这项工作是由牙科的国家研究所和颅面研究所（NIDCR）F32补助（DE021929），以金六福和NIDCR R01授予DE016402到RAS

1，准备钨针<ol><li>切断的钨棒在一半使用钢丝钳使杆不弯曲。 </li><li>通过5中的硼硅玻璃四分之三巴斯德移液管的锥形端部螺纹杆。 </li><li>留下约3/4的杆附着的出玻璃，附着在杆的移液管尖的小珠“热熔性粘合剂”（HMA），这是用于胶枪将胶粘的。 HMA中应迅速融化在酒精火焰，小心不要烫伤胶水，并产生黑烟。 </li><li>利用一对钳子的，弯曲的钨棒的端部（大约1/4的上端）直到45°的角度为止。 </li><li>握住巴斯德吸管，将约1/8的钨棒的前端的进丙烷喷灯的火焰。保持尖端稳定，准确地垂直于火焰。 </li><li>拔出注射针的火焰瞬间童综合的一个微小的橙色斑点10苍蝇落针。 </li></ol> 2，准备斯佩曼移液器<ol><li>使用本生灯，加热巴斯德吸管的窄端，直到玻璃不久以后锥度开始融化。从火焰中删除和拉尖，直到吸管变成密封。可以肯定，仍存在具有大约0.7-1.0毫米的外径渐缩部分的一部分。打破密封端关闭从锥形长约4厘米。 </li><li>连接橡胶管的周围2英尺（60厘米）到移液管的宽（开口）端。吹在橡胶管的另一端，以确保没有空气可以通过巴斯德吸移管的锥形部分。 </li><li>使用丙烷燃料筒用附属的铅笔焰炬，加热略低于锥形的开始上移液管的一侧上的椭圆形区域。通过橡胶管与压力的微量（压力约需要说的莱特开始时的量吹出空气很平缓，并不断R“P”在会话级）。当玻璃变得柔软的一面，并开始向外扣，迅速从火焰取出吸管吹硬稳定通过橡胶管。这会导致玻璃的加热部分，以形成香肠状的气泡可能弹出，这是好的。如果气泡太小，尝试熔化和再吹玻璃。最后打开的窗口中的玻璃应在0.25（6.35毫米）宽的（12.7毫米）0.5长。 </li><li>指着锥形端朝上，刮泡成，同时通过油管轻轻一吹，以防止玻璃碎片进入吸管的玻璃废物容器。 </li><li>使用丙烷火焰，燃烧掉泡沫和消防抛光打开的窗口两侧的剩余边缘。注意不要融化吸管的轴。 </li><li>使用金​​刚石点铅笔，从窗口得分移液管的锥形端部中（30mm）的约1.25和折断的前端用钳子使吸管开口裁切整齐（丢弃移液器碎或不均匀的提示）。 </li><li>使用镊子和从醇燃烧器的火焰的边缘上，在（9.5毫米）的从尖端弯曲的吸管0.375的狭窄部分，以约60°的角度，使得所述弯曲部是在垂直平面内时，窗开口是为右手使用和11:00，以方便左手使用1点的位置。这一步是在一个无风的外壳最好进行。 </li><li>火抛光用解剖显微镜下的酒精火焰尖端。千万不要以关闭尖端而是保持首轮，没有任何收缩顺利。 </li><li>切1中（25毫米）一块橡胶管，喷管件的用70％乙醇，并在移液管的轴滑动，管子，直到窗口开口被完全盖住。此功能为斯佩曼吸管的“隔膜”。 </li><li>放置一个乳胶2毫升球在移液管的底部开口。灯泡保持在斯佩曼吸管的负压。 </li><li>练习使用斯佩曼吸管，从一个培养皿有明显斑点在显微镜下转约100-200微米，直径层析珠的另一显着位置。 </li><li>要清洁斯佩曼移液管，取出橡胶球和地点吸管，尖向下，在一个高脚烧杯内衬棉花或纱布在底部，装有蒸馏水和玻璃器皿洗涤剂。 </li></ol> 3，消毒和孵化蛋<ol><li>擦蛋用70％乙醇，然后将鸡蛋塑料蛋盘。 </li><li>在37.5℃和85-87％设定鸡蛋在加热的孵化器。 </li><li>卵孵化，直到他们达到HH9.5，鹌鹑和48小时的鸭子约26小时。 </li></ol> 4。窗口蛋<ol><li>从用钳子鸡蛋的顶部取下一小片外壳，小心不要刺破内壳米embrane。 </li><li>由1寸毫针使用注射器用18 G，戳一个洞在蛋的尖端最窄，收回约1-2毫升的蛋白中。 </li><li>将透明胶带多年来在外壳孔和穿刺标记。 </li><li>用弯剪（通过透明胶带）剪下一个“窗口”沿着蛋的顶面。 </li></ol> 5，可视化胚胎和准备手术<ol><li>用玻璃棒轻轻刷过胚胎少量中性红（0.02克/毫升汉克的BSS）。 </li><li>切卵黄膜和使用火焰削尖的钨针在胚胎拉出。 </li><li>通过将透明胶带在窗口重新密封的蛋。 </li></ol> 6，供体组织的供体胚胎分离<ol><li>从供体胚胎的蛋窗口中删除磁带。 </li><li>从供体胚胎的外胚层相邻切神经褶，使狭缝在两个SID神经管的ES，使用火焰削尖的钨针（如上所述制备）。 </li><li>然后，贯穿神经管在移植物的期望的前部和后部的水平，并从神经管的其余部分分开的接枝。 </li><li>从使用一个斯佩曼吸管或其他类型的微量的供体胚胎中取出神经褶。 </li><li>然后，从窗口卸下磁带上的主机蛋和使用斯佩曼移液管放置供体神经褶沿着相邻的神经管将接收移植的区域中的宿主胚。 </li></ol> 7，分离出宿主组织与移植供体组织<ol><li>从主机胚胎的神经管分离的神经褶，因为完成了捐助。小心取出一个同样大小的移植物为供体组织。 </li><li>这轻轻的宿主组织推动远离胚胎。 </li><li>然后，用钝钨针，或一米的圆尖cropipette，轻轻移动捐助神经折叠成宿主神经管，保持适当的前 - 后和背腹方向。确保移植物是藏在沿线两侧，但千万不要去戳或损坏下层组织。为了保持原来的方向音符的轨迹从中性红的供体组织或差分染色任何不对称。我们还光笔施主胚胎与随附切除的组织，以及嵌合后立即手术。 </li><li>无菌盐水中应加入鸡蛋，如果主机胚胎显示了干燥的迹象。 </li><li>现在，重新仔细密封并标记蛋，然后轻轻返回到高湿度培养箱中（70-80％），其中所述嵌合胚胎可发育至所需的阶段进行分析。 </li></ol>嵌合体的8。收藏<ol><li>一定要收集新鲜的冷塞拉的固定液嵌合体胚胎，并立即将它们放置在RO克尔在4℃的O / N固定。这将允许更多的抗鹌鹑抗体敏感的检测鹌鹑细胞。 </li><li>通过RT-qPCR的基因表达分析，直接在液氮中之前提取RNA冷冻胚胎。 </li></ol>

