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，显性负TP53或的两侧装接loxP PTEN等位基因。加入的诱发突变的模型可以更好地代表人类癌症（见1.3）。可从美国国家癌症研究所鼠标信息库（ <a href="http://mouse.ncifcrf.gov/" target="_blank">http://mouse.ncifcrf.gov/</a> ）的几个睡美人“转座子，转座子小鼠。 </li><li>品种有条件的转座小鼠，小鼠的转座子最终获得如果可能的两个等位基因纯合子小鼠。品种纯合子后代小鼠表达Cre重组酶产生的的三重转基因的实验小鼠（ 图2）。注：获得纯合子小鼠并不总是可能的或理想的。例如，纯合的p53基因的显性阴性小鼠比杂合子小鼠繁殖要困难得多。此外，它是有帮助的，以确保窝按照孟德尔遗传学确认育种计划成功。 </li><li>如果使用易患背景，第一个种小鼠的转座的易感等位基因的小鼠。同时，种小鼠表达Cre重组酶的小鼠，携带转座子。创建如果可能的话每个等位基因的纯合子小鼠。最后，从以前的两个育种计划培育纯合的后代产生四倍的转基因实验ANIMALS。 </li><li>在另外的实验动物，生成控制小鼠同伙。对照组通常由只继承三个等位基因中的两个（ 图2）从实验育种窝。易感背景的情况下，包括三个满分的四个等位基因的小鼠也将是必要的。 </li><li>监视每天肿瘤的发生发展和/或濒死的实验和对照组。按照您的机构动物护理和使用委员会（IACUC）的要求，为畜牧业。此外，这是典型的以确定的时代中，你将牺牲的动物，如果它尚未成为垂死的。十八个月是一个典型的年龄在小鼠安乐死。 </li></ol> 2。剖检转基因动物<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td>当然密封电感TION商会</td><td>脑树科学</td><td> EZ-177 </td></tr><tr><td>离心管</td><td> Bioexpress </td><td> C-3355-2 </td></tr><tr><td> 10％的缓冲福尔马林</td><td> Sigma Aldrich公司</td><td> HT501128-4L </td></tr></tbody></table>所需的表1中。试剂。 <ol><li>当鼠标是垂死的或确定的寿命已经走到了尽头，安乐死，动物用CO 2室后，IACUC指引。一般来说，在一个干净的气室中，放置在动物打开的气体罐，并调整调节器读取不高于5磅每平方英寸。填写慢慢地减少鼻腔和眼睛的刺激和厌恶CO 2。确保动物的心脏不再跳动，并且不响应时，感动的眼睛。 </li><li>处死小鼠，用70％乙醇的喷雾外部。 </li><li>将鼠标剖检板，并用解剖针固定。用剪刀的ð镊子打开鼠标和删除器官。确定哪些机构应收集项目依赖。然而，它建议收集脾脏和肝脏，因为这些机构往往提供了重要的洞察为什么鼠标是垂死的。还收集的胸骨骨髓的潜力分析。此外，它始终是有利于收集任何器官或组织出现异常。 </li><li>应分为四个部分所收集的组织和肿瘤。其中一个部分被固定在10％福尔马林缓冲液中18小时，然后用70％的乙醇。这部分可用于H＆E染色和/或IHC。剩下的三个部分，通常是50至100毫克的大小，是在液氮中快速冷冻。这些可用于DNA，RNA和蛋白质分离。 </li><li>根据IACUC指引处理的尸体和剩余的组织。 </li></ol> 3。从肿瘤中分离基因组DNA的<table border="1"><tbody><tr><td> &lt;STRONG&gt;的试剂名称</td><td> 公司 </td><td> 目录编号 </td></tr><tr><td>蛋白质沉淀的解决方案</td><td> Qiagen公司</td><td> 158910 </td></tr><tr><td>细胞裂解液</td><td> Qiagen公司</td><td> 158906 </td></tr><tr><td>蛋白酶K </td><td> Qiagen公司</td><td> 158920 </td></tr><tr><td>核糖核酸酶A </td><td> Qiagen公司</td><td> 158924 </td></tr><tr><td> TE缓冲液</td><td> Promega公司</td><td> V6232 </td></tr></tbody></table>所需的表2中。试剂。 <ol><li>细切末冰冻肿瘤样品（50〜100毫克），使用干净的剃刀刀片。 </li><li>地点剁碎组织在1.5 ml离心管中，加入1 ml的细胞裂解液5μL蛋白酶K溶液（20毫克/毫升）。 </li><li>涡彻底。 </li><li>孵育在55℃摇床以250rpm设置为至少4小时，优选过夜。 </li><li>加入1μlRNA酶A溶液（10毫克/毫升），并倒转管25倍。 </li><li>在37℃下孵育30分钟。 </li><li>冷却至室温，在冰上放置3分钟。 </li><li>加入333μL蛋白沉淀的解决方案和涡大力。 </li><li>离心14000转10分钟。蛋白颗粒。如果颗粒非常小，涡再次，冰上孵育5分钟，然后再重新离心机。 </li><li>倒掉上清液含DNA到一个干净的15ml离心管中。 </li><li>加入等体积的100％异丙醇混合轻轻倒转50次。 </li><li>在3,500 rpm转速下离心3分钟。 </li><li>弃去上清液。 </li><li>加入1毫升70％乙醇，并倒转管数次洗涤沉淀。 </li><li>随着乙醇至1.5 mL离心管中，将漂洗过的。 </li><li>离心机在14,000 rpm离心5分钟，在4℃下</li><li>倒出乙醇和反转管空气干燥（约5至30分钟内）。 </li><li>重悬DNA P埃利特取决于DNA沉淀大小在50至500μl的TE中。 </li><li>可选在37°C至重悬DNA。 </li><li>贮存在4℃或-20℃，用于长期存储。 </li></ol> 4。连接器介导的PCR的基因组DNA的肿瘤<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td> BFAI </td><td> New England Biolabs公司</td><td> R0568S </td></tr><tr><td> NlaIII </td><td> New England Biolabs公司</td><td> R0125S </td></tr><tr><td> MinElute 96孔板</td><td> Qiagen公司</td><td> 28051 </td></tr><tr><td> QIAvac 96真空歧管</td><td> Qiagen公司</td><td> 19504 </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></tr><tr><td> T4 DNA连接酶与5倍的连接缓冲</td><td> Invitrogen公司</td><html><td> 15224-041 </td></tr><tr><td> BamHI位</td><td> New England Biolabs公司</td><td> R0136S </td></tr><tr><td> 25毫米的dNTPs </td><td> Bioexpress </td><td> C-5014-200 </td></tr><tr><td>铂金Taq酶</td><td> Invitrogen公司</td><td> 10966-034 </td></tr><tr><td>快速启动Taq DNA聚合酶</td><td>罗氏公司</td><td> 12032929001 </td></tr><tr><td>琼脂糖</td><td> Promega公司</td><td> V3121 </td></tr><tr><td> TAE缓冲液</td><td> Promega公司</td><td> V4271 </td></tr><tr><td> TE缓冲液</td><td> Promega公司</td><td> V6232 </td></tr><tr><td>溴化乙锭</td><td> Promega公司</td><td> H5041 </td></tr></tbody></table>所需的表3中。试剂。 接头序列 BfaI的链接器+ 5&#39;GTAATACGACTCACTATAGGGCTCCGCTTAAGGGAC 3&#39; BFAI连接器-5&#39;磷酸TAGTCCCTTAAGCGGAG 3&#39;NH2 “NlaIII连接器+ 5&#39;GTAATACGACTCACTATAGGGCTCCGCTTAAGGGACCATG 3&#39; NlaIII连接器5&#39;磷酸GTCCCTTAAGCGGAGCC 3 - “ 引物序列小学PCR右侧（NlaIII）：SPL IRDR R1 1 GCTTGTGGAAGGCTACTCGAAATGTTTGACCC 小学PCR左前方侧（BFAI）：SPL IRDR L1 1 CTGGAATTTTCCAAGCTGTTTAAAGGCACAGTCAAC 左侧和右侧的主PCR反向：SPL链接1 GTAATACGACTCACTATAGGGC 二次PCR正向右侧：Illumina的条形码二次引物5&#39;AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNNNNNaGgtgtatgtaaacttccgacttcaa（N为代表的12个bp的条形码，在上壳体中的序列的实施例所需的Illumina平台，并在较低的情况下的序列将绑定到转座子）。 二次PCR左前方侧的Illumina公司的条码次生：例次生的引物5&#39;AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNNNNNaAgtgtatgtaaacttccgacttcaa（N为代表的12个bp的条形码，在上壳体中的序列所需的Illumina平台，并在较低的情况下的序列将绑定到转座子）。 二次PCR反向左侧和右侧的连接器嵌套反向引物5&#39;CAAGCAGAAGACGGCATACGAGCTCTTCCGATCTAGGGCTCCGCTTAAGGGAC <ol><li>退火的+和 - BFAI连接器和NlaII的连接器连接器连接器的混合50微升+（100μM）与50μl的连接器（100μM）和1.5 ml的离心管中，加入2μl氯化钠（5 M）。在95°C加热块5分钟。关闭加热块和更缓慢冷却至室温（这需要一个小时或更长时间）。冷却后，退火的连接子可被存储在一个-20℃的冰箱中，直到需要。 </li><li>准备两个等分的肿瘤DNA，每个含有1微克的DNA的等分试样。一个等份，将被消化机智公顷限制性内切酶，将削减接近“正确的”的转座子。