我们希望感谢质粒的礼物蒙特卡罗韦斯特小林诚和亚切克Topczewski的的原位杂交和基因微量注射。我们是感谢Steph艾哈德在视频中出现的。我们也愿意承认，在视频中显示的共焦显微镜威廉古森斯援助。泰勒Schwend提供视频图像，并协助罗德尼·戴尔准备视频。我们也承认他们与斑马鱼的饲养工作，的CMRC动物保健人员。这项工作是由美国国立卫生研究院授予R21的AA13596政制事务局局长。

1。乙醇处理的胚胎<ol><li>提出了从野生型交配而得的斑马鱼胚胎在28°C在胚胎水5毫升。 （60 mg / L的即时海洋的Milli-Q水）。野生型胚胎的不同菌株具有略微不同的公差乙醇暴露9。 </li><li>暴露于乙醇所需的时间长度，在圆顶阶段（〜4.3小时后受精，HPF）发起长达24小时的脉冲。这起步阶段，大致相当于哺乳动物胚胎的植入，只是前原肠阶段。酒精浓度使用范围从0.2％-2.5％，体积/体积。试验表明，在水溶液中之间的25-50％结束了在胚胎发育10，11。胚胎保持在28°C所需的时​​间长度。乙醇暴露后所需要的长度，被删除，取而代之的乙醇水溶液三个定期斑马鱼水清洗，并保持FOR的所需的时间长度。发病时间，剂量，对乙醇的脉冲长度取决于缺陷的特异性。 </li></ol> 2。收集胚胎的RNA 原位杂交，抗体，和软骨染色<ol><li>收集的定量PCR基因，年龄相匹配的胚胎收集Eppendorf管中。去除斑马鱼水后，添加与TCEP的裂解缓冲。机动粉碎机用于裂解液中分离胚胎。这随后被传递通过预清除列，其中删除任何未解离大块。最终的解决方案包含了所有的裂解过程中释放的大分子。然后提取RNA是由一列运行这一解决方案，其次是清洗和DNase处理。然后使用水或由制造商（5&#39;总理）提供的洗脱液洗脱。的RNA可以转化为cDNA，使用标准的qPCR技术或微阵列分析。 </li><li> 在原位杂交和抗体染色，胚胎从6日至24 HPF被固定在4％多聚甲醛在一夜之间在4°C其次是3清洗在PBS + 0.1％吐温（PBT）的粘在一起，以保持胚胎。然后转移胚胎通过一系列3甲醇：PBT的洗到100％的甲醇，在这一点，他们可以在-20°C存储这些胚胎可用于原位杂交或抗体染色。甲醇处理后，所以这个警告必须考虑和您的特异性抗体测试，以确定它是否将甲醇处理后，一些抗体不起作用。 </li><li>软骨染色，胚胎需要提高到5或6天受精后（DPF）。然后，他们被固定在4％多聚甲醛过夜4°C间，在PBT洗。 </li></ol> 3。评估乙醇诱导基因表达的变化和发展后果<ol><li要确定哪些基因表达水平改变，双方的qPCR 和原位杂交检测靶基因。 qPCR可以实现使用与集成DNA技术（IDT）的软件，然后用3个独立的生物样品的验证，设计引物。一旦引物，已验证，定量PCR进行基因从收集RNA样品。每个样品运行在一式三份。 PCR反应色度-4的PCR仪（BioRad公司）或类似的PCR仪与多色检测系统上运行白金的SYBR Green qPCR的超级组合（Invitrogen公司）。除了正在审讯的基因，这是关键，引物控制用于平衡cDNA或RNA制备的小差异。对于我们的斑马鱼胚胎中，我们使用GAPDH：5&#39;GAAGGTGGGAAACTGGTCAT3和5&#39;TTGCACCACCCTTAATGTGA3“。被分析的基因GAPDH水平正常化，用2 Pfaffl法变异的基因表达相对定量计算<sup- CT，显示的数据作为实验相对于野生型12倍的差异。正常的计算，假定所有基因每个周期重复两次的DNA，从而表现为比例超过参照基因变化的实验基因的变化数据，为功率计算在整数“2”表示，这是一个完美的PCR反应中看到每个周期的DNA加倍效率。 Pfaffl法取代通用的“2”实验者选择的PCR引物产生真正的效率。它反映了在PCR反应的差异，更准确地反映。完整的计算公式为： <table border="0"><tbody><tr><td> （实验基因效率）（CT控制样品处理样品的CT） </td></tr><tr><td> -------------------------------------------------- ----------------------------------- </td></tr><tr><td> （参照基因的效率）（CT对控制样本- CT处理样品） </td></tr></tbody></table></li><li> 在原位杂交，地高辛（DIG）标记riboprobes的构造含有部分感兴趣的基因质粒。年龄相匹配的胚胎暴露riboprobes使用13种标准条件和检测碱性磷酸酶允许使用显色反应（NBT / BCIP）的标记riboprobes的位置加上反掏抗体。胚胎，然后使用徕卡解剖显微镜（ 图1B-G）的可视化。 </li><li>评估形态发生发展，胚胎收集所需的时间，并在解剖显微镜下观察。活胚胎体节形状，成像14。眼间距离和身体长度，胚胎固定在煤灰。在所有情况下，所需区域的图像被捕获在一个固定的放大倍率和测量，解剖显微镜使用Adobe Photoshop softwar北京时间10,14。 </li><li>检查治疗发展斑马鱼幼虫的软骨结构的影响，我们用阿尔新蓝染色。固定斑马鱼幼虫被视为与阿尔新蓝溶于80％乙醇溶液：20％冰醋酸几个小时或过夜（酸酒精）。幼虫脱色酸酒精清洗，然后被转移到1％KOH进一步清除色素细胞的过氧化氢溶液：3％。斑马鱼需要至少4天显示软骨结构，这些都更好地定义在5或6天的鱼。 </li><li>用吖啶橙（AO）的评估在活胚胎细胞死亡。 AO是不是渗透到活细胞的细胞，受损的细胞膜，包括那些发生凋亡和坏死细胞的DNA结合。活胚胎与5毫克/毫升敖在PBS孵育1小时，3 PBS洗涤。胚胎，然后利用共聚焦显微镜成像。数字的Z-SER而IES图像结合创建复合材料。 </li></ol> 4。操纵斑马鱼胚胎<ol><li>减少乙醇暴露的基因鉴定后，有可能尝试使用注射的设计，以取代丢失的基因表达，以取代他们。皑皑的mRNA转录从线性质粒DNA使用RNA聚合酶体外转录试剂盒，根据制造商的指示（mMessage机，应用生物系统公司）。皮克/ NL的RNA 25-200之间被注入到1-2细胞阶段，斑马鱼的卵，在解决了0.1米氯化钾。胚胎，然后恢复之前，用乙醇视为以上（ 图5）。 </li><li>对于任何基因转录增加乙醇暴露，他们可以减少，或“敲出”用反义吗啉技术。