Name: identification of sleeping beauty transposon insertions in solid tumors using linker-mediated pcr
Uploaded: 2013-02-01T13:00:00
Description: Watch this Scientific Journal Video about Identification of Sleeping Beauty Transposon Insertions in Solid Tumors using Linker-mediated PCR at JoVE.com

作者想感谢的布兰登Moriarity，大卫Largaespada，在美国明尼苏达大学的文森特景，并在美国爱荷华大学的亚当·杜佩在发展上述协议提供的援助。

1。转基因动物育种和老化<ol><li>选择适当的转基因小鼠，为您的实验株。一个典型的实验中，将使用3转基因株系;一个条件的转座鼠标，鼠标窝藏之多联体转座子，和一个鼠标表达Cre重组酶的细胞被认为是所需的癌症细胞的起源。如果被建模的癌症有一个已知的突变，在一个大的患者的百分比，它可能是可取的，以包括在开始这种突变。这些“诱发”突变的例子包括活化的KRAS基因，显性?...

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	<li>Fearon, E. R., Vogelstein, B., 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=A+genetic+model+for+colorectal+tumorigenesis.">A genetic model for colorectal tumorigenesis.</a> Cell. 61, 759-767 (1990).</li><li>Wood, L. 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=The+Genomic+Landscapes+of+Human+Breast+and+Colorectal+Cancers.">The Genomic Landscapes of Human Breast and Colorectal Cancers.</a> Science. 318, 1108-1113 (2007).</li><li>Ivics, Z., Hackett, P. B., Plasterk, R. H., Izsv&#225;k, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Reconstruction+of+Sleeping+Beauty,+a+Tc1-like+Transposon+from+Fish,+and+Its+Transposition+in+Human+Cells.">Molecular Reconstruction of Sleeping Beauty, a Tc1-like Transposon from Fish, and Its Transposition in Human Cells.</a> Cell. 91, 501-510 (1997).</li><li>Starr, T. K., Largaespada, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+gene+discovery+using+the+Sleeping+Beauty+transposon.">Cancer gene discovery using the Sleeping Beauty transposon.</a> Cell Cycle. 4, 1744-1748 (2005).</li><li>Mueller, P. R., Wold, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Vivo+Footprinting+of+a+Muscle+Specific+Enhancer+by+Ligation+Mediated+PCR.">In Vivo Footprinting of a Muscle Specific Enhancer by Ligation Mediated PCR.</a> Science. 246, 780-786 (1989).</li><li>Bergemann, T. L., 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=New+methods+for+finding+common+insertion+sites+and+co-occurring+common+insertion+sites+in+transposon-+and+virus-based+genetic+screens.">New methods for finding common insertion sites and co-occurring common insertion sites in transposon- and virus-based genetic screens.</a> Nucleic acids research. 40, 3822-3833 (2012).</li><li>Copeland, N. G., Jenkins, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Harnessing+transposons+for+cancer+gene+discovery.">Harnessing transposons for cancer gene discovery.</a> Nature Reviews Cancer. 10, 696-706 (2010).</li><li>Collier, L. S., Carlson, C. M., Ravimohan, S., Dupuy, A. J., Largaespada, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+gene+discovery+in+solid+tumours+using+transposon-based+somatic+mutagenesis+in+the+mouse.">Cancer gene discovery in solid tumours using transposon-based somatic mutagenesis in the mouse.</a> Nature. 436, 272-276 (2005).</li><li>Starr, T. K., 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=A+transposon-based+genetic+screen+in+mice+identifies+genes+altered+in+colorectal+cancer.">A transposon-based genetic screen in mice identifies genes altered in colorectal cancer.</a> Science. 323, 1747-1750 (2009).</li><li>Keng, V. W., 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=A+conditional+transposon-based+insertional+mutagenesis+screen+for+genes+associated+with+mouse+hepatocellular+carcinoma.">A conditional transposon-based insertional mutagenesis screen for genes associated with mouse hepatocellular carcinoma.</a> Nature. 27, 264-274 (2009).</li><li>Dupuy, A. J., Akagi, K., Largaespada, D. A., Copeland, N. G., Jenkins, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammalian+mutagenesis+using+a+highly+mobile+somatic+Sleeping+Beauty+transposon+system.">Mammalian mutagenesis using a highly mobile somatic Sleeping Beauty transposon system.</a> Nature. 436, 221-226 (2005).</li></ol>

使用睡美人转座子系统的正向遗传筛选提供了一种方法识别基因突变，导致癌症的。通过选择合适的启动子控制Cre重组酶在任何诱发突变，SB屏幕识别已知的和新的候选癌基因。 的SB屏幕的成功在很大程度上依赖于用于创建屏幕中选择的小鼠。对于转座子，我们建议使用两种不同的小鼠品系不同的染色体上携带的联体致癌转座子7。选择的组织特异性表...

