因频率在健康对照人群），这些被发现给予良性的表型，在14/17测试（&gt; 82％），和敏感性被证明是98％ 体内数据和遗传参数足以吨之间的一致性O属性等位基因作为在一个BBS血统因果。17此外，使用三个体内措施（外包跟踪，现场打分，ISH形态测量） 在体外用免疫印迹和细胞定位研究验证观察到的表型效应。虽然解释这些结果前需要了解至少一种疾病的发病机制，这个例子中提供的证据，以证明我们的协议的实用性和鲁棒性。以来，我们已经证实了我们的在体内得分与多个其他线路在一个公正的突变筛查和功能实验证据分析研究TTC21B的 ，逆行intraflagellar的转运蛋白。 主导疾病肢带型肌营养不良症（LGMD）类肌营养不良症是一种常染色体显性遗传，造成缓慢前进中在臀部和肩膀的肌肉无力。我和这个基因chanistically组异质性疾病所造成的显性和隐性突变跨越多个细胞膜，肌节，细胞质和细胞核蛋白。基于磁共振成像肌受累的临床表型和证据的呈现，我们调查的主导的原因在芬兰，美国和意大利的家庭19 LGMD发现。在映射的轨迹位置的候选人测序揭示突变在DNAJB6，至少两个剪接异构体（细胞核和细胞质）在人类基因编码的共同分子伴侣HSP70家族表示。要获得DNAJB6功能和其相关LGMD的进一步深入了解，我们在斑马鱼研究其作用在肌肉的完整性。的斑马鱼（dnajb6b）直向同源物的RT-PCR方法检测到表达为早五体节阶段，然后通过注射的胚胎与拼接阻塞的吗啉代。在受精后48小时，蒙面的得分表明支队慢纤维插入点。这种表型的特异性进行测试的第二非重叠MO，并随后与野生型人类DNAJB6 mRNA的救出。 要查询如何损失DNAJB6功能导致肌肉的完整性的缺陷，我们介绍了发现的错义突变的患者为人类转录两种异构体，并将其注射到斑马鱼的胚胎。虽然注射WT人类mRNA的产生没有明显的表型，这些变化phenocopied工程时，在细胞质中的MO的作用效果的损失，但不是核的异构体。其次是共注射等摩尔量的突变体和野生型的mRNA，这表明增强肌肉表型的严重程度，提出了显性效应。为了测试这个概念，改变突变体和野生型基因的摩尔比进行了进一步的注射。与预测相一致，较WT诱导胚胎致死突变的mRNA的过量，而Ñ​​过量生产的WT逐步增加救援。在这之后， 在体外实验中，确定oligimerization性能和可能的蛋白质的相互作用。这些表明，突变损害细胞质DNAJB6的抗凝集活性和干扰的突变型和野生营业额，以及与另一个的分子，BAG3，这也是相关的病理机制的LGMD，由于LOF BAG3导致的儿科形式肌营养不良症20。我们然后问是否BAG3可以调节表型诱导斑马鱼突变DNAJB6b的的。 ，虽然注射WT BAG3独自制作没有表型，共注射与DNAJB6突变体显着增加的表型的严重程度，这是BAG3这些突变体19介导的致病性中起重要作用。 <table border="1" fo:keep-together.within-page="always"><tbody><tr><td></td><td> 瞬态斑马鱼模型 </td> &lt;TD&gt; 突变斑马鱼线 </td><td> 转基因小鼠线 </td></tr><tr><td> 人类表型的发病年龄正在调查 </td><td><ul><li>产前</li><li>新生儿</li><li>小儿科的</li></ul></td><td><ul><li>产前</li><li>新生儿</li><li>小儿科的</li></ul></td><td><ul><li>产前</li><li>新生儿</li><li>小儿科的</li><li>成人</li></ul></td></tr><tr><td> 可行的表型 </td><td><ul><li>颅面</li><li>肌肉发达</li><li>发信号</li><li>心脏的</li><li>肾</li><li>血管</li></ul></td><td><ul><li>颅面</li><li>肌肉发达</li><li>发信号</li><li>心脏的</li><li>肾</li><li>血管</li></ul></td><td><ul><li>颅面</li><li>肌肉发达</li><li>发信号</li><li>心脏的</li><li>肾</li><li>血管</li><li>感觉的</li><li>骨骼</li><li>内脏</li><li>行为</li><li>呼吸</li><li>生殖</li><li>内分泌</li><li>代谢</li></ul></td></tr><tr><td> 直到使用时间 （从出生） </td><td> 1-7天</td><td> &gt; 3个月</td><td> &gt; 6个月</td></tr><tr><td> 吞吐量 </td><td>中高</td><td>低</td><td>低</td></tr><tr><td> 优点 </td><td><ul><li>测试特定突变的能力</li><li>能够产生连锁反应的型号</li><li>能够使用吗啉击倒</li><li>低维护成本</li></ul></td><td><ul><li>减的实验变异</li></ul></td><td><ul><li>关于建立更紧密进化关系</li><li>器官结构的保护</li><li>基因的能力率敲在模型</li></ul></td></tr></tbody></table> 表1中。 体内模型中的比较 。 <img alt="表2" src="/files/ftp_upload/50338/50338table2.jpg" fo:content-width="7in" fo:src="/files/ftp_upload/50338/50338table2highres.jpg"/> 表2中。下测试所提出的协议的实施例中体内人类dysmorphologies建模。各种表型。可采用一系列的表型的读数和可视化技术的基于无序的类型。 <a href="https://www.jove.com/files/ftp_upload/50338/50338table2highres.jpg" target="_blank">点击这里查看更大的表。</a> <img alt="图1" src="/files/ftp_upload/50338/50338fig1.jpg" /> 如图1所示。 在体内功能测试同义变种。所有功能测试系统的方法ELES模型未知或假设的意义。其次是由一系列的（共）注射野生型和突变型人mRNA的基因敲除通过吗啉代显微注射。统计分析表型的结果告知等位基因致病性和分子功能。简言之， 丧失功能测试:如果击倒产生突变体和野生型表达的表型可以等价获救的等位基因可能是良性的（绿盒）。如果击倒表型突变救援击倒表型没有区别，等位基因可能是一个功能性空（黄色框）。如果拦截表型的突变拯救比MO的显着水平，但比野生差，等位基因可能是一个hypomorph（绿色方块）。 对于显性的测试:如果突变的mRNA注射相当于野生型的mRNA的等位基因可能是良性或丧失功能，或可能已经失败的实验（绿盒）。如果突变的mRNA注射因迪斯<html>区别开从MO拦截，基因产物的功能，有可能以某种方式改变。为了区分在功能上的变化，滴定突变体与野生型的mRNA的mRNA。如果该滴定的结果是无法区分野生型的mRNA的单独突变蛋白产品使用的野生型蛋白作为底物，从而指示一个显性阴性表型（蓝色框）。如果滴定的结果是无法区分的单突变的mRNA，突变蛋白质产物不再具有与野生型的功能相同，因此，很可能的增益函数（蓝色框）。