第二份将被消化，用限制性内切酶切割的“左”的转座子。这样做是为了扩增每个转座子插入位点的机会最大化。 </li><li>使用“右”侧酶NlaII消化肿瘤DNA中的一个等份，消化肿瘤DNA在其他等分，使用“左”侧酶BFAI：将1μl的酶，DNA，5微升10倍的NEB缓冲液＃4（ 1μg）和DDH 2 O至总体积为50μl。隔夜精华DNA在37℃水浴中或孵化器。 </li><li>加热使酶失活（BFAI = 80°下进行20分钟，NlaIII = 65℃下进行20分钟）。 </li><li>清洁MinElute 96孔板上，通过将样品在消化的样品，将板在真空歧管和真空中约15分钟，直到井看起来干燥。 </li><li>删除板从真空歧管和印迹底的96孔板用纸巾除去所有液体。ð </li><li> ，每孔加入30微升DDH 2 O 2分钟抖上了轨道振动筛上设置高，或吸管向上和向下20〜40倍。 </li><li> MinElute 96孔板中的井（30微升）的整个体积转移到干净的96孔板中。 </li><li>步骤4.8和步骤4.1连接器从消化的DNA进行连接器的连接反应。 MIX 10微升消化DNA，4μL5倍的连接缓冲液，5.5μL的退火连接器（950微克/微升），0.5μLT4连接酶（5 U /μl）。 </li><li>孵育4小时至过夜，在16℃下</li><li>加热灭活T4 DNA连接酶在65℃下10分钟。 </li><li>清理结扎使用Minelute 96孔培养板，真空歧管中所述的步骤4.5 - 4.6。 </li><li>重悬在30μlH 2 O，并转移到一个干净的96孔板。 </li><li>为了摧毁剩余的联体转座子进行第二次DNA消化。这是通过切割DNA的第二次使用的酶切割质粒v厄克托被用来创建含有转座子多联体的转基因小鼠的DNA。 </li><li>月刊脱氧核糖核酸使用BamHI（左侧和右侧）：混合连接器连接的DNA的25微升从步骤4.13，5微升10倍NEB缓冲＃3，加入0.5μlBamHI位（20单位/微升），加入0.5μl的BSA（10毫克/毫升），和19微升DDH 2 O。 </li><li>摘要3-6小时或过夜，在37°C。 </li><li>使用转座子和链接器的特异性引物的特异性引物，进行一次PCR。这些措施会有所不同，这取决于您使用的是转座​​子和连接器。的引物的引物表中列出的上述工作为T2/Onc和T2/Onc2转座子。 </li><li>初级PCR：5微升10x缓冲液，2.0微升氯化镁（50毫摩尔），4微升的dNTPs（2.5纳米），1μl引物1（20μM），1μl引物2（20μM），0.25微升白金Taq酶（5 U /微升），3微升消化的DNA与连接器（金额可能有所不同），同比增长50μLDDH 2 O。 </li><li> PCR程序：94℃5分钟，30个循环的94℃30秒，60及d例如，C，持续30秒，72℃90秒。在72℃下进行5分钟的最后延伸。 </li><li>清理的PCR产物用MinElute 96孔板和如上所述的真空歧管。 </li><li>重悬在30μlH 2 O，并转移到一个干净的96孔板。 </li><li>一个1:75的稀释1°PCR稀释到148微升DDH 2 O 2μl的DNA从步骤4.21 </li><li>使用嵌套的版本为的Illumina公司GAIIx机，以及一个12个碱基对的条形码是唯一的处理（见上面的例子，这些引物的引物表）为每个肿瘤样本，也具有所需的序列的引物进行第二次PCR扩增。 </li><li>第二次PCR：10微升氯化镁，缓冲器，具有10倍加入8μl的dNTPs（2.5毫摩尔），1微升的Illumina条形码二次引物（20μM），1微升的Illumina巢1二次引物（20μM），0.25微升的快速启动的Taq（5 U /μl的），2微升DNA从初级PCR摊薄1:75，高达100微升DDH 2 O。 </li><li> PCR程序为：94°℃2分钟，30个循环的94℃30秒，53℃45秒，72℃下为90秒。在72℃下进行5分钟的最后延伸。 </li><li>运行该PCR反应，在1％琼脂糖凝胶含有溴化乙锭（0.5微克/毫升）的45微升。应该有一个代表的许多不同的转座子插入在肿瘤（ 图3）的扩增子的PCR产物的“拖尾”。 </li><li>清理余下的50微升的PCR产物用MinElute 96孔板和如上所述的真空歧管。将50微升DDH 2 O中井和吸尘再清洗一次。 </li><li>重悬在30微升TE中，并转移到一个干净的96孔板中。 </li><li>确定在每个样品中DNA的浓度。在一个单一的1.5毫升微量离心管中，将等量的DNA。这将是在一个车道的Illumina的HiSeq 2000机库，测序。它是可能序列的Illumina的运行在一个单一的车道几百肿瘤。 </li><li>提交单管，含有等量的DNA进行测序的所有的肿瘤。这可以通过您的机构或商业。 </li></ol> 5。转座子插入分析<ol><li>转座子插入数据的分析软件可以通过的SourceForge（ <a href="http://sourceforge.net/projects/tapdancebio/" target="_blank">http://sourceforge.net/projects/tapdancebio/</a> ）免费下载。的程序，称为TapDance，需要Illumina平台的序列文件和条形码库图的文本文件。 </li></ol>