<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（可选） </td></tr><tr><td>当然封口感应室</td><td>脑树科学</td><td> EZ-177 </td><td></td></tr><tr><td>离心管</td><td> Bioexpress </td><td> C-3355-2 </td><td></td></tr><tr><td> 10％的缓冲福尔马林</td><td> Sigma Aldrich公司</td><td> HT501128-4L </td><td></td></tr><tr><td>蛋白质沉淀的解决方案</td><td> Qiagen公司</td><td> 158910 </td><td></td></tr><tr><td>细胞裂解液</td><td> Qiagen公司</td><td> 158906 </td><td></td></tr><tr><td>蛋白酶K </td><td> Qiagen公司</td><td> 158920 </td><td></td></tr><tr><td>核糖核酸酶A </td><td> Qiagen公司</td><td> 158924 </td><td></td></tr><tr><td> TE缓冲液</td> &lt;TD&gt; Promega公司</td><td> V6232 </td><td></td></tr><tr><td> BFAI </td><td> New England Biolabs公司</td><td> R0568S </td><td></td></tr><tr><td> NlaIII </td><td> New England Biolabs公司</td><td> R0125S </td><td></td></tr><tr><td> MinElute 96孔板</td><td> Qiagen公司</td><td> 28051 </td><td></td></tr><tr><td> QIAvac 96真空歧管</td><td> Qiagen公司</td><td> 19504 </td><td></td></tr><tr><td>多微孔板精灵的，120V </td><td>科学工业公司</td><td> SI-4000 </td><td></td></tr><tr><td> T4 DNA连接酶与5倍的连接缓冲</td><td> Invitrogen公司</td><td> 15224-041 </td><td></td></tr><tr><td> BamHI位</td><td> New England Biolabs公司</td><td> R0136S </td><td></td></tr><tr><td>的25mM的dNTPs </td><td> Bioexpress </td><td> C-5014-200 </td><td></td></tr><tr><td>铂金Taq酶</td><td> Invitrogen公司</td><td&gt; 10966-034 </td><td></td></tr><tr><td>快速启动Taq DNA聚合酶</td><td>罗氏公司</td><td> 12032929001 </td><td></td></tr><tr><td>琼脂糖</td><td> Promega公司</td><td> V3121 </td><td></td></tr><tr><td> TAE缓冲液</td><td> Promega公司</td><td> V4271 </td><td></td></tr><tr><td> TE缓冲液</td><td> Promega公司</td><td> V6232 </td><td></td></tr><tr><td>溴化乙锭</td><td> Promega公司</td><td> H5041 </td><td></td></tr></tbody></table>

identification of sleeping beauty transposon insertions in solid tumors using linker-mediated pcr

人类肿瘤基因组学，蛋白质组学，转录组和表观分析表明，有数以千计相匹配的正常组织相比，在每一个癌症基因组的异常。基于这些分析，这是显而易见的，有许多未被发现的遗传的驱动癌症1。不幸的是，这些驱动程序都隐藏在一个更大数量的乘客异常的基因组不直接促进肿瘤的形成。的癌症基因组的另一个方面是，有相当类似类型的肿瘤内的遗传异质性。每个肿瘤能够携带不同的基因突变，肿瘤形成2提供了一个选择性的优势。执行一个公正的正向遗传筛选的小鼠提供的工具，以产生肿瘤，并分析其遗传组成，同时减少乘客突变的背景。 睡美人 （SB）的转座子系统是其中的一个方法3。 SB系统利用移动vectors（转座子）可以被插入在整个基因组的转座酶。被限制到特定的细胞类型，通过使用一个被激活的Cre重组酶的的条件转座等位基因突变。许多小鼠表达Cre重组酶在特定的组织存在。穿越这些线路的的条件转座等位基因（ 如液氧一站式-LOX-SB11），SB只有在系统被激活细胞表达Cre重组酶 。 Cre重组酶切除停止盒块座的等位基因的表达，从而激活内指定的单元格类型的转座子突变。的SB屏幕发起的繁殖三个株的转基因小鼠，使实验小鼠携带一个条件转座等位基因，多联体转座子，并在组织特异性表达Cre重组酶的等位基因。这些老鼠被允许直到肿瘤形成的年龄和他们变得奄奄一息。老鼠是剖检和基因组DNA是从肿瘤中分离。接着，对基因组DNA进行到链接器介导PCR扩增（LM-PCR），含有SB转座子的基因位点的扩增结果。 LM-PCR进行单一的肿瘤，会导致不同的扩增产物的数百名代表的数百个基因的转座子插入位点包含在一个单一的肿瘤4。在所有肿瘤的转座子插入常见的插入位点（CISS）进行了分析和确定适当的统计方法5。在独联体内的极有可能是癌基因或抑癌基因的基因，被认为是候选癌基因。使用SB系统，以确定候选癌基因的优点是：1）可以很容易地位于转座子的基因组中，因为它的序列是已知的，2）转置可以被定向到几乎任何类型的细胞，和3）的转座子是能够增益和损失的功能突变6。该followi纳克协议介绍了如何制定和执行正向遗传学的屏幕采用了SB转座系统，以确定候选癌基因（ 图1）。

