Name: specific marking of hiv-1 positive cells using a rev-dependent lentiviral vector expressing the green fluorescent protein
Uploaded: 2010-09-23T18:30:00
Duration: 9 min 53 s
Description: Watch this Scientific Journal Video about Specific Marking of HIV-1 Positive Cells using a Rev-dependent Lentiviral Vector Expressing the Green Fluorescent Protein at JoVE.com

这项工作是支持部分由公共卫生服务授予1R01AI081568从NIAID的YW M.罗森和第2天公司举办的2009年NYCDC艾滋病骑的捐助者和骑手的慷慨捐赠

1。冯依赖的慢病毒颗粒的生产质粒转染设置：依赖REV - GFP慢病毒载体，PNL - GFP - RRE - SA，描述以前4,5,6。要组装成病毒颗粒，质粒是contransfected到HEK293 T细胞的HIV - 1包装构造，pCMVΔ8.2（请Dider Trono博士提供），质粒携带的VSV - G糖蛋白（pHCMV - G） 。使用磷酸钙法转染进行了。 缓冲区的制备：10 ×哈佛商学院（HEPES缓冲液）溶解5克HEPES，8克氯化钠0.37克氯化钾，1克葡萄糖，0.103克的Na 2 HPO 4（无水）在100毫升H 2 O。分装the10 X哈佛商学院的缓冲区为1毫升分装和储存在-20 ° C在转的时间，转换成10 ×哈佛商学院2个哈佛商学院与H 2 O（1：5稀释），并调整pH值至7.05 - 7.12 。 （10毫升2 ×哈佛商学院，它通常需要大约50μL1M氢氧化钠）。通过传递通过0.22μm的微孔过滤器，过滤消毒2X哈佛商学院的缓冲区。 氯化钙缓冲液是由溶解在10 mM的HEPES 氯化钙2至终浓度为2M 。调节pH值至5.8，过滤消毒的缓冲区，并储存于4 ° C。 程序： <ol><li>转染前一天，种子2 × 10 6 HEK293T细胞在100毫米的Petri盘，增长37细胞在10毫升的DMEM + 10％FBS的隔夜℃，5％的CO 2。 </li><li>第二天，取出细胞培养上清和取代5 mL新鲜的DMEM + 10％FBS，连续4小时的细胞培养。 </li><li>在一个5毫升的聚苯乙烯管，加480μL的2倍哈佛商学院的缓冲区。 </li><li>在第二个5毫升的聚苯乙烯管，添加60μL的2M 氯化钙 2缓冲区，质粒DNA，并转染TE缓冲液（1毫米的Tris，25 mM的EDTA，pH值8.0），一个总体积为480μL 。对于每个基于Petri盘，质粒应在10微克的PNL - GFP - RRE - SA，pCMVΔR8.3，7.5微克和2.5毫克pHCMV - G比例增加。 </li><li>加入质粒DNA - 氯化钙混合滴加到2 x哈佛商学院管，并在室温下孵育30分钟。的罚款，沉淀就会形成。 </li><li>吸管滴加细胞的混合物中加入960微升。在37℃，5％的CO 2过夜孵育。 </li><li>第二天，去除上清液，并添加10毫升新鲜的DMEM + 10％FBS，并不断孵育的细胞在37℃，5％的CO 2一夜之间。 </li><li>在转染后48小时，收获的病毒去除上清，转移到50 mL无菌管。添加到每个培养皿菜新鲜10毫升新鲜的DMEM + 10％FBS，继续培养过夜。病毒上清保存在4 ° C。 </li><li>继续在72小时后转染收获，收集上清。合并所有上清，离心上清在500 XG 15分钟以沉淀，去除细胞碎片。 </li><li>上清通过0.22μm的微孔过滤器收集和筛选。该病毒是通过体积排阻柱进一步集中。 </li></ol> 2。浓缩病毒颗粒，并确定病毒滴度<ol><li>病毒颗粒集中由一个体积排阻柱（100000兆瓦切断，Centricoin）。病毒上清装入列，在4 ° C 15分钟，并在6000 XG离心。 </li><li>浓缩病毒上清，收集，分装，并保存于-80 ° C。 </li><li>由TNF -α治疗的HIV - 1阳性外套细胞株，J1.1 7感染病毒滴度测定。在2.5 × 10 5细胞每毫升（100毫升的总量），细胞培养在96孔板和100毫升为一个星期连续稀释VNL - GFP的RRE - SA的感染。估计粒子的滴度（TCID50）以下的里德和Muench 8方法计数GFP阳性井。 </li></ol> 3。标记HIV - 1阳性细胞的慢病毒颗粒，VNL - GFP - RRE - SA 设置：一个人的CD4 T细胞系，CEM - SS的，是先用HIV - 1感染。在感染后48小时，细胞的慢病毒颗粒，VNL - GFP - RRE - SA superinfected。另外两天的二重感染，GFP阳性细胞流式细胞仪分析。作为对照，HIV - 1未受感染的CEM - SS的细胞是相同的感染VNL - GFP - RRE - SA。 HIV - 1感染的CEM - SS的细胞，只有产生了GFP阳性细胞，但不是HIV - 1的未受感染的CEM - SS的细胞。 程序： <ol><li>感染前的两个小时，加入终浓度为4毫克/毫升的polybrane。计数细胞，并采取2 × 10 5每个感染的细胞。感染200毫微克的HIV - 1的NL4 - 3（P24）2小时细胞。 </li><li>洗涤离心10分钟，重新悬浮细胞在300 XG细胞为2 × 10 5细胞/毫升，和文化在37 ° C 48小时。 </li><li>计数细胞，并采取2 × 10 5％感染的细胞与VNL - GFP - RRE - SA。添加100毫升VNL - GFP - RRE - SA的培养，在37 ° C过夜。洗涤细胞，重悬成2 × 10 5细胞/ mL。 </li><li>执行48小时后二重感染流式细胞仪分析。感染的细胞颗粒和成500毫升1％paraformaldehede室温孵育20分钟，悬浮，然后在FACScan分析仪分析（流式细胞迪肯森）。 </li></ol> 4。代表性的成果如果实验成功地进行，一个庞大的GFP人口将流式细胞仪检测出HIV - 1感染细胞CEM - SS的，在控制，而GFP阳性细胞不会被检测到HIV - 1的未受感染的CEM - SS细胞。 如果实验成功执行，冯依赖的慢病毒载体，VNL - GFP - RRE - SA，将允许在活细胞数量的艾滋病毒复制非常具体的检测，测量通过绿色荧光蛋白（GFP）的表达。在这个例子中，收获CEM - SS的细胞染色的PE标记的大鼠单克隆抗体对CD24的鼠标，HSA的，然后在HSA和绿色荧光蛋白表达的流式细胞仪分析。 <img src="/files/ftp_upload/2198/2198fig1.jpg" alt="图1" /> 图1。如下所示，一个庞大的GFP人口VNL - GFP - RRE - SA（图1c）superinfected的HIV - 1感染细胞中检测到。与此相反，没有检测到GFP阳性细胞为HIV - 1的未受感染的CEM - SS的细胞中没有慢病毒重叠（图1a）或HIV - 1慢病毒重叠（图1b）未受感染的细胞。