如果没有表型呈现从MO拦截，但不与野生型的mRNA的存在，可能会出现进一步的实验，WT人类mRNA的滴定，以尽量减少表型，也可以用来作为一个新的设定点。野生型和突变型人mRNA的进一步的共注射可以评价根据抢救能力的突变体（粉红色框）。ET ="_blank"&gt;点击这里查看大图。 <img alt="图2" src="/files/ftp_upload/50338/50338fig2.jpg" fo:content-width="4in" fo:src="/files/ftp_upload/50338/50338fig2highres.jpg"/> 图2。定量和定性的评价MKS1突变检测在人类中MKS1 morphant的胚胎发育缺陷。根据严重性分为三组，表型。每个类的实例所示（a）中 ，普遍存在于胚胎世代组（n = 100-160胚胎）的表列（图中未示出）。 MO注射胚胎I类表型正常形态，但有严重较短，蛋黄相比过多的胚胎组织来控制注射的胚胎在相同的体节年龄（8-9体节）。被II类与colo2a1的变薄，短而不发达的头部和尾部结构，另外缺乏体节的定义和对称性。 III类胚胎体节发育不良和畸形的严重延误，起伏notochords，通常没有生存过去的10体节阶段。共注射的人类MKS1 mRNA的救出这些缺陷，MKS1抑制证明特异性表型。胚胎在11体节阶段（±1体节）染色，krox20，PAX2和myoD的核糖探针原位杂交（乙，丙）。表型进行定量测量从第一个到最后一个明显的体节的每个胚胎（箭头）， 在 c定量。图改编许可15。 <img alt="图3" src="/files/ftp_upload/50338/50338fig3.jpg" fo:content-width="3.25in" fo:src="/files/ftp_upload/50338/50338fig3highres.jpg"/> 图3。人类畸形学的体内模型中的例子。 （一）颅面畸形学。控制基因注射胚胎（左）和突变体注射胚胎（右）在5 DPF阿尔辛蓝染色。突变的mRNA注入胚胎显示特别是小，畸形头张开鳃弓和缺失或异常结构包括颅面骨软骨与一般的混乱。（二）微/大头畸形。控制未注射胚胎（左）和kctd13 MO注射胚胎（右）在5 DPF。与colo2a1显示加宽的头部，所看到的眼睛之间的空间21（c）减少血管的完整性。控制未注射胚胎（顶部）和2 DPF在FLI1用荧光显微镜成像英 MO注射胚胎（底部）:绿色荧光蛋白转基因记者线。与colo2a1显示减值发芽间血管和其他血管结构。41（D）单向原位杂交心脏循环改变。野生型胚胎njected（左）显示SPAW表达在左外侧板中胚层，而ccdc39 morphant胚胎表现为双侧（右），在大多数情况下，检测不到的SPAW表达（未显示），43（E）肾囊肿。未注射WT胚胎（顶部）和ift80 morphant的（底部）。的显示与colo2a1大的肾囊肿（箭头），心包水肿（箭头）和卷曲的尾巴。44（F）肾小球滤过减少。荧光可视化控制注射胚胎（顶部）和罗丹明右旋糖酐注射到心脏后ift80 morphant的（底部）24小时。荧光分散在整个血管系统和几乎完全抽空肾脏观察荧光，完全没有控制。 morphant的显示持久的荧光右旋糖酐，提示肾小球滤过减少46（克）肌肉萎缩症。 WT DNAJB6先生NA注射胚胎（顶部）显示正常的慢肌纤维跨越体节通常相邻myosepta之间的如使用防慢肌球蛋白抗体免疫组化确定。突变DNAJBb（底部）显示在一个或多个个体节，局部完成支队从myosepta肌纤维，19（H）体节角。放大控制注射的（顶部）或底部）kif7的与colo2a1（30高倍视野成像的现场侧面的景致。的显示与colo2a1异常形状的体节，占异位刺猬信号在斑马鱼的肌节47 经许可后改编的所有数字。