在育种方案已经确立，饲养者应产生的枯枝落叶，每19-21天。产仔数会有所不同5至12个幼崽，根据育种者的年龄和遗传背景。此外，确保基因型的垃圾遵循孟德尔遗传定律的方式，以确认育种方案是正确和成功的。 在实验过程中是成功的，在实验动物的肿瘤发生率应该是显着大于在对照组动物的肿瘤发生率。如果没有“诱发”突变被包含在实验中应该有几个没有在控制动物的...

Watch this Scientific Journal Video about Identification of Sleeping Beauty Transposon Insertions in Solid Tumors using Linker-mediated PCR at JoVE.com

Identification of Sleeping Beauty Transposon Insertions in Solid Tumors using Linker-mediated PCR

鉴定未知的驱动程序致癌作用的一种方法，用一种公正的方法。该方法使用<em&gt;睡美人</em&gt;转座子作为随机诱变剂，向特定的组织。肿瘤的形成转座子插入驱动的新型癌基因和肿瘤抑制基因的基因组作图

鉴定<em&gt;睡美人</em&gt;转座子插入连接器介导的PCR实体瘤

Genomic, proteomic, transcriptomic, and epigenomic analyses of human tumors indicate that there are thousands of anomalies within each cancer genome ...

Research

JoVE Journal

Medicine

Identification of Sleeping Beauty Transposon Insertions in Solid Tumors using Linker-mediated PCR

Establishment and Propagation of Human Retinoblastoma Tumors in Immune Deficient Mice

在免疫缺陷小鼠的建立和传播人类视网膜母细胞瘤肿瘤

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited ...

科研

医学

Evaluation of Nanoparticle Uptake in Tumors in Real Time Using Intravital Imaging

使用实时活体成像在肿瘤纳米粒子吸收的评价

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle ...

Heterogeneity Mapping of Protein Expression in Tumors using Quantitative Immunofluorescence

在使用定量免疫肿瘤的异质性蛋白表达的映射

Morphologic heterogeneity within an individual tumor is well-recognized by histopathologists in surgical practice. While this often takes the form of ...

MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR (qPCR)

MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR qPCR

实时定量PCR（qPCR的microRNA检测在前列腺肿瘤）

MicroRNAs (miRNAs) are single-stranded, 18&#x2013;24 nucleotide long, non-coding RNA molecules. They are involved in virtually every cellular process ...

In vitro Organoid Culture of Primary Mouse Colon Tumors

In vitro Organoid Culture of Primary Mouse Colon Tumors

在体外化培养基层小鼠结肠肿瘤

Several  human and murine colon cancer cell lines have been established, physiologic  integrity of colon tumors such as multiple cell layers, basal-apical ...

Cerebrospinal Fluid MicroRNA Profiling Using Quantitative Real Time PCR

脑脊液的microRNA分析采用实时定量PCR技术

MicroRNAs (miRNAs) constitute a potent layer of gene regulation by guiding RISC to target sites located on mRNAs and, consequently, by modulating their ...

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

在脑肿瘤和关节炎的用荧光SAPC-DOPS纳米囊泡体光学成像

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent ...

Quantitative Mass Spectrometric Profiling of Cancer-cell Proteomes Derived From Liquid and Solid Tumors

对源自液体和固体肿瘤癌症细胞定量蛋白质组质谱剖析

In-depth analyses of cancer cell proteomes are needed to elucidate oncogenic pathomechanisms, as well as to identify potential drug targets and diagnostic ...

Spatial Measurements of Perfusion, Interstitial Fluid Pressure and Liposomes Accumulation in Solid Tumors

灌注的空间测量，实体肿瘤间质流体压力和脂质体积累

The heterogeneous intra-tumoral accumulation of liposomes is a critical determinant of their efficacy. Both the chaotic tumor microcirculation and ...

Multicolor 3D Printing of Complex Intracranial Tumors in Neurosurgery

神经外科复杂颅内肿瘤的多色3D打印

Three-dimensional (3D) printing technologies offer the possibility of visualizing patient-specific pathologies in a physical model of correct dimensions. ...