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作者有没有透露。

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 ...

identification-sleeping-beauty-transposon-insertions-solid-tumors

Research

JoVE Journal

Medicine

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

14.0K 次观看.  University of Minnesota, Minneapolis. 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。该方法使用睡美人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

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

Establishment and Propagation of Human Retinoblastoma Tumors in Immune Deficient Mice

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited time. These cultured tumorspheres may exhibit markedly different cellular phenotypes when compared to the original tumors. Demonstration that cultured cells have the capability of forming new tumors is important to ensure that cultured cells model the biology of the original tumor. 
Here we present a protocol for propagating human retinoblastoma tumors in vivo using Rag2-/- immune deficient mice. Cultured human retinoblastoma tumorspheres of low passage or cells obtained from freshly harvested human retinoblastoma tumors injected directly into the vitreous cavity of murine eyes form tumors within 2-4 weeks. These tumors can be harvested and either further passaged into murine eyes in vivo or grown as tumorspheres in vitro. Propagation has been successfully carried out for at least three passages thus establishing a continuing source of human retinoblastoma tissue for further experimentation. 
Wesley S. Bond and Lalita Wadhwa are co-first authors.

A method is described to propagate human retinoblastoma tumors in mice. Tumor cells are directly injected into the eyes of immune deficient mice. Secondary tumors have been successfully established using both cells directly harvested from human tumors and cultured tumorspheres.