Watch this Scientific Journal Video about In Vivo Modeling of the Morbid Human Genome using Danio rerio at JoVE.com

In Vivo Modeling of the Morbid Human Genome using Danio rerio

在这里，我们提出了一个系统的方法来开发有关生理，敏感，特异<em&gt;在体内</em&gt;分析用于解释人类病理学变化。瞬态的遗传操作，通过微量注射野生型和突变型人mRNA和吗啉代（MO）的反义寡核苷酸利用显影斑马鱼胚胎的快速检测致病突变，特别是，但不完全是，在人类发育障碍的上下文中的易处理性。

在体内病态的人类基因组建模使用斑马鱼

Here,  we present methods for the development of assays to query potentially clinically  significant nonsynonymous changes using in  vivo complementation ...

in-vivo-modeling-of-the-morbid-human-genome-using-danio-rerio

Research

JoVE Journal

Biology

In Vivo Modeling of the Morbid Human Genome using Danio rerio

20.6K 次观看.  Duke University Medical Center. 在这里，我们提出了一个系统的方法来开发有关生理，敏感，特异在体内分析用于解释人类病理学变化。瞬态的遗传操作，通过微量注射野生型和突变型人mRNA和吗啉代（MO）的反义寡核苷酸利用显影斑马鱼胚胎的快速检测致病突变，特别是，但不完全是，在人类发育障碍的上下文中的易处理性。

视频: In Vivo Modeling of the Morbid Human Genome using Danio rerio

Mosaic Zebrafish Transgenesis for Functional Genomic Analysis of Candidate Cooperative Genes in Tumor Pathogenesis

Comprehensive genomic analysis has uncovered surprisingly large numbers of genetic alterations in various types of cancers. To robustly and efficiently identify oncogenic &#8220;drivers&#8221; among these tumors and define their complex relationships with concurrent genetic alterations during tumor pathogenesis remains a daunting task. Recently, zebrafish have emerged as an important animal model for studying human diseases, largely because of their ease of maintenance, high fecundity, obvious advantages for in vivo imaging, high conservation of oncogenes and their molecular pathways, susceptibility to tumorigenesis and, most importantly, the availability of transgenic techniques suitable for use in the fish. Transgenic zebrafish models of cancer have been widely used to dissect oncogenic pathways in diverse tumor types. However, developing a stable transgenic fish model is both tedious and time-consuming, and it is even more difficult and more time-consuming to dissect the cooperation of multiple genes in disease pathogenesis using this approach, which requires the generation of multiple transgenic lines with overexpression of the individual genes of interest followed by complicated breeding of these stable transgenic lines. Hence, use of a mosaic transient transgenic approach in zebrafish offers unique advantages for functional genomic analysis in vivo. Briefly, candidate transgenes can be coinjected into one-cell-stage wild-type or transgenic zebrafish embryos and allowed to integrate together into each somatic cell in a mosaic pattern that leads to mixed genotypes in the same primarily injected animal. This permits one to investigate in a faster and less expensive manner whether and how the candidate genes can collaborate with each other to drive tumorigenesis. By transient overexpression of activated ALK in the transgenic fish overexpressing MYCN, we demonstrate here the cooperation of these two oncogenes in the pathogenesis of a pediatric cancer, neuroblastoma that has resisted most forms of contemporary treatment.

The goal of this study is to demonstrate how the mosaic transgenesis strategy can be used in zebrafish to rapidly and efficiently assess the relative contributions of multiple oncogenes in tumor initiation and progression in vivo.

马赛克斑马鱼转基因的候选基因合作在肿瘤发病机制的功能基因组分析

Comprehensive genomic analysis has uncovered surprisingly large numbers of genetic alterations in various types of cancers.  To robustly and efficiently ...

科研

发育生物学

Efficient Production and Identification of CRISPR/Cas9-generated Gene Knockouts in the Model System Danio rerio

Characterization of the clustered, regularly interspaced, short, palindromic repeat (CRISPR) system of Streptococcus pyogenes has enabled the development of a customizable platform to rapidly generate gene modifications in a wide variety of organisms, including zebrafish. CRISPR-based genome editing uses a single guide RNA (sgRNA) to target a CRISPR-associated (Cas) endonuclease to a genomic DNA (gDNA) target of interest, where the Cas endonuclease generates a double-strand break (DSB). Repair of DSBs by error-prone mechanisms lead to insertions and/or deletions (indels). This can cause frameshift mutations that often introduce a premature stop codon within the coding sequence, thus creating a protein-null allele. CRISPR-based genome engineering requires only a few molecular components and is easily introduced into zebrafish embryos by microinjection. This protocol describes the methods used to generate CRISPR reagents for zebrafish microinjection and to identify fish exhibiting germline transmission of CRISPR-modified genes. These methods include in vitro transcription of sgRNAs, microinjection of CRISPR reagents, identification of indels induced at the target site using a PCR-based method called a heteroduplex mobility assay (HMA), and characterization of the indels using both a low throughput and a powerful next-generation sequencing (NGS)-based approach that can analyze multiple PCR products collected from heterozygous fish. This protocol is streamlined to minimize both the number of fish required and the types of equipment needed to perform the analyses. Furthermore, this protocol is designed to be amenable for use by laboratory personal of all levels of experience including undergraduates, enabling this powerful tool to be economically employed by any research group interested in performing CRISPR-based genomic modification in zebrafish.

