Name: transposon mediated integration of plasmid dna into the subventricular zone of neonatal mice to generate novel models of glioblastoma
Uploaded: 2015-02-22T14:00:00
Description: Watch this Scientific Journal Video about Transposon Mediated Integration of Plasmid DNA into the Subventricular Zone of Neonatal Mice to Generate Novel Models of Glioblastoma at JoVE.com

We thank Dr. John Ohlfest and Dr. Stacey Decker13 for the generous gift of plasmids and for the training provided to master this method. We thank Marta Dzaman for help with standardizing the Hematoxylin-Eosin staining procedure of paraffin-embedded sections. We also thank Molly Dahlgren for perusing this manuscript and providing helpful suggestions. This work is supported by NIH/NINDS grants to MGC and PRL, Leah&#8217;s Happy Hearts Foundation grant to MCG and PRL. Alex&#8217;s Lemonade Foundation young Investigator Award and the St. Baldrick&#8217;s Foundation Fellowship to CK.

注：所有动物的协议已获得密歇根州委员会的大学动物的使用和护理（UCUCA）。 1.克隆利息的顺序进入新睡美人转座子注意：要插入的基因或使用睡美人转座系统的抑制因素（如shRNA的），克隆感兴趣的序列插入或PKT pT2期质粒的中坚力量。直接克隆这样的调控元件，感兴趣和标记基因仍然受到反向重复（IR / DR）两侧。克隆PDGFβ成PKT骨干下文详述的例子（参...

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在这篇文章中，我们详细介绍了产生GBM的新车型使用的致癌质粒DNA SB transposase-介导的融入周围的新生小鼠脑室下区细胞的多功能和可重复的方法。我们提出了一个协议，以产生转座子的质粒与感兴趣的新基因，说明如何监测肿瘤的进展在活的动物，以及如何表征这些肿瘤的组织病理学和免疫组织化学特征。 由于我们的实验室 （ 图3）和其他13</su...