作者什么都没有透露。

神经嵴是一个短暂的胚胎细胞群的广泛迁移整个胚胎，并分化成不同类型的细胞，包括软骨细胞和成骨细胞，有助于颅面骨架。移植神经嵴的鹌鹑，鸭嵌合系统，大大促进了我们的调控颅面骨骼发育的组织相互作用和信号通路的理解做出了贡献。然而，由于神经嵴的巨大潜力，也产生平滑肌细胞，脂肪细胞，黑素细胞，雪旺氏细胞和神经元，鹌鹑，鸭嵌合体系统具有巨大的潜力，未来的应用，尤其是在干细胞生物学的迅猛发展相结合和再生医学。由于鹌鹑，鸭都是商业育成品种，随时提供相对廉价的受精卵可从各种农场。因此，这种技术应便于技术与工程实践她广泛的预算和设施的空间内运行。 虽然这种技术是非常强大的，依然存在着一些局限性。像其他外科技术，鹌鹑，鸭嵌合体的质量和可行性依赖于研究者的手术技巧，因此，会有实验，相比其他机型，如那些使用鼠标之间有更多间和个体内变异遗传学。此外，也有变化的发展和个人胚胎阶段的速率有助于该重复性和各移植的成功。禽胚胎也很容易脱水和在手术过程中，因此重要的步骤包括保持光照水平低，在显微镜到最低限度下的时间，用胶带尽可能密封蛋，和高湿度在手术后培养箱避免干燥。 在嵌合体的生存能力，U条款sually之间50-75％存活，虽然这些百分比可以降低老年收集阶段。手术中一个典型的4-6小时会议，有经验的外科医生可以产生10-15嵌合体。在移植的成功也很大程度上取决于工具的质量。好工具导致更一致，可重复的结果。使用丙烷焊炬，使钨针允许进行非常尖锐的针头。用火炬的类型使一个很大的区别，因为它控制着火焰的大小。电解锐化也可以使用，但这种方法并不甚至接近生产针一样锋利。使用钨棒代替假脱机线，使针可制成直的。 该斯佩曼微量，而耗时，很难做出，是组织转移的理想仪器。吸管可以用不同尺寸的开口被定制，并且可以重复使用。对于加兹登的一个关键因素嘎斯佩曼微量是尖端接触胚胎的表面之前，有在吸管一些流体。一些流体将总是流出来时接触被用弯液面以上的胚胎。压在膜片上使得流体和组织移植物，以非常精确地喷出，而稍微向上让膜片上轻轻地吸供体组织移植入吸管。保持位的正压力在膜片保持供体移植组织在移液管的转移过程中的尖端，并在膜片一点额外的压力使得供体移植组织被故意置于主机。 用于存储和消毒过程中保护斯佩曼吸管，从广角端取下灯泡，并小心将锥形尖端插入灯泡。放置斯佩曼吸管在玻璃试管中，用铝箔覆盖的顶部，并高压灭菌手术前。经过多个移液器重新ADY再次进行手术消毒，颠倒移液器，几乎它们加热到沸腾的蒸馏水中并玻璃器皿洗涤剂相同的溶液中，然后用蒸馏水反复冲洗。高压灭菌移液器在各自管。橡胶管，形成在隔膜和橡胶球应几个绝育手术后进行更换或当他们变得昏暗，僵硬。 许多本协议的组成部分涉及危险设备。例如，对于制作斯佩曼微管的过程包括三个类型的火焰，以及加热，拉，吹，弯曲，切割，抛光玻璃。因此，佩戴适当的个人防护装备（PPE），增加安全性，如护目镜和实验室外套是至关重要的。此外，因为很多人患有，或有发展，鸡蛋过敏，处理鸡蛋的时候总是使用手套的潜力。这些预防措施一点，鹌鹑，鸭嵌合系统ISA安全，高效，而且比较方便的方法，有许多未来的应用。

