在编制本介绍我们感谢他们的帮助埃文斯实验室成员。原来，这里使用的morpholinos博士奥黛丽Holtzinger表示验证。 TE是由来自美国国立卫生研究院（HL064282 HL056182）的赠款支持。按照研究所动物护理和使用委员会规定的准则和法规威尔康乃尔医学院，进行动物实验。

我们的目标是测试规范心肌祖细胞的两个转录因子的功能冗余。由两个gata5和gata6相关基因编码的因素和我们使用的是斑马鱼的模型，以了解他们的相关功能。我们的战略是阻止基因的功能，使用morpholinos 2 。我们将一个或成受精卵基因注入morpholinos，并与来自鸡蛋注射morpholinos同时的胚胎胚胎的表型。为了简化表型的评价，我们使用携带基因对心肌细胞的GFP从鱼的卵。 第1步。准备用于显微注射。 a）建立养殖对鱼晚上，在注射之前，单身男性和单身女性成鱼（3-18个月的年龄）被放置在对分频器隔开，直到第二天早上饲养员坦克。我们通常会成立10-20对鱼，这些都是每周一次交配，无论鸡蛋是否将使用，维护繁殖力。在这个实验中，我们使用了记者的应变，cmlc2转基因：EGFP，因为它提供了一个简单的读数，找出与众不同的心肌。第二天早晨，当灯都打开，水是改变，而分频器允许鱼对交配和产生卵子坦克的拉。蛋产量可监控，并可能立即启动，或经过一个小时或更长时间内，根据鱼。通常情况下，不间断的交配后约20分钟，鸡蛋是用吸管或过滤器收集到100毫米的Petri系统水菜浇。重要的是要监察鸡蛋的生产，以保证之间的1-4细胞阶段的胚胎注入。一次释放几双，这是可以错开产蛋量，从而最大限度地提高鸡蛋的数量可以在早期发育阶段的注射。一双好鱼可能会产生超过200个胚胎，它使在同一时间收集数以千计的鸡蛋没有实际意义，因为一个单一的研究者将无法注入速度不够快，超出4细胞阶段发展之前，他们。鱼受到刺激滋生的光周期，所以这是一个实验，需要尽早开始，和鸡蛋中午过后通常不会获得。 二）准备针<ol><li>使用微管牵引器和一个3.5“玻璃毛细管产生两针，使用下列条件：热= 626，拉= 60，速度= 60，时间= 250，我们用开放的核心不包含灯丝的针，因为。我们面前填写吸痰。 </li><li>然后轻轻地打破每一针的尖端是用干净的刀片或镊子，并随后在30度角为20秒左右的斜角使用一个EG - 4微型磨床。这磨尖，重要的是，如果你通过绒毛膜注入。 </li><li>重要的是要取得一致的针针尖大小直径约15微米（使用网格的显微镜目镜，能够校准1-2 NL注射量为4单位校准）。 </li><li>针应提前做好准备，安全地存储在一个安全的位置，每个单独校准开始之前注射，以保证一致的注射量（详见D节）。如果你是幸运的，你可能能够为整个实验过程中使用的单针。然而，一针可以很容易地打破在一个实验中，你不想使用时间拉新针。 </li></ol> c）在注射板<ol><li>准备100毫升2％琼脂糖系统水的解决方案，在微波炉中煮沸。 </li><li>凉爽的触感，倒到一个100mm的培养皿倒盖12毫升。 </li><li>插入2显微镜幻灯片，在大约45度角倾斜，到双方的盖子。一旦琼脂糖已经凝固，轻轻一拉两个幻灯片远离盖子。 </li><li>这将创建胚胎将被放置，并随后注入的低谷。多个板块可以提前准备的时间。它们可以无限期地储存在4 ° C加入了一层70％的乙醇后，加入原板的底部，并用封口膜密封。请记住，使用前清洗。 </li></ol> D）的显微注射站和针校准<ol><li>打开压缩氮气源的PLI - 100微量。喷油器应调整为一个恒定的流量压力18 PSI。 </li><li>在插入一根针，保证的显微操纵器是在一个适当的位置，让广泛的运动，并插入针不会罢工表。插入一个针头准备（见C部分）为确保有一个密封的显微。不小心打破针。 </li><li>观察针尖在显微镜下，以确保它不是堵塞，然后再进行校准。 </li><li>广场上的封口膜的小片，注射液DDH 2 O的一个下降（2-3μL）和填充使用填充按钮拉进针约1.5 μL。确保您已完全解决方案中的针尖，并通过显微镜观察液体吸，以避免任何气泡绘图。 </li><li>将下降到微米电网的矿物油。 </li><li>凭经验调整注射的时间，以确保墨滴大小，直径约为1.5微米。这将提供一个〜1.8 NL的注射量。实际体积可以调整到您所选择的，但在实际意义上说，它是很难准确校准低于1 NL卷，胚胎可能不会容忍上述2 NL卷。无论音量大小，您选择，保持它包括控制您的所有注射​​相同，并调整您的解决方案（而不是注射量）浓度滴定的注射啉量。 </li><li>重新校准是必要的时间，因为每次更换针针尖大小会有所不同。 </li></ol>第2步。针对两个不同的基因morpholinos的验证 Morpholinos设计为目标的翻译起始位点或目标从而阻止蛋白质的合成或适当的mRNA加工的基因的剪接位点。当首次基因分析中，我们使用morpholinos这两种类型的测试，如果它们产生相同的表型，说明具体击倒。剪接受体阻滞剂的一个好处是，你可以验证通过RT - PCR，如果不可用的靶基因的抗体，这是特别有用的正常谈话内容击倒。我们将说明gata5和gata6使用morpholinos（gata5 5&#39;UTR莫序列的基因，如何取得一致的心脏表型是：5&#39; - AAGATAAAGCCAGGCTCGAATACAT - 3&#39;; gata5剪接位点MO序列：5&#39; - TCTTAAGATTTTTACCTATACTGGA - 3 &#39;; gata6 5 &#39;UTR区莫序列：5&#39; - AGCTGTTATCACCCAGGTCCATCCA - 3&#39;）。 <ol><li>基因工具，LLC（Philomath，或）获得Morpholinos。基因工具将帮助您选择最佳的目标序列，如果你问他们的支持人员。你只需要向他们发送的加入，他们将建议。然而，没有保证，吗啉工作，这就是为什么你需要做好准备，以验证活动。您也可以检查吗啉是从OpenBiosystems。基因工具提供合成morpholinos股票OpenBiosystems。虽然又是不能保证它会工作，他们以较低的成本出售数量较少，这可能有助于支付费用，用于测试新的目标。一定要爆你吗啉对转录;我们使用的UCSC的斑马鱼的基因组浏览器（genome.ucsc.edu）BLAT功能。当然，如果啉已验证文献中（严格），建议您购买相同的序列。 </li><li>吗啉是溶解在无菌DDH 2 0到终浓度为1毫米，并在室温下储存。不要冻结啉的解决方案，低温条件下，由于某种原因有时会导致他们聚集和沉淀。 Morpholinos蛋白酶或核酸不敏感，所以只要您添加的水是无菌的，他们应在室温下非常安全的。 </li><li>注射前，孵化啉在65℃，5分钟，以确保它完全溶解，并允许冷却到室温。不要放在冰样本。保持在室温，直到注射吗啉。 </li><li>滴定一个吗啉，我们通常准备连续稀释的股权集中在2 0 DDH，产量为1毫米的工作浓度，0.5毫米，0.25毫米和0.125毫米。根据序列，使用的注射量1.8 NL，这代表约20ng，10ng，5ng，每次注射啉2.5ng。这仅仅是一个起点，因为每个啉的宽容和疗效会有所不同，并不能预测。大多数morpholinos不会被容忍，远高于20纳克，但有些可以有效剂量远低于1纳克。我们的做法是定义的集中表现型“高原”。换句话说，如果定义的表型没有看到使用2.5毫微克，但类似的使用任何金额达到或超过5纳克，我们会选择一个门槛金额为5毫微克。当然，它可能是有用的，以2.5 ng和5.0纳克之间的未来滴定，以确保您已超过阈值的重复性工作，而不是权利“边界”。 </li><li>填写连接和校准针（见上文）的最低工作浓度（最稀）吗啉的解决方案。避免过度充盈针，因为它只会浪费材料。如果您填写只有1μL，有500注射足够的解决方案。 </li><li>使用一次性吸管，转让受精卵细胞胚胎显微注射板槽。删除多余的水分，以确保胚胎秋天注射槽的底部。他们不应该浮动，而是坚持琼脂糖床。 </li><li>注射板的位置，使显微可以通过蛋​​壳和蛋黄到注射针尖下降。要定位单个胚胎，操纵注射板等，注射针的尖端推或拉胚到正确位置。要小心，不要打破针尖或损坏胚胎！这需要一些练习，但是，作为一项规则，您使用简单的显微注射针头移动到胚胎，然后注射后，相当快速，干净，。移动板用另一只手（左手，如果你用你的右手注入）的胚胎被带进注射的位置。对于一个新的学习，它是有益的实践中使用含有0.25％酚红的一个解决方案，以可视化注入的解决方案。信心也获得通过注射的解决方案，含有荧光标记的微球（Molecular Probes公司的F - 8794），以确认解决方案是注入胚胎绒毛膜间的空间，而不是。然而，实践了几个回合后，这些艾滋病通常不再需要。这是一个好主意，偶尔注入空气，只是为了确认，针头不堵塞，被注入的量适当。 </li><li>注射的胚胎调回一个100mm的Petri系统水盘，在28.5C培养。注射浓度最低的工作后，可以驱逐任何剩余的解决方案和未来最高工作吗啉的浓度用于填补针。重复每个啉的浓度。虽然它是针使用水冲洗，测试鲜明啉时，我们更愿意改变针头和校准。一定要保持一个队列uninjected胚胎，以验证特定批次的胚胎是否健康和正常的。 </li></ol>第3步。在个别的阈值剂量注射morpholinos 进行上述滴定的目标是为每个啉定义的阈值剂量。在最好的情况下，基本上100％的morphants会显示一个定义的表型，如果目标基因的基本功能。但是，可以预期一些变异因误注入工件。随着实践中，这不应该超过10％。胚胎（包括uninjected控制）的部分批次可能无法生存，在这种情况下的实验，可能需要要打折扣。同时，也有生物变异，因为个人的胚胎可能或多或少宽容的击倒。最后，一​​些morpholinos可能只是没有足够的效率，实现了强大的（渗透）击倒。如果表型产生的胚胎的比例很低，但重复性，它可能是值得测试鲜明啉。本实验的目标是，以确定是否被视为一个全新的表型时，这两个基因同时有针对性。我们不能简单地寻找有morphant表型的胚胎比例增加，但产生一个独特的表型，是不是达到或超过阈值水平，而在测试时的单个基因。 <ol><li>准备测试以前，在每一个人啉产生了定义的表型的阈浓度morpholinos组合的混合物。例如，如果每个基因的门槛为5毫微克，准备工作解决方案将包含5纳克每5纳克（10 ng总）。由于胚胎可能不会容忍远远超过20纳克的总吗啉，它是重要的，根据以前的实验中，使用少量每个足以有效地针对每一个基因。 </li><li>控制应包括与每个啉单组合在相同浓度，注入胚胎套。注射液体积应保持不变（如1.8 NL）。记者应变的一个最大的好处是，你可以随时评估表型（如心肌细胞分化），并多次。如果胚胎阶段匹配的胚胎和控制进行评估原位杂交基因表达，可以固定在注射后不同时间。我们的实验中，我们将GFP的表达，在约24 HPF，评估作为心肌细胞分化的替代品。 </li></ol>第4步。 Ë估价表型<ol><li>应监测注射的胚胎，注射后，多次在白天，以消除任何死亡或濒临死亡的胚胎，因为这些可以妥协的，其余morphants的的可行性。为了评估报告基因的表达，胚胎研究使用解剖显微镜配备有荧光的能力。我们使用尼康SMZ1500显微镜下，用1倍或1.6倍的目标和0.75倍的放大倍率范围从11.25x。用荧光过滤器时，务必打开隔膜完全开放的环境，以发挥最大的光强度。另外，一定要使用正确的过滤器，根据记者基因（GFP，RFP等）。 </li><li>胚胎通常镇静剂tricaine磺酸4mg/ml股票的水系统使用一个1 / 20稀释。存储在- 20C冻结的股票。另外，胚胎可以安乐死使用的20倍tricaine股票。即使他们仍然在绒毛膜，胚胎通常可以筛选。然而，尤其是当它们将被拍到，这是一个好主意，用锋利的钳取出chorions。胚胎可以被看作后放置在抑郁症的幻灯片，在tricaine解决方案，或能更好地固定摆成一个小降3％甲基纤维素的摄影。 </li><li>观察在注射morpholinos组合相比，单morphants胚胎的表型。在这里，我们正在寻找GFP表达原始心管的格局。在记者株GFP的表达通过cmlc2的推动者，这使得心脏的规范和心管形态发生的详细分析的心肌细胞。 </li></ol>代表性的成果在我们的实验中，gata5和gata6单morphants显示重复性的心脏表型注射时的阈值水平，虽然有相当大的生物变异 3,4 。例如，大部分gata5 morphants产生裂心，在两个区域位于GFP的表达。出现这种情况，因为心脏祖细胞无法正确迁移保险丝在中线形成心管。然而，一些gata5 morphants产生有缺陷的心管，在少数（5％）有一个GFP +细胞的数量显着减少。我们认为，这反映了生物的变异，但也可能是由于这样的事实，不等同于“空”的突变morpholinos的。 同样，gata6 morphants所有显示心脏表型，但有一系列的表型，包括少数的双歧加热，和不安的心，是形态相比uninjected控制胚胎。这是不寻常的心脏形态表型变异，因为心脏形成是一个动态的过程，至少心脏表型的一些可能是由于从胚胎形成的缺陷间接的。然而，重要的是，gata5和gata6 morphants心脏形态缺陷的范围是一致的和可重复性，以及大部分的胚胎产生大量的GFP +心肌。 在形成了鲜明的对比gata5和gata6单morphants，这两个因素枯竭的胚胎完全丧失了GFP的表达，与心肌细胞丧失一致。因此，gata5和gata6每个人心脏的形态发生非冗余的功能，但是这两个基因在功能上是有区别的心肌细胞的生成要求的冗余。我们先前的研究表明了最早的心肌标志物，包括nkx2.5损失，提示心肌规范3要求。