反义吗啉代（AMOS）是由该公司设计的基因工具，阻止RNA翻译成蛋白质，或BLOCK的成熟和RNA剪接，根据实验需要。这种行动吗啉代活性蛋白的数量限制为目标基因，可以测量使用anitibodies如果他们亏。 RNA剪接吗啉代的效率可以测量采用RT-PCR检测剪接和未拼接的成绩单的相对含量。吗啉的剂量可以导致剂量依赖性的活动15。阿莫斯稀释工作浓度在Danieau解决方案（纳克的浓度滴定PG）（58毫米氯化钠，0.7毫米氯化钾， 硫酸镁 0.4毫米，0.6毫米的Ca（NO 3）2，5毫米肝素钠，pH值7.6）。注射,1-2细胞阶段的地方，然后胚胎被允许之前受到乙醇处理上述发展。 </li></ol> 5。代表结果斑马鱼胚胎暴露于乙醇结果在一些发育和遗传缺陷有关表型的发现在其他脊椎动物。我们已经证明了轴组织发育异常，包括脊索（数据未显示14）。乙醇生产的发育迟缓，显然是导致适当的脊索伸长受阻，导致缩短偶尔打乱的脊索（数据未显示14）。这种早期的延迟和脊索缺陷可能导致在体节的后异常（ 图2），这已经失去了他们强烈的雪佛龙的形状，在未经处理的控制部分，是接近U形体节时看到的表型特征声波信号减少14。体节的角度，可以采用标准的软件，从92.1±4.6°，在未经处理的控制为122.6±6.6°的乙醇处理的胚胎（P &lt;0.001）的角度增加。 另一个乙醇暴露后果可能downstrea的m的早期发育迟缓，后来脊索缺陷的胚胎长度缩短（ 图3）。即使测量胚胎考虑到乙醇暴露产生的曲率，乙醇暴露导致缩短主干，是依赖于剂量的乙醇胚胎暴露（ 图3E 14）。这一发现是持续暴露在发展16，17乙醇的儿童在青春期前短身材发现的类似，这表明斑马鱼模型是了解人类出生缺陷有关。 FAS的典型特征之一，是一个典型的相，其中包括减少下颌，小眼开，并顺利人中。这些功能大部分都在减少中线组织有关。它已被证明在动物模型中，重度乙醇暴露导致更显着的表型，包括synopthalmia和独眼。当世博会增加剂量乙醇唱斑马鱼胚胎，人工距离（IOD）以剂量依赖的方式降低（ 图4）与人类出生缺陷的性质是一致的。独眼只发现具有非常高的剂量，但低剂量产生的IOD（ 图4）在显着减少，表明这种动物模型有类似的缺陷面部中线发展10。 为了了解机制在身体和面对生产乙醇暴露的缺陷，我们试图建立基因表达的变化发生在发展初期，可合理预期，有助于以后的发育缺陷。我们做这两种方式，表达从胚胎提取可以定量评价了乙醇处理的胚胎中的基因表达水平较对照组（ 图1A）。此外，我们可以执行原位杂交基因的格局看，不管与否的信号下降（ 图1B-G的 18）。在这个例子中，两个基因的表达，减少乙醇暴露后，GLI1和six3b。当我们在原位为six3b模式研究，我们发现在该基因的表达的空间范围，在暴露于乙醇胚胎（ 图1，EG）的减少。类似的减少被发现的基因GSC（ 图BD）。都six3b和GSC表示在注定有助于颅面中线组织的组织。所以减少8 HPF这些基因的发现后， 如图4所示，在IOD的减少是一致的。 一旦确定乙醇影响的基因，也有机制，通过他们的表现可以增加或减少。在这个特别的EXAmple，我们已经表明下降的qPCR 2基因（GLI1及six3b）。我们选择注入基因，以确定如果扭转这些基因的变化，可以改善预后。对于这个实验，而不是注射GLI1，我们选择了注入SHH，配体，增加GLI1水平。为six3b，我们能够注入six3b本身。我们没有发现任何变化，当我们注入six3b（数据未显示），但能够从根本上挽救的补充，SHH注射（ 图5）在斑马鱼胚胎的总缺陷。 <img alt="图1" src="/files/ftp_upload/3704/3704fig1.jpg" /> 图1。乙醇暴露改变了早期的模式和选定的发育基因的基因表达水平。一）胚胎在8hpf qPCR的结果。这两个基因的结果显示：six3b和GLI1。平均为三个独立的实验结果表明，乙醇剂量为2.5％。这两个基因的方式，是从控制（t检验，P &lt;0.05）显著减少。 BG） 在 8个HPF斑马鱼胚胎的原位杂交。改变GSC和six3b模式都在这个时间点与对照组相比。 （劳克斯等2007年的许可改性）。 <img alt="图2" src="/files/ftp_upload/3704/3704fig2.jpg" /> 图2。体节的发展改变了处理的胚胎。体节的结构和角度研究在48 HPF。 （一），在控制胚胎体节有雪佛龙的形状和尖尖角。乙醇暴露斑马鱼有更多的I或U形的体节，少锐利的角度。 （改性劳克斯和Ahlgren 2009的权限）。 <img alt="图3" src="/files/ftp_upload/3704/3704fig3.jpg" /> 图3。乙醇处理显着降低在斑马鱼的体长。在受精后5天未处理和乙醇暴露胚胎的长度测量。在所有处理的胚胎被视为一个长度显着减少（ANOVA，P &lt;0.001）。屋宇署。在总长度的1.0％和1.5％的乙醇处理的胚胎有一个轻微但显着减少，而更大的减少了2.0％和2.5％的乙醇剂量的胚胎。 E.要控制在每个离合器的大小差异，每个实验的控制胚胎正常化至100和100的百分比表示的兄弟处理的胚胎。 （改性劳克斯和Ahlgren 2009的权限）。 <img alt="图4" src="/files/ftp_upload/3704/3704fig4.jpg" /> 图4。减少在眼内的距离（IOD）在斑马鱼胚胎中的乙醇暴露的结果。一）正面看法的胚胎表明正常和融合的眼表型。乙）乙醇的不同的给药剂量为3个小时在原肠水库在ULTS下降IOD值时，鱼类24小时后检查。熔融的眼睛和独眼只看到在最高剂量测试（2.4％）。 *表示在IOD显着减少，与对照组相比。 （从2004年Ahlgren，许可修改）。 <img alt="图5" src="/files/ftp_upload/3704/3704fig5.jpg" /> 图5。SHH-N 的基因注射抢救斑马鱼暴露乙醇生产总值的缺陷。一个个细胞的胚胎注入100皮克/ NL SHH-N的基因注入胚胎的一半暴露乙醇剂量的标准，从4.3至24 HPF。胚胎进行了分析5 DPF。 2.0％的乙醇处理的胚胎表现出背侧弯曲，短尾巴，我形体节和眼的缺陷，包括独眼。这些表型的救援被认为是在注射胚胎（93％）71/76。在这些胚胎，身体直，体节是雪佛龙形，眼睛是完全分开的。