<ol>
	<li>Garcia, J. A., Wu, F. K., Mitsuyasu, R., Gaynor, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+of+cellular+proteins+involved+in+the+transcriptional+regulation+of+the+human+immunodeficiency+virus.">Interactions of cellular proteins involved in the transcriptional regulation of the human immunodeficiency virus.</a> EMBO Journal. 6, 3761-3770 (1987).</li><li>Merzouki, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIV-1+gp120/160+expressing+cells+upregulate+HIV-1+LTR+directed+gene+expression+in+a+cell+line+transfected+with+HIV-1+LTR-reporter+gene+constructs.">HIV-1 gp120/160 expressing cells upregulate HIV-1 LTR directed gene expression in a cell line transfected with HIV-1 LTR-reporter gene constructs.</a> Cell Mol Biol (Noisy-le-grand. 41, 445-452 (1995).</li><li>Aguilar-Cordova, E., Chinen, J., Donehower, L., Lewis, D. E., Belmont, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+sensitive+reporter+cell+line+for+HIV-1+tat+activity,+HIV-1+inhibitors,+and+T+cell+activation+effects.">A sensitive reporter cell line for HIV-1 tat activity, HIV-1 inhibitors, and T cell activation effects.</a> AIDS Res Hum Retroviruses. 10, 295-301 (1994).</li><li>Wu, Y., Beddall, M. H., Marsh, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rev-dependent+lentiviral+expression+vector.">Rev-dependent lentiviral expression vector.</a> Retrovirology. 4, 12-12 (2007).</li><li>Wu, Y., Beddall, M. H., Marsh, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rev-dependent+indicator+T+cell+line.">Rev-dependent indicator T cell line.</a> Current HIV Research. 5, 395-403 (2007).</li><li>Young, J., Tang, Z., Yu, Q., Yu, D., Wu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+killing+of+HIV-1-positive+macrophages+and+T+Cells+by+the+Rev-dependent+lentivirus+carrying+anthrolysin+O+from+Bacillus+anthracis.">Selective killing of HIV-1-positive macrophages and T Cells by the Rev-dependent lentivirus carrying anthrolysin O from Bacillus anthracis.</a> Retrovirology. 5, 36-36 (2008).</li><li>Perez, V. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+HIV-1-infected+T+cell+clone+defective+in+IL-2+production+and+Ca2++mobilization+after+CD3+stimulation.">An HIV-1-infected T cell clone defective in IL-2 production and Ca2+ mobilization after CD3 stimulation.</a> Journal of Immunology. 147, 3145-3148 (1991).</li><li>Reed, L. J., Muench, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+of+estimating+fifty+percent+endpoints.">A simple method of estimating fifty percent endpoints.</a> American Journal of Hygiene. 27, 493-497 (1938).</li></ol>