<ol>
	<li>Heicklen-Klein, A., McReynolds, L. J., Evans, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+the+zebrafish+model+to+study+GATA+transcription+factors.">Using the zebrafish model to study GATA transcription factors.</a> Semin Cell Dev Biol. 16, 95-106 (2005).</li><li>Bill, B. R., Petzold, A. M., Clark, K. J., Schimmenti, L. A., Ekker, 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=A+primer+for+morpholino+use+in+zebrafish.">A primer for morpholino use in zebrafish.</a> Zebrafish. 6, 69-77 (2009).</li><li>Holtzinger, A., Evans, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gata5+and+Gata6+are+functionally+redundant+in+zebrafish+for+specification+of+cardiomyocytes.">Gata5 and Gata6 are functionally redundant in zebrafish for specification of cardiomyocytes.</a> Dev Biol. 312, 613-622 (2007).</li><li>Peterkin, T., Gibson, A., Patient, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Redundancy+and+evolution+of+GATA+factor+requirements+in+development+of+the+myocardium.">Redundancy and evolution of GATA factor requirements in development of the myocardium.</a> Dev Biol. 311, 623-635 (2007).</li><li>Holtzinger, A., Evans, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gata4+regulates+the+formation+of+multiple+organs.">Gata4 regulates the formation of multiple organs.</a> Development. 132, 4005-4014 (2005).</li><li>Sorrentino, R. P., Gajewski, K. M., Schulz, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GATA+factors+in+Drosophila+heart+and+blood+cell+development.">GATA factors in Drosophila heart and blood cell development.</a> Semin Cell Dev Biol. 16, 107-116 (2005).</li></ol>

在这里描述的实验，我们结合两个morpholinos，每个单独产生不同表型的范围，并发现一个完全不同的表型时，他们一起合作注入。当然，我们需要摆在首位，做实验的理由。我们怀疑这两个基因可能为早期功能补偿对方在这种情况下心脏规范，。这是因为基因高度相关（相同的DNA序列结合的能力）和重叠在胚胎发育 5的模式表示。此外，在果蝇遗传实验表明，GATA转录因子应为心脏规...