面部骨骼，这也构成神经嵴和中胚层间充质由外胚层和内胚层上皮1-11包围的多个面部过程的生长和融合的发展。每个进程中形态发生的事件是由间质细胞和上皮细胞周围12-16之间不同的信号相互制约。改变这些信令交互和/或其下游效应有助于疾病的表型，也可能是相关的颅面骨骼17,18的演变。因此，阐明组织相互作用的时间和性质有很大的潜力，以提高我们的面部骨骼的发育和进化生物学的理解。 利用嵌合体胚胎的研究组织相互作用有着悠久的历史，在发育生物学。这种方法是率先由Hans斯佩曼和他的实验室谁由不同种两栖动物的胚胎移植与组织发现胚胎“组织者”。斯佩曼是显微外科技术的手工技能，补充了他的发展专门的工具，尤其是斯佩曼吸管的高手。尤汉堡包是一名研究生在汉斯斯佩曼在弗赖堡的实验室在20世纪20年代，也就是当被执行的原版移植实验，导致斯佩曼的诺贝尔奖。当汉堡包搬到华盛顿大学圣路易斯分校在1935年，他详细制作斯佩曼微量在他的实验胚胎学 19 的手动过程。德鲁节点N是一名研究生在汉堡的实验室在华盛顿大学，直到1972年搬到马萨诸塞州阿姆赫斯特大学，然后到康乃尔大学后，节点N继续制造和使用斯佩曼微量他涉及鹌鹑鸡嵌合体移植手术。＆＃160;当一名研究生，培养与德鲁节点N在康奈尔大学1995年至1998年，作者（富施耐德）之一以下协议作出斯佩曼微管是基于写的汉堡包和节点N的描述，并包括由后续修改施耐德。 对颅面部发育的研究，尤其是对理解神经嵴细胞的捐款使用鹌鹑鸡嵌合体是在20世纪70年代初开创的节点N和乐Douarin，在Le Douarin 等 20审阅。这种方法已被广泛采用在许多研究和大量的其他调查1，4，5，21-38。增长和鹌鹑和鸡的形态相当于率使他们在理想的移植细胞命运和谱系追踪研究。因为鹌鹑和小鸡之间的相似性但是，形态变化IND由供体细胞uced是难以破译。相比之下，其他禽类嵌合系统已包括家鸭，以此来研究，使胚胎解剖学独特39-50机制。更具体地说，鹌鹑，鸭嵌合体系统提供了挑剔的捐赠者的主机，反之亦然对影响多重效益。首先，鹌鹑，鸭胚是不同的车身尺寸和形状，它提供了一个直接的方法通过测定基因表达（ 图1 A和​​B）的差分领域探索的模式形成供体或特定于主机的机制。其次，鹌鹑，鸭胚胎发育有很大的不同速率，与鹌鹑孵化17天，28天，鸭孵化。移植的神经嵴保持其固有的成熟率主机环境内，因此，鉴定的基因表达的形态随时间的变化，组织的相互作用，组织发生，并且能够51-57。最后，将抗鹌鹑核抗体（Q¢PN）允许供体和宿主细胞的贡献是永久区别于彼此由认识到广泛表达在鹌鹑细胞，但来自鸭细胞中存在的蛋白。