Efficient Production and Identification of CRISPR/Cas9-generated Gene Knockouts in the Model System Danio rerio

Targeted genome editing in the model system Danio rerio (zebrafish) has been greatly facilitated by the emergence of CRISPR-based approaches. Herein, we describe a streamlined, robust protocol for generation and identification of CRISPR-derived nonsense alleles that incorporates the heteroduplex mobility assay and identification of mutations using next-generation sequencing.

斑马斑马模型系统中 CRISPR/Cas9-generated 基因挖空的高效生产与鉴定

Characterization of the clustered, regularly interspaced, short, palindromic repeat (CRISPR) system of Streptococcus pyogenes has enabled the development ...

遗传学

High-throughput DNA Extraction and Genotyping of 3dpf Zebrafish Larvae by Fin Clipping

Zebrafish (Danio rerio) possess orthologues for 84% of the genes known to be associated with human diseases. In addition, these animals have a short generation time, are easy to handle, display a high reproductive rate, low cost, and are easily amenable to genetic manipulations by microinjection of DNA in embryos. Recent advances in gene editing tools are enabling precise introduction of mutations and transgenes in zebrafish. Disease modeling in zebrafish often leads to larval phenotypes and early death which can be challenging to interpret if genotypes are unknown. This early identification of genotypes is also needed in experiments requiring sample pooling, such as in gene expression or mass spectrometry studies. However, extensive genotypic screening is limited by traditional methods, which in most labs are performed only on adult zebrafish or in postmortem larvae. We addressed this problem by adapting a method for the isolation of PCR-ready genomic DNA from live zebrafish larvae that can be achieved as early as 72 h post-fertilization (hpf). This time and cost-effective technique, improved from a previously published genotyping protocol, allows the identification of genotypes from microscopic fin biopsies. The fins quickly regenerate as the larvae develop. Researchers are then able to select and raise the desired genotypes to adulthood by utilizing this high-throughput PCR-based genotyping procedure.

Zebrafish have been used as reliable genetic model organisms in biomedical research, especially with the advent of gene-editing technologies. When larval phenotypes are expected, DNA extraction and genotype identification can be challenging. Here, we describe an efficient genotyping procedure for zebrafish larvae, by tail clipping, as early as 72-h post-fertilization.

3dpf 斑马鱼幼虫的高通量 DNA 提取及基因分型研究

Zebrafish (Danio rerio) possess orthologues for 84% of the genes known to be associated with human diseases. In addition, these animals have a short ...

Drug Treatment and In Vivo Imaging of Osteoblast-Osteoclast Interactions in a Medaka Fish Osteoporosis Model

Bone-forming osteoblasts interact with bone-resorbing osteoclasts to coordinate the turnover of bone matrix and to control skeletal homeostasis. Medaka and zebrafish larvae are widely used to analyze the behavior of bone cells during bone formation, degeneration, and repair. Their optical clarity allows the visualization of fluorescently labeled bone cells and fluorescent dyes bound to the mineralized skeletal matrix. Our lab has generated transgenic medaka fish that express the osteoclast-inducing factor Receptor Activator of Nuclear-factor &#954;B Ligand (RANKL) under the control of a heat&#160;shock-inducible promoter. Ectopic expression of RANKL results in the excess formation of activated osteoclasts, which can be visualized in reporter lines with nlGFP expression under the control of the cathepsin K (ctsk) promoter. RANKL induction and ectopic osteoclast formation leads to severe osteoporosis-like phenotypes. Compound transgenic medaka lines that express ctsk:nlGFP in osteoclasts, as well as mCherry under the control of the osterix (osx) promoter in premature osteoblasts, can be used to study the interaction of both cell types. This facilitates the in vivo observation of cellular behavior under conditions of bone degeneration and repair. Here, we describe the use of this system to test a drug commonly used in human osteoporosis therapy and describe a protocol for live imaging. The medaka model complements studies in cell culture and mice, and offers a novel system for the in vivo analysis of drug action in the skeletal system.