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

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle uptake in tumors at the microscopic level1,2,3, highlighting the utility of a suitable xenograft model in which to perform detailed uptake analyses. Here, we use high-resolution intravital imaging to evaluate nanoparticle uptake in human tumor xenografts in a modified, shell-less chicken embryo model. The chicken embryo model is particularly well-suited for these in vivo analyses because it supports the growth of human tumors, is relatively inexpensive and does not require anesthetization or surgery 4,5. Tumor cells form fully vascularized xenografts within 7 days when implanted into the chorioallantoic membrane (CAM) 6. The resulting tumors are visualized by non-invasive real-time, high-resolution imaging that can be maintained for up to 72 hours with little impact on either the host or tumor systems. Nanoparticles with a wide range of sizes and formulations administered distal to the tumor can be visualized and quantified as they flow through the bloodstream, extravasate from leaky tumor vasculature, and accumulate at the tumor site. We describe here the analysis of nanoparticles derived from Cowpea mosaic virus (CPMV) decorated with near-infrared fluorescent dyes and/or polyethylene glycol polymers (PEG) 7, 8, 9,10,11. Upon intravenous administration, these viral nanoparticles are rapidly internalized by endothelial cells, resulting in global labeling of the vasculature both outside and within the tumor7,12. PEGylation of the viral nanoparticles increases their plasma half-life, extends their time in the circulation, and ultimately enhances their accumulation in tumors via the enhanced permeability and retention (EPR) effect 7, 10,11. The rate and extent of accumulation of nanoparticles in a tumor is measured over time using image analysis software. This technique provides a method to both visualize and quantify nanoparticle dynamics in human tumors.

We present a novel approach to quantify nanoparticle localization in the vasculature of human xenografted tumors using dynamic, real-time intravital imaging in an avian embryo model.

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

Morphologic heterogeneity within an individual tumor is well-recognized by histopathologists in surgical practice. While this often takes the form of areas of distinct differentiation into recognized histological subtypes, or different pathological grade, often there are more subtle differences in phenotype which defy accurate classification (Figure 1). Ultimately, since morphology is dictated by the underlying molecular phenotype, areas with visible differences are likely to be accompanied by differences in the expression of proteins which orchestrate cellular function and behavior, and therefore, appearance. The significance of visible and invisible (molecular) heterogeneity for prognosis is unknown, but recent evidence suggests that, at least at the genetic level, heterogeneity exists in the primary tumor1,2, and some of these sub-clones give rise to metastatic (and therefore lethal) disease. 
Moreover, some proteins are measured as biomarkers because they are the targets of therapy (for instance ER and HER2 for tamoxifen and trastuzumab (Herceptin), respectively). If these proteins show variable expression within a tumor then therapeutic responses may also be variable. The widely used histopathologic scoring schemes for immunohistochemistry either ignore, or numerically homogenize the quantification of protein expression. Similarly, in destructive techniques, where the tumor samples are homogenized (such as gene expression profiling), quantitative information can be elucidated, but spatial information is lost. Genetic heterogeneity mapping approaches in pancreatic cancer have relied either on generation of a single cell suspension3, or on macrodissection4. A recent study has used quantum dots in order to map morphologic and molecular heterogeneity in prostate cancer tissue5, providing proof of principle that morphology and molecular mapping is feasible, but falling short of quantifying the heterogeneity. Since immunohistochemistry is, at best, only semi-quantitative and subject to intra- and inter-observer bias, more sensitive and quantitative methodologies are required in order to accurately map and quantify tissue heterogeneity in situ.
We have developed and applied an experimental and statistical methodology in order to systematically quantify the heterogeneity of protein expression in whole tissue sections of tumors, based on the Automated QUantitative Analysis (AQUA) system6. Tissue sections are labeled with specific antibodies directed against cytokeratins and targets of interest, coupled to fluorophore-labeled secondary antibodies. Slides are imaged using a whole-slide fluorescence scanner. Images are subdivided into hundreds to thousands of tiles, and each tile is then assigned an AQUA score which is a measure of protein concentration within the epithelial (tumor) component of the tissue. Heatmaps are generated to represent tissue expression of the proteins and a heterogeneity score assigned, using a statistical measure of heterogeneity originally used in ecology, based on the Simpson's biodiversity index7.
To date there have been no attempts to systematically map and quantify this variability in tandem with protein expression, in histological preparations. Here, we illustrate the first use of the method applied to ER and HER2 biomarker expression in ovarian cancer. Using this method paves the way for analyzing heterogeneity as an independent variable in studies of biomarker expression in translational studies, in order to establish the significance of heterogeneity in prognosis and prediction of responses to therapy.