胶质母细胞瘤（GBM）是最常见的（60％）的原发性脑成人肿瘤，具有15-21个月的中位生存当与外科手术，放射治疗和化疗1治疗。新疗法的GBM势在必行。实验性疗法需要测试在动物模型中能充分地再现了人类疾病的显着特征。策略诱导GBM在啮齿类动物中包括化学诱变烷化剂，生殖细胞或体基因变异，或移植使用神经胶质瘤细胞系2。最常用的模型采用的神经胶质瘤细胞系的植入，或者原位，进入脑或皮下使用同源细胞在动物中具有相同基因型;或异种细胞，最常用的人的GBM细胞系中，植入免疫受损小鼠中3。移植提供了许多优势，为颅内肿瘤的研究：方便重复性，standardized增长率，死亡和肿瘤的定位时间。然而，这些模型有局限性，由于用于植入和准确地再现组织能力有限的人工，微创手术的方法提供人类GBM（IV级WHO）的特点：伪pallisading坏死，核atypias，弥漫性浸润，微血管增生和肾小球样的血管的形成异常4-7。感应GBM的通过改变体细胞的基因组中与致癌基因DNA，无论是用病毒载体8-12，或与转座子介导的整合13，再现更接近人类疾病的病因和概括人类GBM的组织病理学特征。 从历史上看，CNS的肿瘤已经基于原点的感知单元，其被观察到的分类，将是一个预测因子为生存14。 GBM中分为原发性和继发性。新出现的证据TODAY指向主要胶质母细胞瘤15的高度异质性。二级GBM（15％），低级别星形细胞瘤（WHO I级），并anaplasic星形细胞瘤恶变的结果（WHO II级），与较早出现的疾病，预后较好和基因表达的“前神经”模式相关，而初级GBM（85％）表现出迟发性，预后差，胶质（古典），神经或间质的表达模式。无论是基因表达的这些模式相关的肿瘤起源细胞的实际仍在积极调查。积累的数据表明，与GBM的相关基因突变的组合预测生存。例如，heterozygocity损失染色体1P / 19Q，IDH1基因突变，PDGFRα扩增，对（LOH）与二级GBM，前神经表达模式和更好的预后相关，而EGFR表达，缺口和Sonic hedgehog通路的激活和NF1和PTEN的损失和p53的突变相关的神经，古典，或间质的主要GBM和预后更差16,17。大规模测序项目的出现和可用于测试许多患者标本的积累带来了丰富的新的信息相对于基因突变和途径牵连GBM发生发展和个体化用药，在治疗可以专门针对的可能性患者的遗传异常。最终，以评估这些突变和途径以系统的方式的预测值，并在每一种情况下，测试可能的治疗，需要的GBM的动物模型中与预先确定的遗传改变。基因组DNA的转座子介导的整合提供了一个可行的方法。 睡美人转座子制，Tc1的/ 航海类转座子的成员，被“唤醒”（构建）的多步骤多项式从鲑转座酶基因，这成为休眠超过10万年前18,19 SS位点特异性诱变的。在本质上，通过特定序列（反向重复/直接重复：IR / DR）的侧翼DNA的转座子可由睡美人转座活性的方式整合入基因组中“切割和粘贴”的方式。转座识别的IR部位的端部，切除转座子随机整合入碱基T和A，其在转（ 图1a）被复制，在转座子的每个末端碱基之间的另一DNA位点。睡美人转座由三个结构域，转座子结合结构域，核定位序列和催化结构域的。四个转座分子需要转座子的两端汇集并允许换位，但是，如果是本转座的过多的分子，它们可以二聚化和tetramerize抑制换位反应20。一个高效的换位反应需要转座到转座子的最佳比例。编码转座的DNA可被传递在相同的质粒用转座子（顺式），或在不同的质粒（反式）。为了确保转座和座子之间的最佳比率，有足够的活性的启动子可被选择用于转座的表达（为“顺式”的模式），或在注射液中的质粒可以被优化的比例（对于“反式”模式）。睡美人座子系统可成功地用于功能基因组学，插入诱变，转基因和体细胞基因治疗21。作为一种合成的结构重新设计，从休眠鲑变体功能分子，睡美人转座不绑定到人类或其他哺乳动物的其他20个座子。自从被发现以后，分子工程有enhanc编了SB座子系统通过改变在IR序列和另外的TATA二核苷酸侧翼的座子，导致中PT2座子的转座功效。这些转座子已经优化了SB的结合到IR部位和增加的切除的效果。在SB转座也经历了显著的改善;在本文中所呈现的实验中使用的转座是SB100X，通过DNA改组的策略，随后通过在哺乳动物细胞中22大规模遗传筛选产生的活动过度的转座。 在本报告中，我们提出了一种快速，灵活和可再现的方法来诱导固有GBM小鼠非病毒，转座子介导的整合的质粒DNA进入新生小鼠23的亚脑室区的细胞。我们提出了一个简单的方法来创建转座子与新基因适合用于使用睡美人转座基因组整合和演示如何监视质粒的摄取和使用生物发光疾病进展。我们也刻画转载本模型GBM的组织学和免疫组化特点。此外，我们提出了一个快速的方法，以产生从这些肿瘤原发性GBM细胞系。睡美人模型，其中肿瘤细胞从原来的动物诱导的，允许候选GBM基因的诱导和肿瘤进展中的作用的功能性评估。此系统也适用于良好的新颖的GBM治疗，包括在免疫感受态小鼠的免疫疗法的检测，而不需要侵入性炎症的外科手术，其可以改变局部微环境。