<table border="0" cellpadding="0" cellspacing="0" width="746">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:169px;">1x PBS</td>
			<td style="width:180px;">TEK</td>
			<td style="width:145px;">TEKZR114</td>
			<td style="width:252px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:169px;">Hank&#8217;s BSS w/o Phenol Red</td>
			<td style="width:180px;">Invitrogen</td>
			<td style="width:145px;">14025-092</td>
			<td style="width:252px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:169px;">Neutral Red</td>
			<td style="width:180px;">Sigma Aldrich</td>
			<td style="width:145px;">N4638-5G</td>
			<td style="width:252px;">.22&#181;m filter-sterilized</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:169px;">18G Needles</td>
			<td style="width:180px;">BD</td>
			<td style="width:145px;">305195</td>
			<td style="width:252px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:169px;">5 ml syringe</td>
			<td style="width:180px;">BD</td>
			<td style="width:145px;">309646</td>
			<td style="width:252px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:169px;">No. 5 Dumont forceps</td>
			<td style="width:180px;">Fine Science Tools</td>
			<td style="width:145px;">11252-20</td>
			<td style="width:252px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:169px;">Straight Scissors</td>
			<td style="width:180px;">Fine Science Tools</td>
			<td style="width:145px;">14028-10</td>
			<td style="width:252px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:169px;">Curved Scissors</td>
			<td style="width:180px;">Fine Science Tools</td>
			<td style="width:145px;">14029-10</td>
			<td style="width:252px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:169px;">Spemann Pipet</td>
			<td style="width:180px;"></td>
			<td style="width:145px;"></td>
			<td style="width:252px;">Hand-made in lab</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:169px;">Egg holder</td>
			<td style="width:180px;"></td>
			<td style="width:145px;"></td>
			<td style="width:252px;">Glass ashtray and modeling clay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:169px;">Alcohol burner</td>
			<td style="width:180px;">Fisher</td>
			<td style="width:145px;">04-245-1</td>
			<td style="width:252px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:169px;">Transparent tape</td>
			<td style="width:180px;">3M Scotch</td>
			<td style="width:145px;">600</td>
			<td style="width:252px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:169px;">Glass Stirring Rod</td>
			<td style="width:180px;">Fisher</td>
			<td style="width:145px;">11-380C</td>
			<td style="width:252px;">Tip is narrowed and rounded using a flame</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:169px;">Tungston wire (.004 x 3 inches)</td>
			<td style="width:180px;">A-M Systems</td>
			<td style="width:145px;">7190</td>
			<td style="width:252px;">Tip is flame-sharpened in a propane torch</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bunsun burner</td>
			<td>Fisher Scientific</td>
			<td>&#160;S49117</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pasteur pipette</td>
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			<td style="width:252px;">amber, thin wall natural rubber; wall thickness: 0.0625 inches/1.6 mm; O.D.: 0.375 inches/9.5 mm; I.D.: 0.25 inches/6.4mm</td>
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			<td height="21" style="height:21px;">Diamond point pencil</td>
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assessing species-specific contributions to craniofacial development using quail-duck chimeras

嵌合体胚胎的产生是研究细胞命运，组织的相互作用，以及具体物种的组织学和脊椎动物胚胎的形态发展的贡献普遍和有效的方法。特别是，利用嵌合体胚胎的建立在引导颅面复合体的物种特异性形态学神经嵴的重要性。本文所述的方法利用2禽类物种，鸭和鹌鹑，具有显着不同的颅面形态。这种方法大大方便了在颅面复合种属特异性格局分子和细胞调控的研究。在鹌鹑，鸭嵌合体胚胎的实验已经发现神经嵴介导的组织相互作用和调节物种特异性的颅面骨骼，肌肉和体壁图案细胞自主的行为。神经嵴衍生工具的巨大差异表明显著潜力为鹌鹑，鸭嵌合系统，以了解脊椎动物的发育，疾病和进化的未来应用。

前进一步的分析，需要移植的效率进行测定。对于组织学，形态学，或基因表达的组织样本进行分析，鹌鹑细胞应通过免疫组化运用Q¢PN抗体作为描述36检测。用于RNA分析，物种特异性的兴趣组织的贡献可以使用基于PCR的策略58来计算。经过移植的疗效已得到验证，进一步的形态学和分子成果的措施可以在嵌合体进行评估。供体神经嵴细胞与周围宿主源性的组织背后适当的组织发生和颅面复合体的形态之间的相互作用先前已经被广泛地研究。特别地，神经嵴间充质引导面的具体物种的形态7，13，51，59，羽毛图案52，肌肉图案56 </s<html>向上&gt;，和软骨53，57至宿主基因表达的调控。例如，神经嵴间充质通过在时间上调节BMP4表达55使然，当骨形成下颌骨。 最近，研究都集中在发生在颅面发育早期组织相互作用。在这方面，利用鹌鹑鸭嵌合体的实验表明宿主胚胎通过确定形态的边界影响神经嵴迁移。也就是说，在嵌合quck胚胎，捐赠鹌鹑神经嵴细胞迁移到了鸭状图案（ 图1D）主机鸭下颌弓。尽管对神经嵴人口的规模该主机贡献，供体神经嵴继续产生一个下颌骨架是鹌鹑般的尺寸和形状（ 图1E）。 Ë1“FO：内容宽度=”6英寸“FO：SRC =”/ files/ftp_upload/51534/51534fig1highres.jpg“SRC =”/ files/ftp_upload/51534/51534fig1.jpg“/&gt; 图1。鹌鹑，鸭嵌合系统。生成（A）鹌鹑和（B）鸭头骨显示的大小和形状相当大的差异，因此，非常适合使用的嵌合系统来研究颅面部发育（从TOKIA 等 56修改）（C）的实验设计单边嵌合quck胚胎阶段匹配HH9.5鹌鹑，鸭胚。在神经褶从鹌鹑胚胎的一侧取出并移植到鸭胚胎的等效片神经折后已被删除（D）鹌鹑的供体细胞（绿色）可以跟随在嵌合体使用抗鹌鹑抗体（Q ¢PN）所示的HH12嵌合体胚胎的腹面观。（F）在quck下颌骨在HH38，鹌鹑供体来源的梅克尔7，S软骨比所观察到的对侧鸭宿主衍生的美克尔软骨，这是较大的和弯曲的短和直。重印时田等人，开发。生物，306，377（2007）与爱思唯尔许可。