Here we describe a method to quantify molecular heterogeneity in histological sections of tumor material using quantitative immunofluorescence, image analysis, and a statistical measure of heterogeneity. The method is intended for use in clinical biomarker development and analysis.

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)

MicroRNAs (miRNAs) are single-stranded, 18&#x2013;24 nucleotide long, non-coding RNA molecules. They are involved in virtually every cellular process including development1, apoptosis2, and cell cycle regulation3. MiRNAs are estimated to regulate the expression of 30% to 90% of human genes4 by binding to their target messenger RNAs (mRNAs)5. Widespread dysregulation of miRNAs has been reported in various diseases and cancer subtypes6. Due to their prevalence and unique structure, these small molecules are likely to be the next generation of biomarkers, therapeutic agents and/or targets.
Methods used to investigate miRNA expression include SYBR green I dye- based as well as Taqman-probe based qPCR. If miRNAs are to be effectively used in the clinical setting, it is imperative that their detection in fresh and/or archived clinical samples be accurate, reproducible, and specific. qPCR has been widely used for validating expression of miRNAs in whole genome analyses such as microarray studies7. The samples used in this protocol were from patients who underwent radical prostatectomy for clinically localized prostate cancer; however other tissues and cell lines can be substituted in. Prostate specimens were snap-frozen in liquid nitrogen after resection. Clinical variables and follow-up information for each patient were collected for subsequent analysis8.
Quantification of miRNA levels in prostate tumor samples. The main steps in qPCR analysis of tumors are: Total RNA extraction, cDNA synthesis, and detection of qPCR products using miRNA-specific primers. Total RNA, which includes mRNA, miRNA, and other small RNAs were extracted from specimens using TRIzol reagent. Qiagen's miScript System was used to synthesize cDNA and perform qPCR (Figure 1). Endogenous miRNAs are not polyadenylated, therefore during the reverse transcription process, a poly(A) polymerase polyadenylates the miRNA. The miRNA is used as a template to synthesize cDNA using oligo-dT and Reverse Transcriptase. A universal tag sequence on the 5' end of oligo-dT primers facilitates the amplification of cDNA in the PCR step. PCR product amplification is detected by the level of fluorescence emitted by SYBR Green, a dye which intercalates into double stranded DNA. Specific miRNA primers, along with a Universal Primer that binds to the universal tag sequence will amplify specific miRNA sequences. 
The miScript Primer Assays are available for over a thousand human-specific miRNAs, and hundreds of murine-specific miRNAs. Relative quantification method was used here to quantify the expression of miRNAs. To correct for variability amongst different samples, expression levels of a target miRNA is normalized to the expression levels of a reference gene. The choice of a gene on which to normalize the expression of targets is critical in relative quantification method of analysis. Examples of reference genes typically used in this capacity are the small RNAs RNU6B, RNU44, and RNU48 as they are considered to be stably expressed across most samples. In this protocol, RNU6B is used as the reference gene.

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

Quantitative Real Time polymerase chain reaction (qPCR) is a rapid and sensitive method to investigate the expression levels of various microRNA (miRNA) molecules in tumor samples. Using this method expression of hundreds of different miRNA molecules can be amplified, quantified, and analyzed from the same cDNA template.