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		<th>Catalogue Number</th>
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<td>Fish breeder tanks and dividers</td>
<td>&nbsp;</td>
<td>Aquatic Habitats</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Fish nets</td>
<td>&nbsp;</td>
<td>Aquatic Habitats</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>System water</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>60mg &#x201C;Instant Ocean&#x201D; per liter dH2O</td>
</tr>
<tr>
<td>100mm Petri dishes</td>
<td>&nbsp;</td>
<td>BD Falcon</td>
<td>351029</td>
<td>&nbsp;</td>
<tr>
<td>Micropipette needle puller</td>
<td>&nbsp;</td>
<td>Sutter instruments, Co.</td>
<td>P-97 Flaming/Brown</td>
<td>&nbsp;</td>
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<td>3.5" glass capillaries</td>
<td>&nbsp;</td>
<td>Drummond</td>
<td>3-000-203-G/X</td>
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<td>Razor blade</td>
<td>&nbsp;</td>
<td>Personna</td>
<td>94-120-71</td>
<td>&nbsp;</td>
<tr>
<td>Micro grinder</td>
<td>&nbsp;</td>
<td>Narishige, Japan</td>
<td>EG-4</td>
<td>&nbsp;</td>
<tr>
<td>Gridded microscope eyepiece</td>
<td>&nbsp;</td>
<td>Premiere</td>
<td>MF-02</td>
<td>&nbsp;</td>
<tr>
<td>Micrometer</td>
<td>&nbsp;</td>
<td>Ward&#x2019;s Natural Science</td>
<td>94 W 9910</td>
<td>&nbsp;</td>
<tr>
<td>Ultrapure agarose</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15510-027</td>
<td>&nbsp;</td>
<tr>
<td>Spatula</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>14-357Q</td>
<td>&nbsp;</td>
<tr>
<td>Scale</td>
<td>&nbsp;</td>
<td>Mettler Toledo</td>
<td>AB54-S/FACT</td>
<td>&nbsp;</td>
<tr>
<td>Disposable weigh dishes</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>2-202-B</td>
<td>&nbsp;</td>
<tr>
<td>Conventional microwave</td>
<td>&nbsp;</td>
<td>General Electric</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Erlenmeyer flask</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>4980</td>
<td>&nbsp;</td>
<tr>
<td>Microscope slides</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>12-552</td>
<td>&nbsp;</td>
<tr>
<td>Graduated cylinder</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>08-572-6D</td>
<td>&nbsp;</td>
<tr>
<td>Automatic pipette man</td>
<td>&nbsp;</td>
<td>BrandTech</td>
<td>2026333</td>
<td>&nbsp;</td>
<tr>
<td>70% ethanol wash solution</td>
<td>&nbsp;</td>
<td>Tarr</td>
<td>801VWR</td>
<td>&nbsp;</td>
<tr>
<td>N2 tank</td>
<td>&nbsp;</td>
<td>Tech Air</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Microinjector</td>
<td>&nbsp;</td>
<td>Harvard apparatus</td>
<td>PLI-100</td>
<td>&nbsp;</td>
<tr>
<td>Micromanipulator</td>
<td>&nbsp;</td>
<td>Micro Instruments</td>
<td> LTD</td>
<td>&nbsp;</td>
<tr>
<td>Dissecting microscope</td>
<td>&nbsp;</td>
<td>Nikon</td>
<td>SMZ1500</td>
<td>&nbsp;</td>
<tr>
<td>Parafilm</td>
<td>&nbsp;</td>
<td>American National Can</td>
<td>PM-992</td>
<td>&nbsp;</td>
<tr>
<td>Mineral oil</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M5904</td>
<td>&nbsp;</td>
<tr>
<td>Kimwipes</td>
<td>&nbsp;</td>
<td>Kimberly-Clark</td>
<td>34155</td>
<td>&nbsp;</td>
<tr>
<td>gata5 splice site morpholino</td>
<td>&nbsp;</td>
<td>Genetools, LLC</td>
<td>&nbsp;</td>
<td>5'-TCTTAAGATTTTTACCTATACTGGA-3'</td>
</tr>
<tr>
<td>gata5 ATG Blocker</td>
<td>&nbsp;</td>
<td>Genetools, LLC</td>
<td>&nbsp;</td>
<td>5'-AAGATAAAGCCAGGCTCGAATACAT-3'</td>
</tr>
<tr>
<td>gata6 Long Isoform ATG Blocker</td>
<td>&nbsp;</td>
<td>Genetools, LLC</td>
<td>&nbsp;</td>
<td>5'-AGCTGTTATCACCCAGGTCCATCCA-3'</td>
</tr>
<tr>
<td>Disposable Pasteur Pipette</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>13-678-20B</td>
<td>&nbsp;</td>
<tr>
<td>Tricaine Methanesulfonate</td>
<td>&nbsp;</td>
<td>Argent Chemical Laboratories</td>
<td>MS-222</td>
<td>&nbsp;</td>
<tr>
<td>Methycellulose</td>
<td>&nbsp;</td>
<td>ICN Biomedicals</td>
<td>155495</td>
<td>&nbsp;</td>
<tr>
<td>Heat Block</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>11-718-4</td>
<td>&nbsp;</td>
<tr>
<td>Sharp forceps</td>
<td>&nbsp;</td>
<td>Dumont</td>
<td>&nbsp;</td>
<td>Size 55</td>
</tr>
<tr>
<td>Depression Microslides</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>48339-009</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