<table border="0" cellpadding="0" cellspacing="0" width="952">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">Name of Material/ Equipment</td>
			<td style="width:199px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:267px;">Comments/Description</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:367px;">Stoelting&#39;s Lab Standard with Rat and Mouse Adaptors</td>
			<td style="width:199px;">Stoelting</td>
			<td style="width:119px;">51670</td>
			<td style="width:267px;">Other companies produce similar frames, any of them with small mouse adaptors are suitable&#160;</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:367px;">Quintessential Stereotaxic Injector (QSI)</td>
			<td style="width:199px;">Stoelting</td>
			<td style="width:119px;">53311</td>
			<td style="width:267px;">Injections can be made without it, ,but&#160;&#160;&#160; the automatic injector allows for increased reproducibility and convenience</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:367px;">10 &#956;L 700 series hand fitted MICROLITER syringe</td>
			<td style="width:199px;">Hamilton</td>
			<td style="width:119px;">Model 701</td>
			<td style="width:267px;">This syringe model will deliver from 0.5 to 500 ul of solution reliably. Other syrnges (fro example 5 ul, Model 75) may also be used</td>
		</tr>
		<tr height="120">
			<td height="120" style="height:120px;width:367px;">30 gauge gauge hypodermic needle 
			(1.25&#34;, 15o bevel)&#160;</td>
			<td style="width:199px;">Hamilton</td>
			<td style="width:119px;">S/O# 197462</td>
			<td style="width:267px;">This needle is ideal to pierce the skin and the skull of a neonatal mouse without the need of other invasive procedures. If it gets dull (doesn&#39;t easily enter the skin), it needs to be replaced.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">in vivo-jetPEI</td>
			<td style="width:199px;">Polyplus Transfection</td>
			<td style="width:119px;">201-10G</td>
			<td style="width:267px;">Aliquot in small volumes and keep at -20oC&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">D-Luciferin, Potassium Salt</td>
			<td style="width:199px;">Goldbio.com</td>
			<td style="width:119px;">LuckK-1g</td>
			<td style="width:267px;">Other sources of firefly lucifierase are just as adequate</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:367px;">Hematoxylin Solution, Harris Modified</td>
			<td style="width:199px;">Sigma Aldrich</td>
			<td style="width:119px;">HHS128-4L</td>
			<td style="width:267px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:367px;">Tissue-Tek O.C.T. Compound</td>
			<td style="width:199px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">62550-01</td>
			<td style="width:267px;">for embedding brains for cryosectioning and immunohistovhemistry</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:367px;">Advanced DMEM/F-12, no glutamine</td>
			<td style="width:199px;">Life Technologies</td>
			<td style="width:119px;">12634-010</td>
			<td style="width:267px;">for the culture of neurospheres</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">B-27&#168; Supplement (50X), serum free&#160;</td>
			<td style="width:199px;">Life Technologies</td>
			<td style="width:119px;">17504-044</td>
			<td style="width:267px;">serum free supplement for the culture of neurospheres</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">N2 Supplement (100x)</td>
			<td style="width:199px;">Life Technologies</td>
			<td style="width:119px;">17502-048</td>
			<td style="width:267px;">serum free supplement for the culture of neurospheres</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">Normocyn</td>
			<td style="width:199px;">InvivoGen</td>
			<td style="width:119px;">ant-nr-1</td>
			<td style="width:267px;">anti-mycoplasma agent for the culture of neurospheres</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">Recombinant Human EGF</td>
			<td style="width:199px;">PEPROTECH</td>
			<td style="width:119px;">AF-100-15</td>
			<td style="width:267px;">growth factor supplement for the culture of&#160; neurospheres</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">Recombinant Human FGF-basic</td>
			<td style="width:199px;">PEPROTECH</td>
			<td>100-18B</td>
			<td style="width:267px;">growth factor supplement for the culture of&#160; neurospheres</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:367px;">HyClone HyQTase&#160; Cell Detachment Reagent&#160;</td>
			<td style="width:199px;">Thermo Scientific</td>
			<td style="width:119px;">SV3003001</td>
			<td style="width:267px;">for the dissociation of neurospheres</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Kimble-Chase Kontes Pellet Pestle</td>
			<td style="width:199px;">Fisher Scientific</td>
			<td>K749510-0590</td>
			<td style="width:267px;">for the dissociation of freshly dissected tumors</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;">Falcon Cell Strainers mesh size 70um</td>
			<td style="width:199px;">Fisher Scientific</td>
			<td>08-771-2</td>
			<td style="width:267px;">for generating single cell suspensions of dissociated neurospheres</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:367px;">Xylenes Histological grade</td>
			<td style="width:199px;">Fisher Scientific</td>
			<td style="width:119px;">C8H10</td>
			<td style="width:267px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">Protocol Harris Hematoxylin Mercury free ( acidified)</td>
			<td style="width:199px;">Fisher Scientific</td>
			<td style="width:119px;">245-678</td>
			<td style="width:267px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:367px;">Protocol Eosyn Y Solution ( intensified)</td>
			<td style="width:199px;">Fisher Scientific</td>
			<td style="width:119px;">314-631</td>
			<td style="width:267px;"></td>
		</tr>
	</tbody>
</table>

transposon mediated integration of plasmid dna into the subventricular zone of neonatal mice to generate novel models of glioblastoma

An urgent need exists to test the contribution of new genes to the pathogenesis and progression of human glioblastomas (GBM), the most common primary brain tumor in adults with dismal prognosis. New potential therapies are rapidly emerging from the bench and require systematic testing in experimental models which closely reproduce the salient features of the human disease. Herein we describe in detail a method to induce new models of GBM with transposon-mediated integration of plasmid DNA into cells of the subventricular zone of neonatal mice. We present a simple way to clone new transposons amenable for genomic integration using the Sleeping Beauty transposon system and illustrate how to monitor plasmid uptake and disease progression using bioluminescence, histology and immuno-histochemistry. We also describe a method to create new primary GBM cell lines. Ideally, this report will allow further dissemination of the Sleeping Beauty transposon system among brain tumor researchers, leading to an in depth understanding of GBM pathogenesis and progression and to the timely design and testing of effective therapies for patients.

