We appreciate Dr. Jeong-Soo Lee for sharing the Tg(dbh:EGFP-MYCN) transgenic fish with us in our study. This work was supported by a grant 1K99CA178189-01 from the National Cancer Institute, a fellowship from the Pablove Foundation and the Friends for Life, and young investigator awards from the Alex's Lemonade Stand Foundation and the CureSearch for Children's Cancer Foundation.

注：所有斑马鱼的研究和维护动物都符合梅奥诊所研究所IACUC批准协议＃A41213完成。 1. DNA构建的转基因<ol><li>扩增使用CH211-270H11 BAC克隆（从BACPAC资源中心（BPRC））作为DNA模板，一个5.2-kb的多巴胺β羟化酶（四βH）启动子区8。使用PCR体系适合于长DNA模板长和准确的PCR扩增和随后循环方案进行PCR：2分钟94℃，10个循环的（94℃，15秒，50℃，30秒，68° ℃，8分钟），随后的（94℃，15秒，53℃，30秒，68℃，8分钟），68℃，4分钟（正向引物5&#39;-GGGGACAACTTTGTATAGAAAAGTTGGCGTACTCCCCCTTTTTAGG-3&#39;30个循环和反向引物5&#39;-GGGGACTGCTTTTTTGTACAAACTTGTGTTGCTTTGTCGTCTTTTGA-3&#39;）。 </li><li>创建ðβH-pDONRP4-P1R进入CLONE 8使用商业重组克隆系统，如多站点网关。混合1微升纯化DβHPCR产物（172纳克/微升），1微升（150纳克/微升）pDONRP4-P1R捐助载体（连同其他网关向量从博士炽彬建，大学丰厚的礼品。犹他州） ，4微升TE缓冲液（pH8.0）中与2微升的BP克隆酶酶混合物，孵育1小时，在25℃，然后转化到单次TOP10感受态大肠杆菌大肠杆菌根据制造商的协议。 </li><li>产生对DβH：EGFP-MYCN基因构造步骤1.2中提到8使用重组克隆系统。混合1μl的每个条目克隆的（150毫微克/微升），其包括5.2-kb的DβH子（DβH-pDONR P4-P1R构建体）， 绿色荧光蛋白缺乏终止密码子（PME-EGFP构建体），和人的cDNA MYCN（慷慨礼物从Hogarty博士在宾夕法尼亚州的儿童医院，MYCN-pDONRP2R-P3结构），1微升含I-SceI的识别位点（从C Grabher博士，卡尔斯鲁厄理工学院，卡尔斯鲁厄，德国）的一份厚礼，4微升TE缓冲液的修改目的载体（150纳克/微升）（ pH 8.0）中与2微升的LR克隆酶酶混合物，孵育1小时，在25℃，然后转化到化学感受态大肠杆菌等一杆排名前10位，并按照制造商的协议。 </li><li>使MITF：MITF转基因消化的PNP-MITF载体构建8用NotI和SalI限制性内切酶2小时RT，并亚克隆释放2.65-kb的（从D Raible，大学博士一份厚礼华盛顿。）包含MITF子和斑马鱼MITF基因的编码序列插入含有侧翼Ⅰ-SceⅠ限制性酶切位识别位点修饰的pBluescript载体的NotI和MluI位位点的DNA片段（从辉铁博士慷慨礼物纳克，波士顿大学），根据生产商的方案使用T4DNA连接酶。 注意：为了提高产生稳定的MYCN -expressing线的效率，DβH：EGFP-MYCN和MITF：MITF DNA构建共注射到单细胞阶段的胚胎珍珠珍珠层表示缺乏神经类型的突变鱼脊衍生发展36时黑色素。因此，色素细胞，在注射的胚胎的外观表明MITF的整合：MITF DNA构建到基因组中。它已被证明，两个或三个共注射的DNA构建体可以协整成鱼的基因组31。因此，造成MITF表达色素沉着可以充当一个标记为DβH的整合：EGFP-MYCN基因，并便于识别MYCN稳定转基因线。 </li><li>混合ðβH：EGFP-MYCN和MITF：MITF DNA构建体在3：1的比例与1微升的I-SceI位酶和0.75微升缓冲液15微升反应的总体积。确保DNA的反应的总量不超过750毫微克。 </li><li>开展了I-SceI的消化在室温4小时或O / N。在第二天，在I-SceI-消化的DNA是准备用于显微注射或可以储存在-20℃用于注射的未来。存储的I-SceI位酶在-80℃以小等分试样保持其酶的效率。 </li><li>从C. Grabher博士亚克隆从载体的pcDNA3人ALKF1174L和野生型ALK基因（来自乔治博士一份厚礼在Dana-Farber癌症研究所）7成pENTRY1A载体的EcoRI和NotI位点（一份厚礼，卡尔斯鲁厄理工学院根据制造商的方案，使用T4 DNA连接酶，卡尔斯鲁厄，德国）。 </li><li>产生对DβH：ALKF1174L &lt;/ em&gt;的或DβH：使用重组系统中提到的协议1.2 ALKWT转基因构建体。简单地说，组合三个入门克隆，DβH-pDONRP4-P1R，ALKF1174L- pENTRY1A（或ALKWT -pENTRY1A）和P3E-聚腺苷酸，到含有I-SceI的识别位点（从C Grabher，卡尔斯鲁厄博士一份厚礼修改后的目标向量技术研究所，卡尔斯鲁厄，德国），使用LR克隆酶的酶混合，根据制造商的协议。 </li><li>混合对dβH：ALKF1174L（或DβH：ALKWT）和DβH：mCherry DNA构建以3：1的比例，并作为在协议1.5-1.6描述的I-SceI位酶线性化它们。 </li></ol> 2.显微注射<ol><li>用玻璃微1.0毫米直径的所有注射37。折断的玻璃微量的尖端与注射前剃刀刀片。校准注射量注射用荷兰国际集团H 2 O外一滴矿物油。测量所得的液滴的直径，并调整微量（压力或压力脉冲的持续时间），以确保在注入量为1个细胞期胚胎的总细胞体积的10％以内。 </li><li>添加额外的0.5微升新鲜Ⅰ-SceⅠ限制性酶切位酶（5单位/微升）在5微升含有DNA注射溶液的构建权前的注射，以增加转基因38的效率。注入50-80皮克线性DNA构建成单细胞阶段的胚胎的细胞质中。为了保证注入的成功，混合用0.25％酚红可视化样品。如果细胞变红注射后，它表示微注射是成功的。为了确保高成功率产生转基因鱼，我们通常注入多达500胚胎。 </li><li>为了产生转基因鱼稳定表达MYCN，coinject线性ðβH：EGFP-MYCN和MITF：MITF DNA构建体（以3：1的比例）插入突变体的色素沉着珍珠层斑马鱼在单细胞阶段的胚胎。 MITF的表达作为记者在鱼的基因组的转基因构建体的整合。 </li><li>与DβH对于ALK的野生型或MYCN转基因胚胎的镶嵌过表达，共注入线性ðβH：ALK F1174L（ALKWT或DβH）：mCherry的DNA构建体（以3：1的比例）为单细胞胚胎繁殖F1杂合的Tg（DβH：EGFP-MYCN）而导致的转基因鱼与野生型AB鱼。因此，有一半的后代都是转基因的MCYN，一半是野生型。 </li></ol> 3.屏幕为稳定或马赛克转基因鱼<ol><li>以提高识别的稳定MYCN -expressing线的效率，主要是麻醉注射珍珠层胚三卡因（0.02％）一吨天受精后和屏幕的色素沉着3-5。传输与色素沉着的胚胎到一个新的培养皿中的新鲜鸡蛋的水，并提高他们的性成熟期为进一步筛查创始人鱼，它搭载了ðβH：EGFP-MYCN基因在生殖细胞。 </li><li>认同EGFP-MYCN，异交色素F0成鱼与野生型AB鱼和屏幕EGFP阳性的胚胎在1-2天受精后进行进一步的基因分型的种系遗传的创始人。将单EGFP阳性的F1胚胎成PCR管，并删除所有的液体。添加50微升含有12.5微升4×裂解缓冲液，35微升H 2 O和2.5微升蛋白酶K（10毫克/毫升），以单胚的gDNA提取缓冲液。孵育在55℃下2-3小时的反应，然后孵育98℃10分钟以灭活蛋白酶K为使加入50ml 4倍裂解缓冲液中，添加1M的Tris将500μl（pH为8.4），2.5毫升，浓度1M的氯化钾吨Ø47毫升的H 2 O.注：在F0 MYCN稳定的转基因鱼的饲养与野生型AB鱼，所有的后代都是色素。因此，色素沉着不能作为标记的MYCN-阳性鱼的鉴定。而在MYCN转基因鱼中，EGFP-MYCN融合蛋白被表达在PSNS，包括颈上神经节和交感神经链的顺序节段性神经节和非PSNS多巴胺能神经元的交感神经元，如延髓和颅神经节8。因此，EGFP的表达可以作为标记用于识别MYCN稳定的转基因胚胎。 </li><li>使用2微升的gDNA从着色F1的胚胎为模板提取，引物MYCN -test F1：5&#39;-CTG CTT GAG AAC GAG CTG TG-3&#39;; MYCN -R3：5&#39;-AGG CAT CGT TTG AGG ATC AG-3 &#39;和下面的程序机智小时GC-RICH PCR系统：95℃3分钟1个循环，95℃25个循环，30秒，58℃30秒和72℃3分钟。我们通常基因型从单一的交配14-16色素胚胎确认集成MYCN基因在胚胎色素的存在，超过6创始人鱼过表达MITF和MYCN都用这种方法确定的。 </li><li>提高了绿色荧光蛋白阳性的F1胚胎的其余部分。鳍夹子和使用上述协议，以进一步证实MYCN转基因的整合到鱼基因型它们在2-3个月的年龄。繁殖F1 MYCN稳定的转基因鱼与野生型AB鱼。共注入的线性ðβH：ALK F1174L（或DβH：ALKWT）配DβH：mCherry DNA构建成单细胞胚胎如在协议2.4所述。 </li><li>使用立体氟在协议2.4排序MYCN -expressing在实验胚胎描述escence显微镜和屏幕的主要注射胚胎在1-3天受精后的EGFP-MYCN在PSNS表达。然后，在天受精后的2-5，麻醉用三卡因（0.02％）的胚胎和基于mCherry中的PSN使用立体荧光显微镜表达那些MYCN-正或负的胚再排序。 mCherry的表达作为标记ALK在镶嵌初级注射的动物的组织中的共表达。 注：〜600后代从MYCN稳定的转基因鱼与野生型鱼繁殖所得每组注射线性的DNA构建体， 包括D-βH：ALK F1174L和DβH：mCherry，DβH：ALKWT和DβH：mCherry，或DβH ：mCherry单独分别。 mCherry的马赛克的表达可以在主要注入鱼70-90％，可以观察到。二分之一到二注入的鱼通过第三幼虫阶段的肿瘤手表活了下来。 </li><li>提高全体mCherry + MYCN +和mCherry + MYCN的-根据来自斑马鱼书39的标准协议的胚胎和监测肿瘤发病开始5周受精后。 </li></ol> 4.肿瘤关注的马赛克转基因鱼<ol><li>监测mCherry-积极的主要是注入的鱼，每2周开始5周受精后对肿瘤发生的证据。 </li><li>麻醉鱼用三卡因（0.02％）和画面mCherry的存在-和EGFP -expressing肿瘤中配备了数字视力的DS-U1照相机尼康SMZ-1500立体荧光显微镜下的PSN。 </li><li>要确认肿瘤是否表达ALK基因，分离出mCherry-和EGFP阳性群众ALK基因分型PCR。 </li><li>使用PCR，扩增基​​因组DNAALK P7：5&#39;-AGG CCA GGT GTC CGG AAT GC-3&#39;和ALK P18：5&#39;-TGT CTT CAG GCT GAT GTT GC-3&#39;和下述PCR反应：1周期从形成肿瘤用下面的引物中提取94℃5分钟，30个循环（94℃，30秒，55℃30秒和72℃60秒）。然后，序列的PCR产物与ALK P7引物，以进一步证实突变体或野生型ALK中的mCherry阳性肿瘤的存在。 </li></ol>

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The authors declare that they have no competing financial interests.

在这种代表性的研究中，我们采用瞬时共注射和激活ALK的共表达与mCherry报告基因MYCN -expressing转基因鱼，以表明这些基因合作，显着加速神经母细胞瘤发病的，与我们之前的发现，复合稳定转基因鱼共表达一致无论激活ALK和MYCN 8。这种转基因拼接方法具有几个明显的优势比传统方法。最重要的，它使得能够共表达在主要注射的动物的候选癌基因（F0代）而?...

巨蟹座的标志是病理突变，缺失和染色体的收益随着时间的积累渐进的疾病。这些遗传异常可影响多个细胞过程，从细胞周期，细胞死亡，有活力的细胞骨架强调反应，如缺氧的代谢和组装。因此，肿瘤发生反映多种遗传畸变的跨生物过程的频谱集体行动。最近综合基因组的研究工作，包括全基因组测序，基因组测序，测序的目标，深度测序和全基因组关联研究，已确定在几乎所有类型的肿瘤1-4的越来越多的新的遗传变化。在许多情况下，遗传发生病变一起以非随机方式5-8，表明它们在疾病的发病机制的合作。剖析大阵造成˚F异常表达的基因的致癌作用ROM这些基因组的病变有必要设计新的治疗策略，并了解肿瘤细胞对这些药物的反应，但是这已经被证明是一项艰巨的任务，需要非常健壮的动物模型系统对于高通量功能基因组学分析的情况下进行体内 。 虽然哺乳动物，尤其是啮齿动物，青睐车型在癌症生物学，斑马鱼已开始引起相当大的关注。该硬骨斑马鱼（ 达里奥鱼 ）已被用来作为模式生物自20世纪60年代开发研究，并首先应用于肿瘤的发病机制的研究，在1982年9-11。易于维护，小的车身尺寸，以及高繁殖力使得斑马鱼稳健的模型，进行大规模的正向遗传学的屏幕，以识别对异常和病理表现型10的突变。斑马鱼胚胎的光学透明度是支持更广泛地使用这种癌症模型中，作为另一重要特征它允许体内成像的情况下进行，以找到肿瘤的发展实时9，一种应用程序，它是在啮齿动物12相对困难。最近比较基因组学的斑马鱼参考基因组（Zv9）的分析显示26206个蛋白质编码基因，与具有人直向同源物，其中82％的值与在所述在线孟德尔遗传疾病相关基因在曼（OMIM）数据库13 71％， 14。因此，斑马鱼已经被用于模拟不同类型的人类癌症，包括神经母细胞瘤8中，T细胞急性淋巴细胞白血病（T-ALL）15,16，黑素瘤17,18，尤因氏肉瘤19，横纹肌肉瘤20,21，胰腺癌22，肝细胞癌23和髓系恶性血液病24,25，并已被选定作为癌症模型异种移植研究11,26。 一个稳定的转基因在斑马鱼的方法通常用于研究的增益的功能的基因在正常发育或疾病发病27,28的效果。开发这样的模型（ 图1A），一个注射含有感兴趣由一个组织特异性启动子驱动的为单细胞的野生型胚胎的基因的DNA构建物。注射三至四个月后，当注射的胚胎达到性成熟，它们远交，与野生型鱼以筛选那些表示整合的DNA的构建在其种系，哪些许可证它们作为创始人鱼。许多因素，如拷贝数和转基因的整合位点，影响在稳定的转基因系的转基因的表达。因此，开发一种转基因肿瘤模型中，多个稳定的转基因株系过度单一的基因必须首先生成和筛选表达转基因的可能导致肿瘤的诱导，一个水平线上。但是，如果候选人邻表达ncogene是有毒的生殖细胞，很难通过直接过表达的转基因29，以产生一个稳定的转基因系。因此，这种方法可以是耗时的，具有故障，以产生合适的癌症模型中的高风险。 这里，我们说明根据镶嵌瞬时转基因（ 图1B）的替代策略，比传统的稳定的转基因用于功能基因组研究在体内提供了独特的优势。在这种方法中，一个或多个转基因构建体注射到转基因或野生型胚胎的单细胞阶段。含有转基因的注射DNA构建然后mosaically和随机整合到主注入鱼，导致在个体的鱼30的多个细胞群内混合基因型。此外，多种的DNA共注射构建在单细胞胚胎导致共整合到在随机位点相同的细胞，允许一个TRACE细胞与转基因的表达，并在镶嵌31动物疾病的发病机制研究不同基因之间的相互作用。作为原理的证明，我们暂时下多巴胺β羟化酶（四βH）启动子在野生型鱼和转基因鱼过度MYCN的控制过度mutationally活化ALK（F1174L）配mCherry报告基因在周交感神经系统（PSNS）。 ALK，编码受体酪氨酸激酶，是最常见的突变基因中的高风险神经母细胞瘤5-7,32,33。ALK（F1174L），作为最频繁的和有效的体细胞活化突变之一，是在超过限额MYCN-扩增高危神经母细胞瘤的患者，用MYCN表达协同作用，以加速神经母细胞瘤肿瘤发生在两个稳定的转基因小鼠和转基因斑马鱼的模型8,34,35。马赛克ALK（F1174L）配mCherry在MYCN转基因鱼的瞬态过表达，我们概括在稳定的转基因鱼过量表达既ALK（F1174L）和MYCN观察肿瘤发病的加速度，这表明转基因镶嵌策略可用于快速和有效地评估多个癌基因在肿瘤发生在体内的相对贡献。

<table border="0" cellpadding="0" cellspacing="0" width="663">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Name of Material/ Equipment</td>
			<td style="width:171px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Expand Long Template PCR System&#160;</td>
			<td style="width:171px;">Roche Applied Science, IN</td>
			<td style="width:119px;">11681834001</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pCR-TOPO vector&#160;</td>
			<td style="width:171px;">Invitrogen, CA</td>
			<td style="width:119px;">451641</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">T4 DNA ligase</td>
			<td style="width:171px;">New England Biolabs, MA</td>
			<td style="width:119px;">M0202M</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gateway LR Clonase II enzyme 
			Mix</td>
			<td style="width:171px;">Invitrogen, CA</td>
			<td>11791-100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gateway&#174; BP Clonase&#174; II enzyme mix</td>
			<td style="width:171px;">Invitrogen, CA</td>
			<td>11789-020</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:25px;">GC-RICH PCR System&#160;</td>
			<td style="width:171px;">Roche Applied Science, IN</td>
			<td>12 140 306 001</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">Meganuclease I-SceI&#160;</td>
			<td style="width:171px;">New England Biolabs, MA</td>
			<td>R0694S</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nikon SMZ-1500 stereoscopic fluorescence microscope&#160;</td>
			<td style="width:171px;">Nikon, NY</td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nikon digital sight DS-U1 camera</td>
			<td style="width:171px;">Nikon, NY</td>
			<td style="width:119px;"></td>
		</tr>
	</tbody>
</table>

mosaic zebrafish transgenesis for functional genomic analysis of candidate cooperative genes in tumor pathogenesis

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

调查是否mutationally活化ALK F1174L或野生型ALK的过表达可在神经母细胞瘤诱导MYCN协作，我们过表达或者活化的人ALK或野生型人ALK下的转基因鱼过度MYCN的PSN 对dβH启动子的控制。下列任一构建体，DβH的- ALKF1174L或DβH - ALKWT，被共注射与DβH - mCherry成单细胞的野生型或MYCN转基因胚胎（ 图2A）8。</s...

Watch this Scientific Journal Video about Mosaic Zebrafish Transgenesis for Functional Genomic Analysis of Candidate Cooperative Genes in Tumor Pathogenesis at JoVE.com

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

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

马赛克斑马鱼转基因的候选基因合作在肿瘤发病机制的功能基因组分析

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

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11.0K 次观看.  Mayo Clinic College of Medicine, Center for Individualized Medicine. The goal of this study is to demonstrate how the mosaic transgenesis strategy can be used in zebrafish to rapidly and efficiently assess the relative contributions of multiple oncogenes in tumor initiation and progression in vivo.