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

Several human and murine colon cancer cell lines have been established, physiologic integrity of colon tumors such as multiple cell layers, basal-apical polarity, ability to differentiate, and anoikis are not maintained in colon cancer derived cell lines. The present study demonstrates a method for culturing primary mouse colon tumor organoids adapted from Sato T et al. 1, which retains important physiologic features of colon tumors. This method consists of mouse colon tumor tissue collection, adjacent normal colon epithelium dissociation, colon tumor cells digestion into single cells, embedding colon tumor cells into matrigel, and selective culture based on the principle that tumor cells maintain growth on limiting nutrient conditions compared to normal epithelial cells.
The primary tumor organoids if isolated from genetically modified mice provide a very useful system to assess tumor autonomous function of specific genes. Moreover, the tumor organoids are amenable to genetic manipulation by virus meditated gene delivery; therefore signaling pathways involved in the colon tumorigenesis could also be extensively investigated by overexpression or knockdown. Primary tumor organoids culture provides a physiologic relevant and feasible means to study the mechanisms and therapeutic modalities for colon tumorigenesis.

In vitro Organoid Culture of Primary Mouse Colon Tumors

A simple method to establish primary murine colon tumor organoid is described. This method utilizes the feature that colon tumor cells survive and grow into organoids in media containing limited growth factors, whereas normal colon epithelial do not.

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

MicroRNAs (miRNAs) constitute a potent layer of gene regulation by guiding RISC to target sites located on mRNAs and, consequently, by modulating their translational repression. Changes in miRNA expression have been shown to be involved in the development of all major complex diseases. Furthermore, recent findings showed that miRNAs can be secreted to the extracellular environment and enter the bloodstream and other body fluids where they can circulate with high stability. The function of such circulating miRNAs remains largely elusive, but systematic high throughput approaches, such as miRNA profiling arrays, have lead to the identification of miRNA signatures in several pathological conditions, including neurodegenerative disorders and several types of cancers. In this context, the identification of miRNA expression profile in the cerebrospinal fluid, as reported in our recent study, makes miRNAs attractive candidates for biomarker analysis.
There are several tools available for profiling microRNAs, such as microarrays, quantitative real-time PCR (qPCR), and deep sequencing. Here, we describe a sensitive method to profile microRNAs in cerebrospinal fluids by quantitative real-time PCR. We used the Exiqon microRNA ready-to-use PCR human panels I and II V2.R, which allows detection of 742 unique human microRNAs. We performed the arrays in triplicate runs and we processed and analyzed data using the GenEx Professional 5 software.
Using this protocol, we have successfully profiled microRNAs in various types of cell lines and primary cells, CSF, plasma, and formalin-fixed paraffin-embedded tissues.

We describe a protocol of real time PCR to profile microRNAs in the cerebrospinal fluid (CSF). With the exception of RNA extraction protocols, the procedure can be extended to RNA extracted from other body fluids, cultured cells, or tissue specimens.

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

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent marker. Mouse models (brain tumor and arthritis) were used to evaluate the usefulness of this method. Saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles tagged with CellVue Maroon (CVM) fluorophore were administered intravenously. Animals were then placed in the rotational holder (MARS) of the in vivo imaging system. Images were acquired in 10&#176; steps over 380&#176;. A rectangular&#160;region of interest (ROI) was placed across the full image width at the model disease site. Within the ROI, and for every image, mean fluorescence intensity was computed after background subtraction. In the mouse models studied, the labeled nanovesicles were taken up in both the orthotopic and transgenic brain tumors, and in the arthritic sites (toes and ankles). Curve analysis of the multi angle image ROIs determined the angle with the highest signal. Thus, the optimal angle for imaging each disease site was characterized. The MAROI method applied to imaging of fluorescent compounds is a noninvasive, economical, and precise tool for in vivo quantitative analysis of the disease states in the described mouse models.

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

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo quantitation of a fluorescent marker delivered by saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles. Employing mouse models of cancer and arthritis, we demonstrate how the MAROI signal curve analysis can be used for the precise mapping and biological characterization of disease processes.