为了表征的SB引起的胶质母细胞瘤的组织病理学特征，C57 / BL6新生小鼠注射在P1与质粒编码荧光素酶（pT2期/ SB100x吕克）联合的质粒编码座子用致癌基因DNA， 也就是说 ，NRAS（PT / CAGGS -NRASV12）和SV40 LGT（PT / CMVSV40-LGT）（ 图3c）或质粒编码的短发夹的p53与PDGFβ和GFP报告（pT2shp53 / GFP4 /mPDGFβ）结合NRAS（ 图3d）。对于生物发光一天注射（ 图2a）后的动物进行?...

Watch this Scientific Journal Video about Transposon Mediated Integration of Plasmid DNA into the Subventricular Zone of Neonatal Mice to Generate Novel Models of Glioblastoma at JoVE.com

Transposon Mediated Integration of Plasmid DNA into the Subventricular Zone of Neonatal Mice to Generate Novel Models of Glioblastoma

Here we describe an efficient and versatile protocol to induce, monitor and analyze novel glioblastomas (GBM) using transposon DNA injected into the ventricles of neonatal mice. Cells of the subventricular zone, which take up the plasmid, transform, proliferate and generate tumors with histo-pathological characteristics of human GBM.

质粒DNA转座子介导整合到新生小鼠的脑室下区，以生成胶质母细胞瘤的新模型

An urgent need exists to test the contribution of new genes to the pathogenesis and progression of human glioblastomas (GBM), the most common primary ...

Research

JoVE Journal

Medicine

Use of Animal Model of Sepsis to Evaluate Novel Herbal Therapies

脓毒症动物模型，用于评估新型草药疗法

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection. It has been routinely simulated in animals by several ...

科研

医学

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

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

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 ...

Image-guided Convection-enhanced Delivery into Agarose Gel Models of the Brain

图像引导的对流增强交付到大脑的琼脂糖凝胶模型

Convection-enhanced delivery (CED) has been proposed as a treatment option for a wide range of neurological diseases. Neuroinfusion catheter CED allows ...

Orthotopic Injection of Breast Cancer Cells into the Mammary Fat Pad of Mice to Study Tumor Growth.

注射原位乳腺癌细胞到小鼠乳腺脂肪垫来研究肿瘤的生长。

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the ...

Mouse Kidney Transplantation: Models of Allograft Rejection

鼠肾移植：移植排斥反应模型

Rejection of the transplanted kidney in humans is still a major cause of morbidity and mortality. The mouse model of renal transplantation closely ...

A Piglet Model of Neonatal Hypoxic-Ischemic Encephalopathy

新生儿缺氧缺血性脑病的小猪模型

Birth asphyxia, which causes hypoxic-ischemic encephalopathy (HIE), accounts for 0.66 million deaths worldwide each year, about a quarter of the ...

Surgical Models of Gastroesophageal Reflux with Mice

胃食管反流与小鼠手术模型

Multiple surgical procedures have been reported to induce gastroesophageal reflux in animals. Herein, we report three surgical models with mice aiming to ...

Myocardial Infarction in Neonatal Mice, A Model of Cardiac Regeneration

心肌梗死新生小鼠，心肌再生的模型

Myocardial infarction induced by coronary artery ligation has been used in many animal models as a tool to study the mechanisms of cardiac repair and ...

Quantitative Immunohistochemistry of the Cellular Microenvironment in Patient Glioblastoma Resections

患者胶质母细胞瘤切除中细胞微环境的定量免疫组织化学

With the growing interest in the tumor microenvironment, we set out to develop a method to specifically determine the microenvironment components within ...

In Vivo Photolabeling of Cells in the Colon to Assess Migratory Potential of Hematopoietic Cells in Neonatal Mice

In Vivo Photolabeling of Cells in the Colon to Assess Migratory Potential of Hematopoietic Cells in Neonatal Mice

体内结肠细胞 Photolabeling 对新生小鼠造血细胞迁移潜能的评价

Enteric bacterial communities are established early in life and influence immune cell development and function. The neonatal microbiota is susceptible to ...