Watch this Scientific Journal Video about Assessing Species-specific Contributions To Craniofacial Development Using Quail-duck Chimeras at JoVE.com

Assessing Species-specific Contributions To Craniofacial Development Using Quail-duck Chimeras

本文介绍了一种方法来生成被设计来测试神经嵴和/或其他组织对颅面部发育的种属特异性的贡献嵌合体胚胎。

评估具体物种的贡献颅面开发利用鹌鹑，鸭嵌合体

The generation of chimeric embryos is a widespread and powerful approach to study cell fates, tissue interactions, and species-specific contributions to ...

assessing-species-specific-contributions-to-craniofacial-development

Research

JoVE Journal

Biology

10.8K 次观看.  University of California at San Francisco. 本文介绍了一种方法来生成被设计来测试神经嵴和/或其他组织对颅面部发育的种属特异性的贡献嵌合体胚胎。 

视频: Assessing Species-specific Contributions To Craniofacial Development Using Quail-duck Chimeras

Development of New Therapeutic Applications Using Microfluidics

使用微流体开发新的治疗应用

科研

生物学

In vivo Bioluminescent Imaging of Mammary Tumors Using IVIS Spectrum

4T1 mouse mammary tumor cells can be implanted sub-cutaneously in nu/nu mice to form palpable tumors in 15 to 20 days. This xenograft tumor model system is valuable for the pre-clinical in vivo evaluation of putative antitumor compounds.
The 4T1 cell line has been engineered to constitutively express the firefly luciferase gene (luc2). When mice carrying 4T1-luc2 tumors are injected with Luciferin the tumors emit a visual light signal that can be monitored using a sensitive optical imaging system like the IVIS Spectrum. The photon flux from the tumor is proportional to the number of light emitting cells and the signal can be measured to monitor tumor growth and development. IVIS is calibrated to enable absolute quantitation of the bioluminescent signal and longitudinal studies can be performed over many months and over several orders of signal magnitude without compromising the quantitative result.
Tumor growth can be monitored for several days by bioluminescence before the tumor size becomes palpable or measurable by traditional physical means. This rapid monitoring can provide insight into early events in tumor development or lead to shorter experimental procedures.
Tumor cell death and necrosis due to hypoxia or drug treatment is indicated early by a reduction in the bioluminescent signal. This cell death might not be accompanied by a reduction in tumor size as measured by physical means. The ability to see early events in tumor necrosis has significant impact on the selection and development of therapeutic agents.
Quantitative imaging of tumor growth using IVIS provides precise quantitation and accelerates the experimental process to generate results.

In vivo Bioluminescent Imaging of Mammary Tumors Using IVIS Spectrum

Mammary tumor cells expressing luciferase are implanted subcutaneously in mice and visualized using optical imaging to monitor tumor growth and development non-invasively in a longitudinal study.

使用IVIS频谱的乳腺肿瘤在体内生物发光成像

4T1 mouse mammary tumor cells can be implanted sub-cutaneously in nu/nu mice to form palpable tumors in 15 to 20 days. This xenograft tumor model system ...

Tissue Targeted Embryonic Chimeras: Zebrafish Gastrula Cell Transplantation

Certain fundamental questions in the field of developmental biology can only be answered when cells are placed in novel environments or when small groups of cells in a larger context are altered.  Watching how one cell interacts with and behaves in a unique environment is essential to characterizing cell functions.  Determining how the localized misexpression of a specific protein influences surrounding cells provides insightful information on the roles that protein plays in a variety of developmental processes.  Our lab uses the zebrafish model system to uniquely combine genetic approaches with classical transplantation techniques to generate genotypic or phenotypic chimeras.  We study neuron-glial cell interactions during the formation of forebrain commissures in zebrafish. This video describes a method that allows our lab to investigate the role of astroglial populations in the diencephalon and the roles of specific guidance cues that influence projecting axons as they cross the midline. Due to their transparency zebrafish embryos are ideal models for this type of ectopic cell placement or localized gene misexpression. Tracking transplanted cells can be accomplished using a vital dye or a transgenic fish line expressing a fluorescent protein. We demonstrate here how to prepare donor embryos with a vital dye tracer for transplantation, as well as how to extract and transplant cells from one gastrula staged embryo to another.  We present data showing ectopic GFP+ transgenic cells within the forebrain of zebrafish embryos and characterize the location of these cells with respect to forebrain commissures.  In addition, we show laser scanning confocal timelapse microscopy of Alexa 594 labeled cells transplanted into a GFP+ transgenic host embryo.  These data provide evidence that gastrula staged transplantation enables the targeted positioning of ectopic cells to address a variety of questions in Developmental Biology.

Zebrafish cell transplantation enables the combination of genetics and embryology to generate tissue specific chimeras. This video demonstrates gastrula staged cell transplantations that have allowed our lab to investigate the roles of astroglial populations and specific guidance cues during commissure formation in the forebrain.