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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fetal+alcohol+spectrum+disorders+in+young+adulthood.">Fetal alcohol spectrum disorders in young adulthood.</a> J. Pediatr. 150 (2), 175-179 (2007).</li><li>Strauss, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+the+intrauterine+environment+on+childhood+growth.">Effects of the intrauterine environment on childhood growth.</a> Br. Med. Bull. 53 (1), 81-95 (1997).</li><li>Loucks, E. J., Schwend, T., Ahlgren, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+changes+associated+with+teratogen-induced+cyclopia.">Molecular changes associated with teratogen-induced cyclopia.</a> Birth Defects Res. A. Clin. Mol. Teratol. 79 (9), 642-651 (2007).</li><li>Schwend, T., Loucks, E. J., Ahlgren, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+Gli+activity+in+craniofacial+tissues+of+hedgehog-pathway+reporter+transgenic+zebrafish.">Visualization of Gli activity in craniofacial tissues of hedgehog-pathway reporter transgenic zebrafish.</a> PLoS One. 5 (9), 14396-14396 (2011).</li><li>Chrisman, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gestational+ethanol+exposure+disrupts+the+expression+of+FGF8+and+Sonic+hedgehog+during+limb+patterning.">Gestational ethanol exposure disrupts the expression of FGF8 and Sonic hedgehog during limb patterning.</a> Birth Defects Res. A. Clin. Mol. Teratol. 70 (4), 163-171 (2004).</li></ol>