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

In-depth analyses of cancer cell proteomes are needed to elucidate oncogenic pathomechanisms, as well as to identify potential drug targets and diagnostic biomarkers. However, methods for quantitative proteomic characterization of patient-derived tumors and in particular their cellular subpopulations are largely lacking. Here we describe an experimental set-up that allows quantitative analysis of proteomes of cancer cell subpopulations derived from either liquid or solid tumors. This is achieved by combining cellular enrichment strategies with quantitative Super-SILAC-based mass spectrometry followed by bioinformatic data analysis. To enrich specific cellular subsets, liquid tumors are first immunophenotyped by flow cytometry followed by FACS-sorting; for solid tumors, laser-capture microdissection is used to purify specific cellular subpopulations. In a second step, proteins are extracted from the purified cells and subsequently combined with a tumor-specific, SILAC-labeled spike-in standard that enables protein quantification. The resulting protein mixture is subjected to either gel electrophoresis or Filter Aided Sample Preparation (FASP) followed by tryptic digestion. Finally, tryptic peptides are analyzed using a hybrid quadrupole-orbitrap mass spectrometer, and the data obtained are processed with bioinformatic software suites including MaxQuant. By means of the workflow presented here, up to 8,000 proteins can be identified and quantified in patient-derived samples, and the resulting protein expression profiles can be compared among patients to identify diagnostic proteomic signatures or potential drug targets.

In-depth analyses of cancer cell proteomes facilitate identification of novel drug targets and diagnostic biomarkers. We describe an experimental workflow for quantitative analysis of (phospho-)proteomes in cancer cell subpopulations derived from liquid and solid tumors. This is achieved by combining cellular enrichment strategies with quantitative Super-SILAC-based mass spectrometry.

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

The heterogeneous intra-tumoral accumulation of liposomes is a critical determinant of their efficacy. Both the chaotic tumor microcirculation and elevated IFP are linked to the heterogeneous intra-tumoral distribution of nanotechnology-based drug delivery systems such as liposomes. In the present study, the relationship between tumor microcirculation, elevated IFP, and accumulation of nanoparticles was investigated through in vivo experimentation. This was accomplished by evaluation of the tumor microcirculation using dynamic contrast enhanced computed tomography (DCE-CT) and measurement of tumor IFP using a novel image-guided robotic needle placement system connected to the micro-CT scanner. The intra-tumoral accumulation of liposomes was determined by CT image-based assessment of a nanoparticle liposomal formulation that stably encapsulate the contrast agent iohexol (CT-liposomes). CT imaging allowed for co-localization of the spatial distribution of tumor hemodynamics, IFP and CT-liposome accumulation in an individual subcutaneous xenograft mouse model of breast cancer. Measurements led to the discovery that perfusion and plasma volume fraction are strong mediators of the intra-tumoral distribution of liposomes. Furthermore, the results suggest that IFP plays an indirect role in mediating liposome distribution through modulating blood flow.

The heterogeneous intra-tumoral accumulation of liposomes has been linked to an abnormal tumor microenvironment. Herein methods are presented to measure tumor microcirculation by perfusion imaging and elevated interstitial fluid pressure (IFP) using an image-guided robotic system. Measurements are compared to the intra-tumoral accumulation of liposomes, determined using volumetric micro-CT imaging.

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

Three-dimensional (3D) printing technologies offer the possibility of visualizing patient-specific pathologies in a physical model of correct dimensions. The model can be used for planning and simulating critical steps of a surgical approach. Therefore, it is important that anatomical structures such as blood vessels inside a tumor can be printed to be colored not only on their surface, but throughout their whole volume. During simulation this allows for the removal of certain parts (e.g., with a high-speed drill) and revealing internally located structures of a different color. Thus, diagnostic information from various imaging modalities (e.g., CT, MRI) can be combined in a single compact and tangible object.
However, preparation and printing of such a fully colored anatomical model remains a difficult task. Therefore, a step-by-step guide is provided, demonstrating the fusion of different cross-sectional imaging data sets, segmentation of anatomical structures, and creation of a virtual model. In a second step the virtual model is printed with volumetrically colored anatomical structures using a plaster-based color 3D binder jetting technique. This method allows highly accurate reproduction of patient-specific anatomy as shown in a series of 3D-printed petrous apex chondrosarcomas. Furthermore, the models created can be cut and drilled, revealing internal structures that allow for simulation of surgical procedures.

The protocol describes the fabrication of fully colored three-dimensional prints of patient-specific, anatomical skull models to be used for surgical simulation. The crucial steps of combining different imaging modalities, image segmentation, three-dimensional model extraction, and production of the prints are explained.

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. ...