组织有针对性的胚胎嵌合体：斑马鱼原肠胚细胞移植

Certain fundamental questions in the field of developmental biology can only be answered when cells are placed in novel environments or when small groups ...

Dechorionation of Medaka Embryos and Cell Transplantation for the Generation of Chimeras

Medaka is a small egg-laying freshwater fish that allows both genetic and embryological analyses and is one of the three vertebrate model organisms in which genome-wide phenotype-driven mutant screens were carried out 1. Divergence of functional overlap of related genes between medaka and zebrafish allows identification of novel phenotypes that are unidentifiable in a single species 2, thus medaka and zebrafish are complementary for genetic dissection of the vertebrate genome functions. Manipulation of medaka embryos, such as dechorionation, mounting embryos for imaging and cell transplantation, are key procedures to work on both medaka and zebrafish in a laboratory. Cell transplantation examines cell autonomy of medaka mutations. Chimeras are generated by transplanting labeled cells from donor embryos into unlabeled recipient embryos. Donor cells can be transplanted to specific areas of the recipient embryos based on the fate maps 3 so that clones from transplanted cells can be integrated in the tissue of interest during development. Due to the hard chorion and soft embryos, manipulation of medaka embryos is more involved than in zebrafish. In this video, we show detailed procedures to manipulate medaka embryos.

Due to the hard chorion and soft embryos, manipulation of medaka embryos is more involved than in zebrafish. This video shows step-by-step procedures for how to manipulate medaka embryos, including dechorionation, mounting in agarose for imaging and cell transplantation for the production of chimeras. These procedures are essential to use medaka and zebrafish in a laboratory to take full advantage of their complementary features for the genetic dissection of vertebrate genome functions.

鳉胚胎和细胞移植嵌合体生成Dechorionation

Medaka is a small egg-laying freshwater fish that allows both genetic and embryological analyses and is one of the three vertebrate model organisms in ...

Closed System Cell Culture Protocol Using HYPERStack Vessels with Gas Permeable Material Technology

Large volume adherent cell culture is currently standardized on stacked plate cell growth products when microcarrier beads are not an optimal choice. HYPERStack vessels allow closed system scale up from the current stacked plate products and delivers >2.5X more cells in the same volumetric footprint. The HYPERStack vessels function via gas permeable material which allows gas exchange to occur, therefore eliminating the need for internal headspace within a vessel. The elimination of headspace allows the compartment where cell growth occurs to be minimized to reduce space, allowing more layers of cell growth surface area within the same volumetric footprint.
For many applications such as cell therapy or vaccine production, a closed system is required for cell growth and harvesting. The HYPERStack vessel allows cell and reagent addition and removal via tubing from media bags or other methods.
This protocol will explain the technology behind the gas permeable material used in the HYPERStack vessels, gas diffusion results to meet the metabolic needs of cells, closed system cell growth protocols, and various harvesting methods.

An Introduction into the technology, protocol and handling of the Corning HYPERStack Vessels and accessories used for high yield adherent cell culture. The protocol will show how to use the closed system vessels for increasing cell harvesting over current stacked plate products.

封闭的系统使用透氧材料科技HYPERStack船只的细胞培养协议

Large volume adherent cell culture is currently standardized on stacked plate cell growth products when microcarrier beads are not an optimal choice.   ...

Assessing Signaling Properties of Ectodermal Epithelia During Craniofacial Development

The accessibility of avian embryos has helped experimental embryologists understand the fates of cells during development and the role of tissue interactions that regulate patterning and morphogenesis of vertebrates (e.g., 1, 2, 3, 4). Here, we illustrate a method that exploits this accessibility to test the signaling and patterning properties of ectodermal tissues during facial development. In these experiments, we create quail-chick 5 or mouse-chick 6 chimeras by transplanting the surface cephalic ectoderm that covers the upper jaw from quail or mouse onto either the same region or an ectopic region of chick embryos. The use of quail as donor tissue for transplantation into chicks was developed to take advantage of a nucleolar marker present in quail but not chick cells, thus allowing investigators to distinguish host and donor tissues 7. Similarly, a repetitive element is present in the mouse genome and is expressed ubiquitously, which allows us to distinguish host and donor tissues in mouse-chick chimeras 8. The use of mouse ectoderm as donor tissue will greatly extend our understanding of these tissue interactions, because this will allow us to test the signaling properties of ectoderm derived from various mutant embryos.

This article describes a tissue transplantation technique that was designed to test the signaling and patterning properties of surface cephalic ectoderm during craniofacial development.

评估外胚层上皮细胞在颅面发育的信号属性

The accessibility of avian embryos has helped experimental embryologists understand the fates of cells during development and the role of tissue ...