a reverse genetic approach to test functional redundancy during embryogenesis

通常由胚胎发育过程中基因功能丧失功能的实验，例如，通过有针对性的突变在小鼠（淘汰赛）。在斑马鱼模型，有效的反向遗传技术已经开发使用微量注射特定基因的反义morpholinos。 Morpholinos目标，通过特定的碱基配对和阻止基因的功能mRNA的瞬时抑制翻译或数天的胚胎发育过程中的拼接（击倒）。然而，在脊椎动物，如鼠标或斑马鱼，一些基因的功能可以被遮蔽的损失进行补偿的另一种基因的存在，由于这些方法。这是基因姐姐在同一发展组织共同表达的基因的家庭尤其如此。在斑马鱼，补偿功能，可以在一个相对高通量的方式进行测试，共注射morpholinos目标击倒的这两个基因同时。同样，使用morpholinos，任何两个基因之间的遗传相互作用，可以证明这两个基因敲除在亚阈值水平。例如，morpholinos可滴定等，无论是个人击倒生成一个表型。下，如果这些条件中，双方morpholinos的合作注入导致的表型，是一种遗传性的互动。在这里，我们演示了如何显示在两个相关的转录因子GATA转录因子方面的功能冗余。转录因子GATA因素是必不可少的心脏祖细胞的规范，但是这仅仅是损失Gata5和Gata6透露。我们如何进行显微注射​​实验，验证morpholinos，评估补偿cardiogenesis表型。

Watch this Scientific Journal Video about A Reverse Genetic Approach to Test Functional Redundancy During Embryogenesis at JoVE.com

A Reverse Genetic Approach to Test Functional Redundancy During Embryogenesis

基因的功能，可以掩盖损失功能的实验中，如果有另一种基因的赔偿。斑马鱼模型提供了一个相对高通量的手段来揭示生活胚胎这样的功能冗余。

一个反向遗传学方法测试胚胎发育过程中的功能冗余

Gene function during embryogenesis is typically defined by loss-of-function experiments, for example by targeted mutagenesis (knockout) in the mouse. In ...

a-reverse-genetic-approach-to-test-functional-redundancy-during

Research

JoVE Journal

Biology

11.9K 次观看.  Weill Cornell Medical College of Cornell University. 基因的功能，可以掩盖损失功能的实验中，如果有另一种基因的赔偿。斑马鱼模型提供了一个相对高通量的手段来揭示生活胚胎这样的功能冗余。

视频: A Reverse Genetic Approach to Test Functional Redundancy During Embryogenesis

Light/dark Transition Test for Mice

Although all of the mouse genome sequences have been determined, we do not yet know the functions of most of these genes. Gene-targeting techniques, however, can be used to delete or manipulate a specific gene in mice. The influence of a given gene on a specific behavior can then be determined by conducting behavioral analyses of the mutant mice. As a test for behavioral phenotyping of mutant mice, the light/dark transition test is one of the most widely used tests to measure anxiety-like behavior in mice. The test is based on the natural aversion of mice to brightly illuminated areas and on their spontaneous exploratory behavior in novel environments. The test is sensitive to anxiolytic drug treatment. The apparatus consists of a dark chamber and a brightly illuminated chamber. Mice are allowed to move freely between the two chambers. The number of entries into the bright chamber and the duration of time spent there are indices of bright-space anxiety in mice. To obtain phenotyping results of a strain of mutant mice that can be readily reproduced and compared with those of other mutants, the behavioral test methods should be as identical as possible between laboratories. The procedural differences that exist between laboratories, however, make it difficult to replicate or compare the results among laboratories. Here, we present our protocol for the light/dark transition test as a movie so that the details of the protocol can be demonstrated. In our laboratory, we have assessed more than 60 strains of mutant mice using the protocol shown in the movie. Those data will be disclosed as a part of a public database that we are now constructing.

Visualization of the protocol will facilitate understanding of the details of the entire experimental procedure, allowing for standardization of the protocols used across laboratories and comparisons of the behavioral phenotypes of various strains of mutant mice assessed using this test.

The light/dark transition test is one of the most widely used tests to measure anxiety-like behavior in mice. Here, we present a movie that shows detailed procedures on how we conduct the test.

光/暗转换测试小鼠

Although all of the mouse genome sequences have been determined, we do not yet know the functions of most of these genes. Gene-targeting techniques, ...