我们什么都没有透露。

这里介绍的方法，结果表明只是如何斑马鱼可以用来审问发育缺陷的一角。由于胚胎的辅助，每个离合器，高重复性的结果产卵数量高，这些脊椎动物是致畸研究的理想。鱼也可以操纵含有荧光分子，利用视觉作为一个特定的基因或通路利息19记者。在乙醇的研究，使用剂量出现相比，哺乳动物的血液酒精含量相当高。然而，这些级别反映了什么是水，不是所有的乙醇被传递到胚胎。 两者合计，描述和上面详细介绍的方法表明，斑马鱼的胚胎是乙醇暴露相关的模拟人类出生缺陷非常有用。此外，发现在斑马鱼的基因表达的变化已被证实由他人在哺乳动物模型系统20，表明乙醇的发展影响整个脊椎动物保留。上面显示的技术可用于许多其他环境或制药侮辱，快速，方便地检测潜在的致畸和判断，有助于特定物质的致畸的潜在机制。

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td>组织RNA试剂盒：5首相PerfectPure的</td><td> Fisher Scientific则</td><td> FP2302410 </td></tr><tr><td>白金SYBR绿色的qPCR SuperMix-UDG的</td><td> Invitrogen公司</td><td> 11733038 </td></tr><tr><td> mMessage mMachine高产量上限RNA转录试剂盒</td><td>应用生物系统公司</td><td>取决于你的质粒RNA聚合酶</td></tr></tbody></table>

assessing teratogenic changes in a zebrafish model of fetal alcohol exposure

胎儿酒精综合征（FAS）是一个严重的表现胚胎暴露于乙醇。它提出了缺陷的脸和器官，包括精神发育迟滞，由于紊乱和脑损伤发展的特点。胎儿酒精谱系障碍（FASD）是用于支付出生缺陷的发生是由于产妇饮酒连续任期，并在发生在美国出生的儿童约4％。与育龄妇女的50％食用酒精，一半的怀孕是无计划的，无意的暴露是一个持续的问题2。 ，以最好地理解乙醇产生的破坏，加上产生的模型来测试潜在的干预，我们开发了一个使用斑马鱼胚胎发育乙醇曝光模式。斑马鱼是理想的这种致畸研究3-8。每对奠定了数百个鸡蛋，然后可以不损害成年人的情况下收集鱼。斑马鱼的胚胎是透明的，可以很容易地与任何数量的污渍成像。分析这些胚胎后，暴露在不同剂量和时间期限和应用表明乙醇，乙醇生产总值的发育缺陷与人类的出生缺陷是一致的。这里描述的是用于研究和操纵斑马鱼FAS模型的基本技巧。

Watch this Scientific Journal Video about Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure at JoVE.com

Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure

为了了解乙醇诱导发育损伤的分子机制，我们已经开发的乙醇暴露斑马鱼模型，并正在探索乙醇暴露后发生的物理，细胞和基因改变<sup&gt; 1</sup&gt;。然后，我们来寻求潜在的干预和快速测试他们在这个动物模型。