Electroporation of Craniofacial Mesenchyme

Electroporation is an efficient method of delivering DNA and other charged macromolecules into tissues at precise time points and in precise locations. For example, electroporation has been used with great success to study neural and retinal development in Xenopus, chicken and mouse 1-10. However, it is important to note that in all of these studies, investigators were not targeting soft tissues. Because we are interested in craniofacial development, we adapted a method to target facial mesenchyme.
When we searched the literature, we found, to our surprise, very few reports of successful gene transfer into cartilaginous tissue. The majority of these studies were gene therapy studies, such as siRNA or protein delivery into chondrogenic cell lines, or, animal models of arthritis 11-13. In other systems, such as chicken or mouse, electroporation of facial mesenchyme has been challenging (personal communications, Dept of Craniofacial Development, KCL). We hypothesized that electroporation into procartilaginous and cartilaginous tissues in Xenopus might work better. In our studies, we show that gene transfer into the facial cartilages occurs efficiently at early stages (28), when the facial primordium is still comprised of soft tissue prior to cartilage differentiation.
Xenopus is a very accessible vertebrate system for analysis of craniofacial development. Craniofacial structures are more readily visible in Xenopus than in any other vertebrate model, primarily because Xenopus embryos are fertilized externally, allowing analyses of the earliest stages, and facilitating live imaging at single cell resolution, as well as reuse of the mothers 14. Among vertebrate models developing externally, Xenopus is more useful for craniofacial analysis than zebrafish, as Xenopus larvae are larger and easier to dissect, and the developing facial region is more accessible to imaging than the equivalent region in fish. In addition, Xenopus is evolutionarily closer to humans than zebrafish (&tilde;100 million years closer) 15. Finally, at these stages, Xenopus tadpoles are transparent, and concurrent expression of fluorescent proteins or molecules will allow easy visualization of the developing cartilages. We anticipate that this approach will allow us to rapidly and efficiently test candidate molecules in an in vivo model system.

Craniofacial cartilages develop in close contact with other tissues and are difficult to manipulate in live animals. We are using electroporation to deliver molecular tools during growth of the craniofacial skeleton while bypassing early embryonic effects. This approach will allow us to efficiently test candidate molecules in vivo.

颅面间质细胞的电

Electroporation is an efficient method of delivering DNA and other charged macromolecules into tissues at precise time points and in precise locations. ...

A Semi-quantitative Approach to Assess Biofilm Formation Using Wrinkled Colony Development

Biofilms, or surface-attached communities of cells encapsulated in an extracellular matrix, represent a common lifestyle for many bacteria. Within a biofilm, bacterial cells often exhibit altered physiology, including enhanced resistance to antibiotics and other environmental stresses 1. Additionally, biofilms can play important roles in host-microbe interactions. Biofilms develop when bacteria transition from individual, planktonic cells to form complex, multi-cellular communities 2. In the laboratory, biofilms are studied by assessing the development of specific biofilm phenotypes. A common biofilm phenotype involves the formation of wrinkled or rugose bacterial colonies on solid agar media 3. Wrinkled colony formation provides a particularly simple and useful means to identify and characterize bacterial strains exhibiting altered biofilm phenotypes, and to investigate environmental conditions that impact biofilm formation. Wrinkled colony formation serves as an indicator of biofilm formation in a variety of bacteria, including both Gram-positive bacteria, such as Bacillus subtilis 4, and Gram-negative bacteria, such as Vibrio cholerae 5, Vibrio parahaemolyticus 6, Pseudomonas aeruginosa 7, and Vibrio fischeri 8.
The marine bacterium V. fischeri has become a model for biofilm formation due to the critical role of biofilms during host colonization: biofilms produced by V. fischeri promote its colonization of the Hawaiian bobtail squid Euprymna scolopes 8-10. Importantly, biofilm phenotypes observed in vitro correlate with the ability of V. fischeri cells to effectively colonize host animals: strains impaired for biofilm formation in vitro possess a colonization defect 9,11, while strains exhibiting increased biofilm phenotypes are enhanced for colonization 8,12. V. fischeri therefore provides a simple model system to assess the mechanisms by which bacteria regulate biofilm formation and how biofilms impact host colonization.
In this report, we describe a semi-quantitative method to assess biofilm formation using V. fischeri as a model system. This method involves the careful spotting of bacterial cultures at defined concentrations and volumes onto solid agar media; a spotted culture is synonymous to a single bacterial colony. This 'spotted culture' technique can be utilized to compare gross biofilm phenotypes at single, specified time-points (end-point assays), or to identify and characterize subtle biofilm phenotypes through time-course assays of biofilm development and measurements of the colony diameter, which is influenced by biofilm formation. Thus, this technique provides a semi-quantitative analysis of biofilm formation, permitting evaluation of the timing and patterning of wrinkled colony development and the relative size of the developing structure, characteristics that extend beyond the simple overall morphology.

We provide a simple, semi-quantitative method to investigate biofilm formation in vitro. This method takes advantage of the Zeiss stemi 2000-C Dissecting Microscope (with camera attachment) to monitor both the timing and pattern of biofilm formation, as assessed by the development of wrinkled colonies.

一个半定量的方法来评估生物膜形成皱殖民地的发展

Biofilms, or surface-attached communities of cells encapsulated in an extracellular matrix, represent a common lifestyle for many bacteria. Within a ...