Drug Treatment and In Vivo Imaging of Osteoblast-Osteoclast Interactions in a Medaka Fish Osteoporosis Model

Small laboratory fish have become popular models for bone research on the mechanisms underlying human bone disorders and for the screening of bone-modulating drugs. In this report, we describe a protocol to assess the effect of alendronate on bone cells in medaka larvae with osteoporotic lesions.

戒毒治疗和<em&gt;在体内</em&gt;在青鳉鱼骨质疏松模型成骨破骨细胞相互作用的成像

Bone-forming osteoblasts interact with bone-resorbing osteoclasts to coordinate the turnover of bone matrix and to control skeletal homeostasis. Medaka ...

In Vivo Functional Study of Disease-associated Rare Human Variants Using Drosophila

Advances in sequencing technology have made whole-genome and whole-exome datasets more accessible for both clinical diagnosis and cutting-edge human genetics research. Although a number of in silico algorithms have been developed to predict the pathogenicity of variants identified in these datasets, functional studies are critical to determining how specific genomic variants affect protein function, especially for missense variants. In the Undiagnosed Diseases Network (UDN) and other rare disease research consortia, model organisms (MO) including Drosophila, C. elegans, zebrafish, and mice are actively used to assess the function of putative human disease-causing variants. This protocol describes a method for the functional assessment of rare human variants used in the Model Organisms Screening Center Drosophila Core of the UDN. The workflow begins with gathering human and MO information from multiple public databases, using the MARRVEL web resource to assess whether the variant is likely to contribute to a patient&#39;s condition as well as design effective experiments based on available knowledge and resources. Next, genetic tools (e.g., T2A-GAL4 and UAS-human cDNA lines) are generated to assess the functions of variants of interest in Drosophila. Upon development of these reagents, two-pronged functional assays based on rescue and overexpression experiments can be performed to assess variant function. In the rescue branch, the endogenous fly genes are &#34;humanized&#34;&#160;by replacing the orthologous Drosophila gene with reference or variant human transgenes. In the overexpression branch, the reference and variant human proteins are exogenously driven in a variety of tissues. In both cases, any scorable phenotype (e.g., lethality, eye morphology, electrophysiology) can be used as a read-out, irrespective of the disease of interest. Differences observed between reference and variant alleles suggest a variant-specific effect, and thus likely pathogenicity. This protocol allows rapid, in vivo assessments of putative human disease-causing variants of genes with known and unknown functions.

In Vivo Functional Study of Disease-associated Rare Human Variants Using Drosophila

The goal of this protocol is to outline the design and performance of in vivo experiments in Drosophila melanogaster to assess the functional consequences of rare gene variants associated with human diseases.

在Vivo功能研究的疾病相关的稀有人类变异使用果蝇

Advances in sequencing technology have made whole-genome and whole-exome datasets more accessible for both clinical diagnosis and cutting-edge human ...

Visualization of the Superior Ocular Sulcus during Danio rerio Embryogenesis

Congenital ocular coloboma is a genetic disorder that is typically observed as a cleft in the inferior aspect of the eye resulting from incomplete choroid fissure closure. Recently, the identification of individuals with coloboma in the superior aspect of the iris, retina, and lens led to the discovery of a novel structure, referred to as the superior fissure or superior ocular sulcus (SOS), that is transiently present on the dorsal aspect of the optic cup during vertebrate eye development. Although this structure is conserved across mice, chick, fish, and newt, our current understanding of the SOS is limited. In order to elucidate factors that contribute to its formation and closure, it is imperative to be able to observe it and identify abnormalities, such as delay in the closure of the SOS. Here, we set out to create a standardized series of protocols that can be used to efficiently visualize the SOS by combining widely available microscopy techniques with common molecular biology techniques such as immunofluorescent staining and mRNA overexpression. While this set of protocols focuses on the ability to observe SOS closure delay, it is adaptable to the experimenter&#39;s needs and can be easily modified. Overall, we hope to create an approachable method through which our understanding of the SOS can be advanced to expand the current knowledge of vertebrate eye development.