至于与早期开发的达依赖性表达载体，这里描述的是冯系统利用一个演变艾滋病毒过程。列入冯依赖呈现的LTR表达载体的高度依赖复制艾滋病毒的存在。这个向量的应用报道中，一个艾滋病毒相关的记者病毒，提供了全新的方式来确定艾滋病毒呈阳性的细胞。向量允许活细胞检查，可以表达任何基础或临床试验的基因，并作为一个伪类型的慢病毒已获得大多数细胞类型和组织。

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Biosafety Cabinet</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>37&deg;C 5%CO2 incubator</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Flow cytometer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>FACSCalibur</td>
<td>&nbsp;</td>
<tr>
<td>Centrifuge</td>
<td>&nbsp;</td>
<td>Beckman</td>
<td>Allega&trade;6KR</td>
<td>&nbsp;</td>
<tr>
<td>4&deg;C regrigerator</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>-20&deg;C refrigerator</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td> -80&deg;C refrigerator</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Cell counter</td>
<td>&nbsp;</td>
<td>Nexcelom</td>
<td>Cellometer&trade; AutoT4</td>
<td>&nbsp;</td>
<tr>
<td>HEK293T cell line</td>
<td>&nbsp;</td>
<td>NIH</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>CEM-SS cell line</td>
<td>&nbsp;</td>
<td>NIH</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Jurkat cell line J1.1</td>
<td>&nbsp;</td>
<td>NIH</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>DMEM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11965-118</td>
<td>&nbsp;</td>
<tr>
<td>RPMI 1640</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>21870</td>
<td>&nbsp;</td>
<tr>
<td>HI FBS</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>10438-018</td>
<td>&nbsp;</td>
<tr>
<td>HIV-1NL4-3</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>prepared by tranfsection of HeLa cells with cloned proviral DNA</td>
</tr>
<tr>
<td>pNL-GFP-RRE</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>copmlete deletion of all HIV ORFs of pNL4-3</td>
</tr>
<tr>
<td>pNL-GFP-RRE-SA</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Insert HIV-1 A5 and D4 into pNL-GFP-RRE</td>
</tr>
<tr>
<td>pCMV&Delta;8.2</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>provided by Dr. Dider Trono</td>
</tr>
<tr>
<td>pHCMV-G</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>10 x HBS (Hepes Buffered Saline)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11344-041</td>
<td>&nbsp;</td>
<tr>
<td>2M CaCl2 buffer</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>S-013-154-10</td>
<td>&nbsp;</td>
<tr>
<td>1% paraformaldehy</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>158127</td>
<td>&nbsp;</td>
<tr>
<td>Bleach</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>50ml Falcon tubes</td>
<td>&nbsp;</td>
<td>BD Bioscience</td>
<td>358206</td>
<td>&nbsp;</td>
<tr>
<td>5ml round bottom tubes</td>
<td>&nbsp;</td>
<td>BD Bioscience</td>
<td>352063</td>
<td>&nbsp;</td>
<tr>
<td>0.45&mu;M Millipore filter</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>SLHA033SS</td>
<td>&nbsp;</td>
<tr>
<td>0.20&mu;M Millipore filter</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>SLFG85000</td>
<td>&nbsp;</td>
<tr>
<td>100mm Petri-dish</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>CLS430588</td>
<td>&nbsp;</td>
<tr>
<td>Size-exclusion column (100,000MW cut off)</td>
<td>&nbsp;</td>
<td>SartoriuStedim biotech</td>
<td>VS2041</td>
<td>&nbsp;</td>
<tr>
<td>2ml frozen tube</td>
<td>&nbsp;</td>
<td>Axygen</td>
<td>SCT-200</td>
<td>&nbsp;</td>
<tr>
<td>96-well plate</td>
<td>&nbsp;</td>
<td>BD Bioscience</td>
<td>353227</td>
<td>&nbsp;</td>
<tr>
<td>1.7mL Microcentrifuge Tube</td>
<td>&nbsp;</td>
<td>Axygen</td>
<td>MCT- 175-C</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