科研

生物学

Method for Culture of Early Chick Embryos ex vivo (New Culture)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  the formation and patterning of brain, neural tube, somite and heart primordia. Applications of chick embryo culture include electroporation of DNA or RNA constructs in order to analyze gene function, grafts of growth factor coated beads such as FGFs and BMPs , as well as whole mount in situ hybridization and immunohistochemistry. This video demonstrates the different steps in chick embryo culture; First, the embryo is explanted in saline. Then, the embryo is centered on a glass ring. The membranes surrounding the embryo are lifted along the walls of the ring. The ring is then placed in a culture dish containing a pool of albumine. The culture dish is sealed and placed in a humid chamber, where the embryo is cultured for up to 24 hrs. Finally, the embryo is removed from the ring, fixed and processed for further applications. A troubleshooting guide is also presented.

Method for Culture of Early Chick Embryos ex vivo New Culture

This video demonstrates New culture, a method by which chick embryos are cultured outside the egg for up to 24 hr. This method enables one to study early development (primitive streak to 14 som.), a period corresponding to E7-9 in mouse. Applications of this technique include electroporation, in situ hybridization and immunohistochemistry.

文化早期鸡胚体外（新文化）的方法

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Double Whole Mount in situ Hybridization of Early Chick Embryos

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  gene expression in brain, neural tube, somite and heart primordia formation. This video demonstrates the different steps in 2-color whole mount in situ hybridization; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, the embryo is processed for prehybridization. The embryo is then hybridized with two different probes, one coupled to DIG, and one coupled to FITC.  Following overnight hybridization, the embryo is incubated with DIG coupled antibody. Color reaction for DIG substrate is performed, and the region of interest appears blue. The embryo is then incubated with FITC coupled antibody. The embryo is processed for color reaction with FITC, and the region of interest appears red. Finally, the embryo is fixed and processed for phtograph and sectioning. A troubleshooting guide is also presented.

This video demonstrates 2-color whole mount in situ hybridization, a method by which the spatial and temporal expression pattern of 2 different genes can be visualized in young chick embryos. This method was originally introduced by David Wilkinson, Domingos Henrique, Phil Ingham and David Ish -Horowicz.

双整装原位杂交技术在早期鸡胚

Assay for Neural Induction in the Chick Embryo

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying the formation and initial patterning of the nervous system. This video demonstrates how to graft organizer tissue into a host, a method by which Hensen s node (the organizer in the chick embryo) is grafted to a host competent ectoderm. The organizer graft instructs overlying na ve tissue to adopt a neural fate via neural inducing signals. This mechanism is referred to as neural induction, and constitutes the initial step in the formation of brain and spinal cord in amniotes. This method is essentially used for the characterization of putative neural inducing molecules in chick. This video demonstrates the different steps in the assay for neural induction; First, the donnor embryo is explanted and pinned on a dish. Then, the host embryo is prepared for New culture. The graft is excised and transplanted to the host area pellucida margin. The host is cultured for 18-22 hrs. The assembly is fixed and processed for further applications (e.g. in situ hybridization). This method was originally devised by Waddington 1,2 and Gallera 3,4.

Neural induction is the first step in the formation of the brain. It is a mechanism by which Hensen's node (organizer), instructs adjacent tissue to adopt a neural fate, i.e. to give rise to the nervous system. This video demonstrates an assay for neural induction in chick embryo.

在鸡胚神经诱导的含量为

Elevated Plus Maze for Mice

Although the mouse genome is now completely sequenced, the functions of most of the genes expressed in the brain are not known. The influence of a given gene on a specific behavior can be determined by behavioral analysis of mutant mice. If a target gene is expressed in the brain, behavioral phenotype of the mutant mice could elucidate the genetic mechanism of normal behaviors. The elevated plus maze test is one of the most widely used tests for measuring anxiety-like behavior. The test is based on the natural aversion of mice for open and elevated areas, as well as on their natural spontaneous exploratory behavior in novel environments. The apparatus consists of open arms and closed arms, crossed in the middle perpendicularly to each other, and a center area. Mice are given access to all of the arms and are allowed to move freely between them. The number of entries into the open arms and the time spent in the open arms are used as indices of open space-induced anxiety in mice. Unfortunately, the procedural differences that exist between laboratories make it difficult to duplicate and compare results among laboratories. Here, we present a detailed movie demonstrating our protocol for the elevated plus maze test. In our laboratory, we have assessed more than 90 strains of mutant mice using the protocol shown in the movie. These data will be disclosed as a part of a public database that we are now constructing.
Visualization of the protocol will promote better understanding of the details of the entire experimental procedure, allowing for standardization of the protocols used in different laboratories and comparisons of the behavioral phenotypes of various strains of mutant mice assessed using this test. 


The elevated plus maze test is one of the most widely used tests for measuring anxiety-like behavior in mice. Here, we present a movie showing the detailed procedures for conducting the test.

对小鼠高架十字迷宫

Although the mouse genome is now completely sequenced, the functions of most of the genes expressed in the brain are not known. The influence of a given ...