Visualization of the Superior Ocular Sulcus during Danio rerio Embryogenesis

Here, we present a standardized series of protocols to observe the superior ocular sulcus, a recently-identified, evolutionarily-conserved structure in the vertebrate eye. Using zebrafish larvae, we demonstrate techniques necessary to identify factors that contribute to the formation and closure of the superior ocular sulcus.

达尼奥胚胎发生过程中的上眼硫可视化

Congenital ocular coloboma is a genetic disorder that is typically observed as a cleft in the inferior aspect of the eye resulting from incomplete choroid ...

Real Time and Repeated Measurement of Skeletal Muscle Growth in Individual Live Zebrafish Subjected to Altered Electrical Activity

A number of methods can be used to visualize individual cells throughout the body of live embryonic, larval or juvenile zebrafish. We show that live fish with fluorescently-marked plasma membranes can be scanned in a confocal laser scanning microscope in order to determine the volume of muscle tissue and the number of muscle fibers present. Efficient approaches for the measurement of cell number and size in live animals over time are described and validated against more arduous segmentation methods. Methods are described that permit the control of muscle electrical, and thus contractile, activity. Loss of skeletal muscle contractile activity greatly reduced muscle growth. In larvae, a protocol is described that allows reintroduction of patterned electrical-evoked contractile activity. The described methods minimize the effect of inter-individual variability and will permit analysis of the effect of electrical, genetic, drug, or environmental stimuli on a variety of cellular and physiological growth parameters in the context of the living organism. Long-term follow-up of the measured effects of a defined early-life intervention on individuals can subsequently be performed.

Optical clarity is a major advantage for cell biological and physiological work in zebrafish. Robust methods for measurement of cell growth in individual animals are described that permit novel insights into how growth of skeletal muscle and neighboring tissues are integrated with whole body growth.

实时和重复测量电活动改变的个体活斑马鱼的骨骼肌生长

A number of methods can be used to visualize individual cells throughout the body of live embryonic, larval or juvenile zebrafish. We show that live fish ...

A Doxorubicin-induced Cardiomyopathy Model in Adult Zebrafish

The genetically accessible adult zebrafish (Danio rerio) has been increasingly used as a vertebrate model for understanding human diseases such as cardiomyopathy. Because of its convenience and amenability to high throughput genetic manipulations, the generation of acquired cardiomyopathy models, such as the doxorubicin-induced cardiomyopathy (DIC) model in adult zebrafish, is opening the doors to new research avenues, including discovering cardiomyopathy modifiers via forward genetic screening. Different from the embryonic zebrafish DIC model, both initial acute and later chronic phases of cardiomyopathy can be determined in the adult zebrafish DIC model, enabling the study of stage-dependent signaling mechanisms and therapeutic strategies. However, variable results can be obtained with the current model, even in the hands of experienced investigators. To facilitate future implementation of the DIC model, we present a detailed protocol on how to generate this DIC model in adult zebrafish and describe two alternative ways of intraperitoneal (IP) injection. We further discuss options on how to reduce variations to obtain reliable results and provide suggestions on how to appropriately interpret the results.

A method to generate a doxorubicin-induced cardiomyopathy model in adult zebrafish (Danio rerio) is described here. Two alternative ways of intraperitoneal injection are presented and conditions to reduce variations among different experimental groups are discussed.

阿霉素诱发成年斑马鱼的心肌病模型

The genetically accessible adult zebrafish (Danio rerio) has been increasingly used as a vertebrate model for understanding human diseases such as ...