在斑马鱼模型的胎儿酒精暴露评估致畸的变化

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, ...

assessing-teratogenic-changes-zebrafish-model-fetal-alcohol

Research

JoVE Journal

Medicine

26.1K 次观看.  Children's Memorial Research Center. 为了了解乙醇诱导发育损伤的分子机制，我们已经开发的乙醇暴露斑马鱼模型，并正在探索乙醇暴露后发生的物理，细胞和基因改变 1。然后，我们来寻求潜在的干预和快速测试他们在这个动物模型。

视频: Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure

The Polyvinyl Alcohol Sponge Model Implantation

Wound healing is a complicated, multistep process involving many cell types, growth factors and compounds1-3. Because of this complexity, wound healing studies are most comprehensive when carried out in vivo. There are many in vivo models available to study acute wound healing, including incisional, excisional, dead space, and burns. Dead space models are artificial, porous implants which are used to study tissue formation and the effects of substances on the wound. Some of the commonly used dead space models include polyvinyl alcohol (PVA) sponges, steel wire mesh cylinders, expanded polytetrafluoroethylene (ePTFE) material, and the Cellstick1,2.
Each dead space model has its own limitations based on its material's composition and implantation methods. The steel wire mesh cylinder model has a lag phase of infiltration after implantation and requires a long amount of time before granulation tissue formation begins1. Later stages of wound healing are best analyzed using the ePTFE model1,4. The Cellstick is a cellulose sponge inside a silicon tube model which is typically used for studying human surgery wounds and wound fluid2. The PVA sponge is limited to acute studies because with time it begins to provoke a foreign body response which causes a giant cell reaction in the animal5. Unlike other materials, PVA sponges are easy to insert and remove, made of inert and non-biodegradable materials and yet are soft enough to be sectioned for histological analysis2,5.
In wound healing the PVA sponge is very useful for analyzing granulation tissue formation, collagen deposition, wound fluid composition, and the effects of substances on the healing process1,2,5. In addition to its use in studying a wide array of attributes of wound healing, the PVA sponge has also been used in many other types of studies. It has been utilized to investigate tumor angiogenesis, drug delivery and stem cell survival and engraftment1,2,6,7. With its great alterability, prior extensive use, and reproducible results, the PVA sponge is an ideal model for many studies1,2.
Here, we will describe the preparation, implantation and retrieval of PVA sponge disks (Figure 1) in a mouse model of wound healing.

A useful tool to analyze the effects of drugs, growth factors, and/or manipulated cells in an animal model of wound repair is described. This technique utilizes the properties of a polyvinyl alcohol (PVA) sponge to deliver and contain the desired treatment and also provide a platform to be excised and analyzed.

聚乙烯醇海绵模型植入

Wound healing is a complicated, multistep process involving many cell types, growth factors and compounds1-3. Because of this complexity, wound healing ...

科研

医学

A Novel Surgical Approach for Intratracheal Administration of Bioactive Agents in a Fetal Mouse Model

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and acquired diseases. Apart from congenital or inherited abnormalities with the requirement for long-term expression of the delivered gene, several non-inherited perinatal conditions, where short-term gene expression or pharmacological intervention is sufficient to achieve therapeutic effects, are considered as potential future indications for this kind of approach. Candidate diseases for the application of short-term prenatal therapy could be the transient neonatal deficiency of surfactant protein B causing neonatal respiratory distress syndrome1,2 or hyperoxic injuries of the neonatal lung3. Candidate diseases for permanent therapeutic correction are Cystic Fibrosis (CF)4, genetic variants of surfactant deficiencies5 and &alpha;1-antitrypsin deficiency6.
Generally, an important advantage of prenatal gene therapy is the ability to start therapeutic intervention early in development, at or even prior to clinical manifestations in the patient, thus preventing irreparable damage to the individual. In addition, fetal organs have an increased cell proliferation rate as compared to adult organs, which could allow a more efficient gene or stem cell transfer into the fetus. Furthermore, in utero gene delivery is performed when the individual's immune system is not completely mature. Therefore, transplantation of heterologous cells or supplementation of a non-functional or absent protein with a correct version should not cause immune sensitization to the cell, vector or transgene product, which has recently been proven to be the case with both cellular and genetic therapies7.
In the present study, we investigated the potential to directly target the fetal trachea in a mouse model. This procedure is in use in larger animal models such as rabbits and sheep8, and even in a clinical setting9, but has to date not been performed before in a mouse model. When studying the potential of fetal gene therapy for genetic diseases such as CF, the mouse model is very useful as a first proof-of-concept because of the wide availability of different transgenic mouse strains, the well documented embryogenesis and fetal development, less stringent ethical regulations, short gestation and the large litter size.
Different access routes have been described to target the fetal rodent lung, including intra-amniotic injection10-12, (ultrasound-guided) intrapulmonary injection13,14 and intravenous administration into the yolk sac vessels15,16 or umbilical vein17. Our novel surgical procedure enables researchers to inject the agent of choice directly into the fetal mouse trachea which allows for a more efficient delivery to the airways than existing techniques18.

We developed a novel surgical approach for intratracheal administration of bioactive agents into the mouse fetus. The delivery route is more efficient in targeting the fetal mouse lungs than the commonly used intra-amniotic injection. This procedure has to date not been described in a mouse model.

一种新的手术方法气管内注射生物活性剂在胎鼠模型

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and ...

Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model

Genomic studies of human cancers have yielded a wealth of information about genes that are altered in tumors1,2,3. A challenge arising from these studies is that many genes are altered, and it can be difficult to distinguish genetic alterations that drove tumorigenesis from that those arose incidentally during transformation. To draw this distinction it is beneficial to have an assay that can quantitatively measure the effect of an altered gene on tumor initiation and other processes that enable tumors to persist and disseminate. Here we present a rapid means to screen large numbers of candidate melanoma modifiers in zebrafish using an autochthonous tumor model4 that encompasses steps required for melanoma initiation and maintenance. A key reagent in this assay is the miniCoopR vector, which couples a wild-type copy of the mitfa melanocyte specification factor to a Gateway recombination cassette into which candidate melanoma genes can be recombined5. The miniCoopR vector has a mitfa rescuing minigene which contains the promoter, open reading frame and 3'-untranslated region of the wild-type mitfa gene. It allows us to make constructs using full-length open reading frames of candidate melanoma modifiers. These individual clones can then be injected into single cell Tg(mitfa:BRAFV600E);p53(lf);mitfa(lf)zebrafish embryos. The miniCoopR vector gets integrated by Tol2-mediated transgenesis6 and rescues melanocytes. Because they are physically coupled to the mitfa rescuing minigene, candidate genes are expressed in rescued melanocytes, some of which will transform and develop into tumors. The effect of a candidate gene on melanoma initiation and melanoma cell properties can be measured using melanoma-free survival curves, invasion assays, antibody staining and transplantation assays.

A rapid way to screen for melanoma modifiers using a zebrafish autochthonous tumor model is presented. It takes advantage of the miniCoopR vector which allows for expression of candidate melanoma genes in melanocytes. A method to obtain melanoma-free survival curves, an invasion assay, a protocol for antibody staining of scale melanocytes and a melanoma transplantation assay are described.

对黑色素瘤修饰符一个斑马鱼原地肿瘤模型的筛选

Genomic studies of  human cancers have yielded a wealth of information about genes that are altered  in tumors1,2,3. A challenge arising from these ...

Fetal Echocardiography and Pulsed-wave Doppler Ultrasound in a Rabbit Model of Intrauterine Growth Restriction

Fetal intrauterine growth restriction (IUGR) results in abnormal cardiac function that is apparent antenatally due to advances in fetoplacental Doppler ultrasound and fetal echocardiography. Increasingly, these imaging modalities are being employed clinically to examine cardiac function and assess wellbeing in utero, thereby guiding timing of birth decisions. Here, we used a rabbit model of IUGR that allows analysis of cardiac function in a clinically relevant way. Using isoflurane induced anesthesia, IUGR is surgically created at gestational age day 25 by performing a laparotomy, exposing the bicornuate uterus and then ligating 40-50% of uteroplacental vessels supplying each gestational sac in a single uterine horn. The other horn in the rabbit bicornuate uterus serves as internal control fetuses. Then, after recovery at gestational age day 30 (full term), the same rabbit undergoes examination of fetal cardiac function. Anesthesia is induced with ketamine and xylazine intramuscularly, then maintained by a continuous intravenous infusion of ketamine and xylazine to minimize iatrogenic effects on fetal cardiac function. A repeat laparotomy is performed to expose each gestational sac and a microultrasound examination (VisualSonics VEVO 2100) of fetal cardiac function is performed. Placental insufficiency is evident by a raised pulsatility index or an absent or reversed end diastolic flow of the umbilical artery Doppler waveform. The ductus venosus and middle cerebral artery Doppler is then examined. Fetal echocardiography is performed by recording B mode, M mode and flow velocity waveforms in lateral and apical views. Offline calculations determine standard M-mode cardiac variables, tricuspid and mitral annular plane systolic excursion, speckle tracking and strain analysis, modified myocardial performance index and vascular flow velocity waveforms of interest. This small animal model of IUGR therefore affords examination of in utero cardiac function that is consistent with current clinical practice and is therefore useful in a translational research setting.

We describe examination of fetal cardiac function with contemporary functional fetal echocardiography and fetoplacental Doppler ultrasound using the VisualSonics VEVO 2100 microultrasound in a surgically induced model of intrauterine fetal growth restriction in a rabbit.

在家兔模型宫内生长受限胎儿超声心动图和多普勒超声脉冲波

Fetal intrauterine growth restriction (IUGR) results in abnormal cardiac function that is apparent antenatally due to advances in fetoplacental Doppler ...

Assessing Changes in Volatile General Anesthetic Sensitivity of Mice after Local or Systemic Pharmacological Intervention

One desirable endpoint of general anesthesia is the state of unconsciousness, also known as hypnosis. Defining the hypnotic state in animals is less straightforward than it is in human patients. A widely used behavioral surrogate for hypnosis in rodents is the loss of righting reflex (LORR), or the point at which the animal no longer responds to their innate instinct to avoid the vulnerability of dorsal recumbency. We have developed a system to assess LORR in 24 mice simultaneously while carefully controlling for potential confounds, including temperature fluctuations and varying gas flows. These chambers permit reliable assessment of anesthetic sensitivity as measured by latency to return of the righting reflex (RORR) following a fixed anesthetic exposure. Alternatively, using stepwise increases (or decreases) in anesthetic concentration, the chambers also enable determination of a population's sensitivity to induction (or emergence) as measured by EC50 and Hill slope. Finally, the controlled environmental chambers described here can be adapted for a variety of alternative uses, including inhaled delivery of other drugs, toxicology studies, and simultaneous real-time monitoring of vital signs.

Loss of the righting reflex has long served as a standard behavioral surrogate for unconsciousness, also called hypnosis, in laboratory animals. Alterations in volatile anesthetic sensitivity caused by pharmacological interventions can be detected with a carefully controlled high-throughput assessment system, which may be adapted for delivery of any inhaled therapeutic.

局部或全身药物干预后评估的变化小鼠中挥发性全身麻醉药敏感性

One desirable endpoint of general anesthesia is  the state of unconsciousness, also known as hypnosis. Defining the hypnotic state in animals is  less ...

A Possible Zebrafish Model of Polycystic Kidney Disease: Knockdown of wnt5a Causes Cysts in Zebrafish Kidneys

Polycystic kidney disease (PKD) is one of the most common causes of end-stage kidney disease, a devastating disease for which there is no cure. The molecular mechanisms leading to cyst formation in PKD remain somewhat unclear, but many genes are thought to be involved. Wnt5a is a non-canonical glycoprotein that regulates a wide range of developmental processes. Wnt5a works through the planar cell polarity (PCP) pathway that regulates oriented cell division during renal tubular cell elongation. Defects of the PCP pathway have been found to cause kidney cyst formation. Our paper describes a method for developing a zebrafish cystic kidney disease model by knockdown of the wnt5a gene with wnt5a antisense morpholino (MO) oligonucleotides. Tg(wt1b:GFP) transgenic zebrafish were used to visualize kidney structure and kidney cysts following wnt5a knockdown. Two distinct antisense MOs (AUG - and splice-site) were used and both resulted in curly tail down phenotype and cyst formation after wnt5a knockdown. Injection of mouse Wnt5a mRNA, resistant to the MOs due to a difference in primary base pair structure, rescued the abnormal phenotype, demonstrating that the phenotype was not due to &#8220;off-target&#8221; effects of the morpholino. This work supports the validity of using a zebrafish model to study wnt5a function in the kidney.

A Possible Zebrafish Model of Polycystic Kidney Disease: Knockdown of wnt5a Causes Cysts in Zebrafish Kidneys

We describe a method of generating a possible zebrafish model of polycystic kidney disease. We used Tg(wt1b:GFP) fish to visualize kidney structure. Knockdown of wnt5a was by morpholino injection. Pronephric cyst formation after wnt5a knockdown was observed in this GFP transgenic zebrafish.

多囊肾一种可能的斑马鱼型号：击倒<em&gt; Wnt5a的</em&gt;原因在斑马鱼肾囊肿

Polycystic kidney disease (PKD) is one of the most common causes of end-stage kidney disease, a devastating disease for which there is no cure. The ...

A Mouse Fetal Skin Model of Scarless Wound Repair

Early in utero, but not in postnatal life, cutaneous wounds undergo regeneration and heal without formation of a scar. Scarless fetal wound healing occurs across species but is age dependent. The transition from a scarless to scarring phenotype occurs in the third trimester of pregnancy in humans and around embryonic day 18 (E18) in mice. However, this varies with the size of the wound with larger defects generating a scar at an earlier gestational age. The emergence of lineage tracing and other genetic tools in the mouse has opened promising new avenues for investigation of fetal scarless wound healing. However, given the inherently high rates of morbidity and premature uterine contraction associated with fetal surgery, investigations of fetal scarless wound healing in vivo require a precise and reproducible surgical model. Here we detail a reliable model of fetal scarless wound healing in the dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

During mammalian development, early gestational skin wounds heal without a scar. Here we detail a reliable and reproducible model of fetal scarless wound healing in the cutaneous dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

对无瘢痕修复鼠标胎儿皮肤模型

Early in utero, but not in postnatal life, cutaneous wounds undergo regeneration and heal without formation of a scar. Scarless fetal wound healing occurs ...

Generation of a Chronic Obstructive Pulmonary Disease Model in Mice by Repeated Ozone Exposure

Chronic obstructive pulmonary disease (COPD) is characterized by persistent airflow limitation and lung parenchymal destruction. It has a very high incidence in aging populations. The current conventional therapies for COPD focus mainly on symptom-modifying drugs; thus, the development of new therapies is urgently needed. Qualified animal models of COPD could help to characterize the underlying mechanisms and can be used for new drug screening. Current COPD models, such as lipopolysaccharide (LPS) or the porcine pancreatic elastase (PPE)-induced emphysema model, generate COPD-like lesions in the lungs and airways but do not otherwise resemble the pathogenesis of human COPD. A cigarette smoke (CS)-induced model remains one of the most popular because it not only simulates COPD-like lesions in the respiratory system, but it is also based on one of the main hazardous materials that causes COPD in humans. However, the time-consuming and labor-intensive aspects of the CS-induced model dramatically limit its application in new drug screening. In this study, we successfully generated a new COPD model by exposing mice to high levels of ozone. This model demonstrated the following: 1) decreased forced expiratory volume 25, 50, and 75/forced vital capacity (FEV25/FVC, FEV50/FVC, and FEV75/FVC), indicating the deterioration of lung function; 2) enlarged lung alveoli, with lung parenchymal destruction; 3) reduced fatigue time and distance; and 4) increased inflammation. Taken together, these data demonstrate that the ozone exposure (OE) model is a reliable animal model that is similar to humans because ozone overexposure is one of the etiological factors of COPD. Additionally, it only took 6 - 8 weeks, based on our previous work, to create an OE model, whereas it requires 3 - 12 months to induce the cigarette smoke model, indicating that the OE model might be a good choice for COPD research.

This study describes the successful generation of a new chronic obstructive pulmonary disease (COPD) animal model by repeatedly exposing mice to high concentrations of ozone.

反复臭氧暴露对小鼠慢性阻塞性肺疾病模型的产生

Chronic obstructive pulmonary disease (COPD) is characterized by persistent airflow limitation and lung parenchymal destruction. It has a very high ...

Model Surgical Training: Skills Acquisition in Fetoscopic Laser Photocoagulation of Monochorionic Diamniotic Twin Placenta Using Realistic Simulators

Fetoscopic laser coagulation of arterio-venous anastomoses (AVA) in a monochorionic placenta is the standard of care for twin-twin transfusion syndrome (TTTS), but is technically challenging and can lead to significant complications. Acquiring and maintaining the necessary surgical skills require consistent practice, a critical caseload, and time. Training on realistic surgical simulators can potentially shorten this steep learning curve and enables several proceduralists to acquire procedure-specific skills simultaneously. Here we describe realistic simulators designed to allow the user familiarity with the equipment and specific steps required in the surgical treatment of TTTS, including fetoscopic handling, approaches to anterior and posterior placenta, recognition of anastomoses, and efficient coagulation of vessels. We describe the skills that are especially important in conducting placental laser coagulation that the surgeon can practice on the model and apply in a clinical case. These models can be adapted easily depending on the availability of materials and require standard fetoscopy equipment. Such training systems are complementary to traditional surgical apprenticeships and can be useful aids for fetal medicine units that provide this clinical service.

Practicing the specific skills required for fetoscopic laser coagulation of monochorionic placental anastomoses on realistic models can aid less experienced surgeons in overcoming the steep learning curve associated with this procedure that is now regarded as the standard of care for twin-twin transfusion syndrome.

模型外科训练: 用逼真模拟器 Fetoscopic 激光光凝单绒毛膜 Diamniotic 双胎胎盘的技能习得

Fetoscopic laser coagulation of arterio-venous anastomoses (AVA) in a monochorionic placenta is the standard of care for twin-twin transfusion syndrome ...

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.