Dissection of Hippocampal Dentate Gyrus from Adult Mouse

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its remarkable neuronal cell plasticity, and its involvement in epilepsy, neurodegenerative diseases, and psychiatric disorders. The hippocampus is composed of distinct regions; the dentate gyrus, which comprises mainly granule neurons, and Ammon's horn, which comprises mainly pyramidal neurons, and the two regions are connected by both anatomic and functional circuits. Many different mRNAs and proteins are selectively expressed in the dentate gyrus, and the dentate gyrus is a site of adult neurogenesis; that is, new neurons are continually generated in the adult dentate gyrus. To investigate mRNA and protein expression specific to the dentate gyrus, laser capture microdissection is often used. This method has some limitations, however, such as the need for special apparatuses and complicated handling procedures. In this video-recorded protocol, we demonstrate a dissection technique for removing the dentate gyrus from adult mouse under a stereomicroscope. Dentate gyrus samples prepared using this technique are suitable for any assay, including transcriptomic, proteomic, and cell biology analyses. We confirmed that the dissected tissue is dentate gyrus by conducting real-time PCR of dentate gyrus-specific genes, tryptophan 2,3-dioxygenase (TDO2) and desmoplakin (Dsp), and Ammon's horn enriched genes, Meis-related gene 1b (Mrg1b) and TYRO3 protein tyrosine kinase 3 (Tyro3). The mRNA expressions of TDO2 and Dsp in the dentate gyrus samples were detected at obviously higher levels, whereas Mrg1b and Tyro3 were lower levels, than those in the Ammon's horn samples. To demonstrate the advantage of this method, we performed DNA microarray analysis using samples of whole hippocampus and dentate gyrus. The mRNA expression of TDO2 and Dsp, which are expressed selectively in the dentate gyrus, in the whole hippocampus of alpha-CaMKII+/- mice, exhibited 0.037 and 0.10-fold changes compared to that of wild-type mice, respectively. In the isolated dentate gyrus, however, these expressions exhibited 0.011 and 0.021-fold changes compared to that of wild-type mice, demonstrating that gene expression changes in dentate gyrus can be detected with greater sensitivity. Taken together, this convenient and accurate dissection technique can be reliably used for studies focused on the dentate gyrus.

A dissection technique for removal of the dentate gyrus from adult mouse under a stereomicroscope was demonstrated in this video-recorded protocol.

从成年小鼠海马齿状回的剖析

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its ...

A Practical Approach to Genetic Inducible Fate Mapping: A Visual Guide to Mark and Track Cells In Vivo

Fate maps are generated by marking and tracking cells in vivo to determine how progenitors contribute to specific structures and cell types in developing and adult tissue. An advance in this concept is Genetic Inducible Fate Mapping (GIFM), linking gene expression, cell fate, and cell behaviors in vivo, to create fate maps based on genetic lineage.
GIFM exploits X-CreER lines where X is a gene or set of gene regulatory elements that confers spatial expression of a modified bacteriophage protein, Cre recombinase (CreERT). CreERT contains a modified estrogen receptor ligand binding domain which renders CreERT sequestered in the cytoplasm in the absence of the drug tamoxifen. The binding of tamoxifen releases CreERT, which translocates to the nucleus and mediates recombination between DNA sequences flanked by loxP sites. In GIFM, recombination typically occurs between a loxP flanked Stop cassette preceding a reporter gene such as GFP.
Mice are bred to contain either a region- or cell type-specific CreER and a conditional reporter allele. Untreated mice will not have marking because the Stop cassette in the reporter prevents further transcription of the reporter gene. We administer tamoxifen by oral gavage to timed-pregnant females, which provides temporal control of CreERT release and subsequent translocation to the nucleus removing the Stop cassette from the reporter. Following recombination, the reporter allele is constitutively and heritably expressed. This series of events marks cells such that their genetic history is indelibly recorded. The recombined reporter thus serves as a high fidelity genetic lineage tracer that, once on, is uncoupled from the gene expression initially used to drive CreERT.
We apply GIFM in mouse to study normal development and ascertain the contribution of genetic lineages to adult cell types and tissues. We also use GIFM to follow cells on mutant genetic backgrounds to better understand complex phenotypes that mimic salient features of human genetic disorders.
This video article guides researchers through experimental methods to successfully apply GIFM. We demonstrate the method using our well characterized Wnt1-CreERT;mGFP mice by administering tamoxifen at embryonic day (E)8.5 via oral gavage followed by dissection at E12.5 and analysis by epifluorescence stereomicroscopy. We also demonstrate how to micro-dissect fate mapped domains for explant preparation or FACS analysis and dissect adult fate-mapped brains for whole mount fluorescent imaging. Collectively, these procedures allow researchers to address critical questions in developmental biology and disease models.

A Practical Approach to Genetic Inducible Fate Mapping: A Visual Guide to Mark and Track Cells In Vivo

Genetic Inducible Fate Mapping (GIFM) marks and tracks cells with fine spatial and temporal control in vivo and elucidates how cells from a specific genetic lineage contribute to developing and adult tissues. Demonstrated here are the techniques required to fate map E12.5 mouse embryos for epifluorescent and explant analysis.

一个实用的方法遗传诱导的命运映射：一个视觉引导标记和跟踪细胞在体内</em

Fate maps are generated by marking and tracking cells in vivo to determine how progenitors contribute to specific structures and cell types in developing ...

An Allele-specific Gene Expression Assay to Test the Functional Basis of Genetic Associations

The number of significant genetic associations with common complex traits is constantly increasing. However, most of these associations have not been understood at molecular level. One of the mechanisms mediating the effect of DNA variants on phenotypes is gene expression, which has been shown to be particularly relevant for complex traits1.
This method tests in a cellular context the effect of specific DNA sequences on gene expression. The principle is to measure the relative abundance of transcripts arising from the two alleles of a gene, analysing cells which carry one copy of the DNA sequences associated with disease (the risk variants)2,3. Therefore, the cells used for this method should meet two fundamental genotypic requirements: they have to be heterozygous both for DNA risk variants and for DNA markers, typically coding polymorphisms, which can distinguish transcripts based on their chromosomal origin (Figure 1). DNA risk variants and DNA markers do not need to have the same allele frequency but the phase (haplotypic) relationship of the genetic markers needs to be understood. It is also important to choose cell types which express the gene of interest. This protocol refers specifically to the procedure adopted to extract nucleic acids from fibroblasts but the method is equally applicable to other cells types including primary cells.
DNA and RNA are extracted from the selected cell lines and cDNA is generated. DNA and cDNA are analysed with a primer extension assay, designed to target the coding DNA markers4. The primer extension assay is carried out using the MassARRAY (Sequenom)5 platform according to the manufacturer's specifications. Primer extension products are then analysed by matrix-assisted laser desorption/ionization time of-flight mass spectrometry (MALDI-TOF/MS). Because the selected markers are heterozygous they will generate two peaks on the MS profiles. The area of each peak is proportional to the transcript abundance and can be measured with a function of the MassARRAY Typer software to generate an allelic ratio (allele 1: allele 2) calculation. The allelic ratio obtained for cDNA is normalized using that measured from genomic DNA, where the allelic ratio is expected to be 1:1 to correct for technical artifacts. Markers with a normalised allelic ratio significantly different to 1 indicate that the amount of transcript generated from the two chromosomes in the same cell is different, suggesting that the DNA variants associated with the phenotype have an effect on gene expression. Experimental controls should be used to confirm the results.

Genetic associations often remain unexplained at a functional level. This method aims to assess the effect of phenotype-associated genetic markers on gene expression by analyzing cells heterozygous for transcribed SNPs. The technology allows accurate measurement by MALDI-TOF mass spectrometry to quantify allele-specific primer extension products.

等位基因特异性基因表达检测，测试的遗传协会的功能基础

The number of significant genetic associations with common complex traits is constantly increasing. However, most of these associations have not been ...

Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica

Free-living planarian flatworms have a long history of experimental usage owing to their remarkable regenerative abilities1. Small fragments excised from these animals reform the original body plan following regeneration of missing body structures. For example if a 'trunk' fragment is cut from an intact worm, a new 'head' will regenerate anteriorly and a 'tail' will regenerate posteriorly restoring the original 'head-to-tail' polarity of body structures prior to amputation (Figure 1A).
Regeneration is driven by planarian stem cells, known as 'neoblasts' which differentiate into ~30 different cell types during normal body homeostasis and enforced tissue regeneration. This regenerative process is robust and easy to demonstrate. Owing to the dedication of several pioneering labs, many tools and functional genetic methods have now been optimized for this model system. Consequently, considerable recent progress has been made in understanding and manipulating the molecular events underpinning planarian developmental plasticity2-9.
The planarian model system will be of interest to a broad range of scientists. For neuroscientists, the model affords the opportunity to study the regeneration of an entire nervous system, rather than simply the regrowth/repair of single nerve cell process that typically are the focus of study in many established models. Planarians express a plethora of neurotransmitters10, represent an important system for studying evolution of the central nervous system11, 12 and have behavioral screening potential13, 14. 
Regenerative outcomes are amenable to manipulation by pharmacological and genetic apparoaches. For example, drugs can be screened for effects on regeneration simply by placing body fragments in drug-containing solutions at different time points after amputation. The role of individual genes can be studied using knockdown methods (in vivo RNAi), which can be achieved either through cycles of microinjection or by feeding bacterially-expressed dsRNA constructs8, 9, 15. Both approaches can produce visually striking phenotypes at high penetrance- for example, regeneration of bipolar animals16-21. To facilitate adoption of this model and implementation of such methods, we showcase in this video article protocols for pharmacological and genetic assays (in vivo RNAi by feeding) using the planarian Dugesia japonica.

Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica

An attractive model for studying stem cell differentiation within a live animal is the planarian flatworm. Regeneration is studied by simple amputation experiments that are easily performed in a basic laboratory and are amenable to pharmacological and genetic (in vivo RNAi) manipulation as detailed by protocols in this article.

药理和功能的遗传分析处理涡虫的再生日本三角涡虫</em

Free-living planarian flatworms have a long history of experimental usage owing to their remarkable regenerative abilities1. Small fragments excised from ...

Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an attractive model system to study organ development and tissue-specific signaling. Although hair follicle development is genetically tractable, fast and reproducible analysis of factors essential for this process remains a challenge. Here we describe a procedure to generate targeted overexpression or shRNA-mediated knockdown of factors using lentivirus in a tissue-specific manner. Using a modified version of a hair regeneration model 5, 6, 11, we can achieve robust gain- or loss-of-function analysis in primary mouse keratinocytes or dermal cells to facilitate study of epithelial-mesenchymal signaling pathways that lead to hair follicle morphogenesis. We describe how to isolate fresh primary mouse keratinocytes and dermal cells, which contain dermal papilla cells and their precursors, deliver lentivirus containing either shRNA or cDNA to one of the cell populations, and combine the cells to generate fully formed hair follicles on the backs of nude mice. This approach allows analysis of tissue-specific factors required to generate hair follicles within three weeks and provides a fast and convenient companion to existing genetic models.

Tissue-specific analysis of a hair follicle regeneration model using lentivirus to mediate gain- or loss-of-function.