医学

In vivo Imaging of Fully Active Brain Tissue in Awake Zebrafish Larvae and Juveniles by Skull and Skin Removal

Understanding the ephemeral changes that occur during brain development and maturation requires detailed high-resolution imaging in space and time at cellular and subcellular resolution. Advances in molecular and imaging technologies have allowed us to gain numerous detailed insights into cellular and molecular mechanisms of brain development in the transparent zebrafish embryo. Recently, processes of refinement of neuronal connectivity that occur at later larval stages several weeks after fertilization, which are for example control of social behavior, decision making or motivation-driven behavior, have moved into focus of research. At these stages, pigmentation of the zebrafish skin interferes with light penetration into brain tissue, and solutions for embryonic stages, e.g., pharmacological inhibition of pigmentation, are not feasible anymore.
Therefore, a minimally invasive surgical solution for microscopy access to the brain of awake zebrafish is provided that is derived from electrophysiological&#160;approaches. In teleosts, skin and soft skull cartilage can be carefully removed by micro-peeling these layers, exposing underlying neurons and axonal tracts without damage. This allows for recording neuronal morphology, including synaptic structures and their molecular contents, and the observation of physiological changes such as Ca2+ transients or intracellular transport events. In addition, interrogation of these processes by means of pharmacological inhibition or optogenetic manipulation is feasible. This brain exposure approach provides information about structural and physiological changes in neurons as well as the correlation and interdependence of these events in live brain tissue in the range of minutes or hours. The technique is suitable for in vivo brain imaging of zebrafish larvae up to 30 days post fertilization, the latest developmental stage tested so far. It, thus, provides access to such important questions as synaptic refinement and scaling, axonal and dendritic transport, synaptic targeting of cytoskeletal cargo or local activity-dependent expression. Therefore, a broad use for this mounting and imaging approach can be anticipated.

Here we present a method to image the zebrafish embryonic brain in vivo upto larval and juvenile stages. This microinvasive procedure, adapted from electrophysiological approaches, provides access to cellular and subcellular details of mature neuron and can be combined with optogenetics and neuropharmacological studies for characterizing brain function and drug intervention.

在体内成像完全活跃的脑组织在清醒斑马鱼幼虫和青少年由骷髅和皮肤去除

Understanding the ephemeral changes that occur during brain development and maturation requires detailed high-resolution imaging in space and time at ...

神经科学

A Manual Small Molecule Screen Approaching High-throughput Using Zebrafish Embryos

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology due to their considerable degree of genetic conservation with humans. Chemical genetics entails testing the effect that small molecules have on a biological process and is becoming a popular translational research method to identify therapeutic compounds. Zebrafish are specifically appealing to use for chemical genetics because of their ability to produce large clutches of transparent embryos, which are externally fertilized. Furthermore, zebrafish embryos can be easily drug treated by the simple addition of a compound to the embryo media. Using whole-mount in situ hybridization (WISH), mRNA expression can be clearly visualized within zebrafish embryos. Together, using chemical genetics and WISH, the zebrafish becomes a potent whole organism context in which to determine the cellular and physiological effects of small molecules. Innovative advances have been made in technologies that utilize machine-based screening procedures, however for many labs such options are not accessible or remain cost-prohibitive. The protocol described here explains how to execute a manual high-throughput chemical genetic screen that requires basic resources and can be accomplished by a single individual or small team in an efficient period of time. Thus, this protocol provides a feasible strategy that can be implemented by research groups to perform chemical genetics in zebrafish, which can be useful for gaining fundamental insights into developmental processes, disease mechanisms, and to identify novel compounds and signaling pathways that have medically relevant applications.

The zebrafish is an excellent experimental organism to study vertebrate developmental processes and model human disease. Here, we describe a protocol on how to perform a manual high-throughput chemical screen in zebrafish embryos with a whole-mount in situ hybridization (WISH) read-out.

手册小分子屏幕接近高通量使用斑马鱼胚胎

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology ...