Name: screening for melanoma modifiers using a zebrafish autochthonous tumor model
Uploaded: 2012-11-13T13:00:00
Description: Watch this Scientific Journal Video about Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model at JoVE.com

我们感谢伦纳德一博士在他的实验室，这些技术最初开发区域生态克里斯汀关和已故的简智彬在这项工作中所用的质粒的礼物，詹姆斯·李斯特和大卫Raible抗体染色的援助;和詹姆斯Neiswender显微注射的视频。这项工作是由美国国立卫生研究院授予R00AR056899-03 CJC

1。筛选黑色素瘤的发病修饰符<ol><li>创建网关中间条目的克隆，通过PCR扩增全长的感兴趣的基因（GOI）的开放阅读框和重组到pDONR 221中使用BP克隆酶II（Invitrogen公司）。使用多地点Gateway技术（Invitrogen）中，以重组p5E_mitfa，pME_GOI，Tol2kit＃302 p3E_SV40polyA 6和miniCoopR 5放置在miniCoopR载体（ 图1A）根据mitfa启动子基因的兴趣。 </li><li>随着25的皮克TOL2转座?...

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	<li>Beroukhim, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+landscape+of+somatic+copy-number+alteration+across+human+cancers.">The landscape of somatic copy-number alteration across human cancers.</a> Nature. 463, 899-905 (2010).</li><li>Curtin, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+sets+of+genetic+alterations+in+melanoma.">Distinct sets of genetic alterations in melanoma.</a> N. Engl. J. Med. 353, 2135-2147 (2005).</li><li>Lin, W. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+genomic+diversity+and+tumor+dependency+in+malignant+melanoma.">Modeling genomic diversity and tumor dependency in malignant melanoma.</a> Cancer Res. 68, 664-673 (2008).</li><li>Patton, E. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BRAF+mutations+are+sufficient+to+promote+nevi+formation+and+cooperate+with+p53+in+the+genesis+of+melanoma.">BRAF mutations are sufficient to promote nevi formation and cooperate with p53 in the genesis of melanoma.</a> Curr. Biol. 15, 249-254 (2005).</li><li>Ceol, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+histone+methyltransferase+SETDB1+is+recurrently+amplified+in+melanoma+and+accelerates+its+onset.">The histone methyltransferase SETDB1 is recurrently amplified in melanoma and accelerates its onset.</a> Nature. 471, 513-517 (2011).</li><li>Kwan, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Tol2kit:+a+multisite+gateway-based+construction+kit+for+Tol2+transposon+transgenesis+constructs.">The Tol2kit: a multisite gateway-based construction kit for Tol2 transposon transgenesis constructs.</a> Dev. Dyn. 236, 3088-3099 (2007).</li><li>Rosen, J. N., Sweeney, M. F., Mably, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+of+Zebrafish+Embryos+to+Analyze+Gene+Function.">Microinjection of Zebrafish Embryos to Analyze Gene Function.</a> J. Vis. Exp. (25), e1115 (2009).</li><li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The zebrafish book. A guide of the laboratory use of zebrafish (Danio rerio). , (2000).</li><li>White, R. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transparent+adult+zebrafish+as+a+tool+for+in+vivo+transplantation+analysis.">Transparent adult zebrafish as a tool for in vivo transplantation analysis.</a> Cell Stem Cell. 2, 183-189 (2008).</li><li>Traver, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+and+in+vivo+imaging+of+multilineage+engraftment+in+zebrafish+bloodless+mutants.">Transplantation and in vivo imaging of multilineage engraftment in zebrafish bloodless mutants.</a> Nat. Immunol. 4, 1238-1246 (2003).</li></ol>

miniCoopR方法使表达的基因在斑马鱼的黑素细胞的兴趣。这种方法利用的事实，的斑马鱼mitfa基因的作用细胞自主性。出于这个原因，黑素细胞获救由miniCoopR载体是一定要包含小基因和任何感兴趣的基因，它在物理上耦合。营救黑素细胞是清晰可见，能够得到在被注射的动物，作为单细胞胚胎。特定程度的嵌合体被选中，并可以拿下超过此截止动物肿瘤发病或其他特性。的一大好处是足够的?...

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>网关重组试剂</td><td> Invitrogen公司</td><td></td><td></td></tr><tr><td> miniCoopR </td><td></td><td></td><td>参考文献5 </td></tr><tr><td> Mitfa抗体</td><td></td><td></td><td>参考文献5 </td></tr><tr><td> FITC标记的山羊抗兔IgG抗体</td><td> Invitrogen公司</td><td></td><td></td></tr><tr><td> Vectashield </td><td>矢量实验室</td><td> H-1000 </td><td></td></tr><tr><td> 卡斯帕斑马鱼</td><td></td><td></td><td>参考9 </td></tr><tr><td> 701N 10μl注射器</td><td>汉密尔顿/费舍尔</td><td> 14-824 </td><td> ＆提供; </td></tr><tr><td> 40微米的过滤器</td><td> BD猎鹰/费舍尔</td><td> 352340 </td><td></td></tr><tr><td> FBS </td><td> Invitrogen公司</td><td> 26140079 </td><td></td></tr></tbody></table>

screening for melanoma modifiers using a zebrafish autochthonous tumor model

的人类癌症基因组学研究已经取得了丰富的信息改变的基因，在肿瘤1,2,3。从这些研究中所产生的一个挑战是，许多基因被改变，它可以开车从那些在转换过程中偶然产生的肿瘤的遗传改变，很难区分。作出这样的区分是有好处的分析，可以定量衡量一个改变基因在肿瘤的发生和流程，使肿瘤的坚持和传播的效果。在这里，我们提出了一个快速的方法来筛选大量的候选黑色素瘤修饰符在斑马鱼黑色素瘤的启动和维护所需的步骤，包括一个土生土长的肿瘤模型4。在此分析是一个关键试剂的miniCoopR载体，这对新人的mitfa黑素细胞的规范因素的野生型拷贝到网关重组盒候选人梅尔阿诺马基因可重组5。 miniCoopR矢量具有mitfa的的抢救小基因的其中包含的的野生型mitfa的基因的启动子，开放阅读框和3&#39;-非翻译区的。它使我们能够使用全长度的开放阅读框的候选人黑色素瘤修饰符的结构。个人可以被注入单细胞TG（mitfa：BRAF V600E）p53蛋白（LF）; mitfa（LF）斑马鱼胚胎克隆。矢量被集成miniCoopR TOL2介导的转基因6和救援黑色素细胞。因为它们被物理地连接至的mitfa挽救小基因，候选基因的表达在获救的黑素细胞，其中的一些将改造，并发展成肿瘤。对黑色素瘤的启动和黑色素瘤细胞属性的候选基因的效果，可以使用无黑色素瘤生存曲线，入侵检测，抗体染色和移植试验测定。

一单元格的Tg（mitfa：BRAF V600E）; p53基因（如果）; mitfa（如果）斑马鱼胚胎与miniCoopR矢量含有黑色素瘤癌基因SETDB1 5或EGFP的mitfa启动子的控制下，每个注入。胚胎选择，并允许成熟的黑素细胞的救援。在2个月内黑素细胞救援与动物的年龄大于4平方毫米的选择。在动物中筛选每周黑色素瘤。为成人肿瘤发病率曲线表明，SETDB1癌基因显着加速黑...

Watch this Scientific Journal Video about Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model at JoVE.com

Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model

屏幕使用一个斑马鱼原地肿瘤模型的黑色素瘤的改性剂是一种快速的方式。它利用的miniCoopR允许候选黑色素的黑素细胞中的基因表达的载体。黑色素瘤生存率曲线，入侵检测，协议规模的黑素细胞抗体染色和黑色素瘤移植试验的方法获得。

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 ...

Research

JoVE Journal

Medicine

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

多光子成像在口腔鳞状细胞癌原位小鼠模型的肿瘤细胞的侵袭

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

科研

医学

Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure

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, ...

Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy

人类神经内分泌肿瘤细胞株为研究人类神经内分泌肿瘤治疗的三维模型

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human ...

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

来自患者的细胞培养和分离的CD133<sup&gt; +</sup&gt;假定的肿瘤干细胞的恶性黑色素瘤

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

A Zebrafish Model of Diabetes Mellitus and Metabolic Memory

斑马鱼模型的糖尿病和代谢记忆

Diabetes mellitus currently affects 346 million individuals and this is projected to increase to 400 million by 2030. Evidence from both the laboratory ...

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

一种新型的高分辨率<em&gt;在体内</em&gt;成像技术研究肿瘤生长及治疗颅内结构的动态响应

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a ...

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

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

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 ...

Three-dimensional Co-culture Model for Tumor-stromal Interaction

对于肿瘤基质相互作用的三维共培养模式

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal ...

Isolated Hepatic Perfusion as a Treatment for Liver Metastases of Uveal Melanoma

隔离肝脏灌注作为治疗葡萄膜黑色素瘤肝转移

Isolated hepatic perfusion (IHP) is a procedure where the liver is surgically isolated and perfused with a high concentration of the chemotherapeutic ...

Evaluation of Tumor-infiltrating Leukocyte Subsets in a Subcutaneous Tumor Model

在皮下肿瘤模型的评估肿瘤浸润白细胞亚群

Specialized immune cells that infiltrate the tumor microenvironment regulate the growth and survival of neoplasia.&#160; Malignant cells must elude or ...