Conversion of a Capture ELISA to a Luminex xMAP Assay using a Multiplex Antibody Screening Method

The enzyme-linked immunosorbent assay (ELISA) has long been the primary tool for detection of analytes of interest in biological samples for both life science research and clinical diagnostics. However, ELISA has limitations. It is typically performed in a 96-well microplate, and the wells are coated with capture antibody, requiring a relatively large amount of sample to capture an antigen of interest . The large surface area of the wells and the hydrophobic binding of capture antibody can also lead to non-specific binding and increased background. Additionally, most ELISAs rely upon enzyme-mediated amplification of signal in order to achieve reasonable sensitivity. Such amplification is not always linear and can thus skew results.
In the past 15 years, a new technology has emerged that offers the benefits of the ELISA, but also enables higher throughput, increased flexibility, reduced sample volume, and lower cost, with a similar workflow 1, 2. Luminex xMAP Technology is a microsphere (bead) array platform enabling both monoplex and multiplex assays that can be applied to both protein and nucleic acid applications 3-5. The beads have the capture antibody covalently immobilized on a smaller surface area, requiring less capture antibody and smaller sample volumes, compared to ELISA, and non-specific binding is significantly reduced. Smaller sample volumes are important when working with limiting samples such as cerebrospinal fluid, synovial fluid, etc. 6. Multiplexing the assay further reduces sample volume requirements, enabling multiple results from a single sample.
Recent improvements by Luminex include: the new MAGPIX system, a smaller, less expensive, easier-to-use analyzer; Low-Concentration Magnetic MagPlex Microspheres which eliminate the need for expensive filter plates and come in a working concentration better suited for assay development and low-throughput applications; and the xMAP Antibody Coupling (AbC) Kit, which includes a protocol, reagents, and consumables necessary for coupling beads to the capture antibody of interest. (See Materials section for a detailed list of kit contents.)
In this experiment, we convert a pre-optimized ELISA assay for TNF-alpha cytokine to the xMAP platform and compare the performance of the two methods 7-11. TNF-alpha is a biomarker used in the measurement of inflammatory responses in patients with autoimmune disorders.
We begin by coupling four candidate capture antibodies to four different microsphere sets or regions. When mixed together, these four sets allow for the simultaneous testing of all four candidates with four separate detection antibodies to determine the best antibody pair, saving reagents, sample and time. Two xMAP assays are then constructed with the two most optimal antibody pairs and their performance is compared to that of the original ELISA assay in regards to signal strength, dynamic range, and sensitivity.

An ELISA can be easily converted to a Luminex xMAP assay and, through the benefits of multiplexing, several antibodies can be screened simultaneously to identify an optimum antibody pair, resulting in increased sensitivity and dynamic range, while reducing assay cost.

转换到一个点阵仪XMAP含量，使用多重抗体筛查方法的捕获ELISA

The enzyme-linked immunosorbent assay (ELISA) has long been the primary tool for detection of analytes of interest in biological samples for both life ...

Assessing Murine Resistance Artery Function Using Pressure Myography

Pressure myograph systems are exquisitely useful in the functional assessment of small arteries, pressurized to a suitable transmural pressure. The near physiological condition achieved in pressure myography permits in-depth characterization of intrinsic responses to pharmacological and physiological stimuli, which can be extrapolated to the in vivo behavior of the vascular bed. Pressure myograph has several advantages over conventional wire myographs. For example, smaller resistance vessels can be studied at tightly controlled and physiologically relevant intraluminal pressures. Here, we study the ability of 3rd order mesenteric arteries (3-4 mm long), preconstricted with phenylephrine, to vaso-relax in response to acetylcholine. Mesenteric arteries are mounted on two cannulas connected to a pressurized and sealed system that is maintained at constant pressure of 60 mmHg. The lumen and outer diameter of the vessel are continuously recorded using a video camera, allowing real time quantification of the vasoconstriction and vasorelaxation in response to phenylephrine and acetylcholine, respectively.
	To demonstrate the applicability of pressure myography to study the etiology of cardiovascular disease, we assessed endothelium-dependent vascular function in a murine model of systemic hypertension. Mice deficient in the &alpha;1 subunit of soluble guanylate cyclase (sGC&alpha;1-/-) are hypertensive when on a 129S6 (S6) background (sGC&alpha;1-/-S6) but not when on a C57BL/6 (B6) background (sGC&alpha;1-/-B6). Using pressure myography, we demonstrate that sGC&alpha;1-deficiency results in impaired endothelium-dependent vasorelaxation. The vascular dysfunction is more pronounced in sGC&alpha;1-/-S6 than in sGC&alpha;1-/-B6 mice, likely contributing to the higher blood pressure in sGC&alpha;1-/-S6 than in sGC&alpha;1-/-B6 mice. 
	Pressure myography is a relatively simple, but sensitive and mechanistically useful technique that can be used to assess the effect of various stimuli on vascular contraction and relaxation, thereby augmenting our insight into the mechanisms underlying cardiovascular disease.

In pressure myography, an intact small segment of a vessel is mounted onto two small cannulas and pressurized to a suitable luminal pressure. Here, we describe the method to measure vasorelaxation response of the mouse 3rd order mesenteric arteries in c57 and sGC&alpha;1-/- mice using pressure myography.

评估抗小鼠动脉功能使用压力Myography

Pressure myograph systems are  exquisitely useful in the functional assessment of small arteries, pressurized  to a suitable transmural pressure. The  ...