specific marking of hiv-1 positive cells using a rev-dependent lentiviral vector expressing the green fluorescent protein

大部分的艾滋病毒抗体反应表达载体的基础上艾滋病毒的推动者，长末端重复序列（LTR）的。 LTR的响应早期艾滋病毒的蛋白质，达，也反应细胞活化状态，并整合发生的地方染色活动。这可能会导致艾滋病毒高独立的活动，并已成为制约LTR的记者有用标志艾滋病毒1,2,3阳性细胞。在这里，我们构建了一个表达的慢病毒载体，具有，除了达LTR的响应，许多艾滋病毒的DNA序列，包括冯响应元素和艾滋病毒剪接位点4,5,6。纳入一种慢病毒记者病毒载体，允许艾滋病毒的复制非常具体的检测活细胞群。测定绿色荧光蛋白（GFP）的表达载体的活动。这个向量的应用，这里提供了一个新颖的现有方法的替代方法，如原位PCR或HIV抗原染色，以确定HIV阳性细胞。载体也可以为基础或临床试验，针对艾滋病毒抗体阳性的细胞表达的基因治疗。

Watch this Scientific Journal Video about Specific Marking of HIV-1 Positive Cells using a Rev-dependent Lentiviral Vector Expressing the Green Fluorescent Protein at JoVE.com

Specific Marking of HIV-1 Positive Cells using a Rev-dependent Lentiviral Vector Expressing the Green Fluorescent Protein

我们已经开发出一种慢病毒载体，拥有，除了达反应的LTR，冯应答元件（RRE）报告基因的表达在HIV - 1达和冯依赖的方式，可以调节。向量允许通过活细胞GFP表达的复制艾滋病毒的特异性检测。

HIV - 1使用转速依赖的慢病毒表达载体的绿色荧光蛋白阳性细胞的特定标记

Most of HIV-responsive expression vectors are based on the HIV promoter, the long terminal repeat (LTR). While responsive to an early HIV protein, Tat, ...

Research

JoVE Journal

Immunology and Infection

Genotypic Inference of HIV-1 Tropism Using Population-based Sequencing of V3

V3的HIV - 1取向的基因型推理使用人口为基础的测序

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her ...

科研

免疫与感染

Prediction of HIV-1 Coreceptor Usage (Tropism) by Sequence Analysis using a Genotypic Approach

Prediction of HIV-1 Coreceptor Usage Tropism by Sequence Analysis using a Genotypic Approach

使用基因型的方法预测HIV-1辅助用途（归经）的序列分析

Maraviroc (MVC) is the first licensed antiretroviral drug from the class of coreceptor antagonists. It binds to the host coreceptor CCR5, which is used by ...

In vitro Uncoating of HIV-1 Cores

In vitro Uncoating of HIV-1 Cores

在体外对HIV - 1核心脱壳

The genome of the retroviruses is encased in a capsid surrounded by a lipid envelope. For lentiviruses, such as HIV-1, the conical capsid shell is ...

New Tools to Expand Regulatory T Cells from HIV-1-infected Individuals

新工具展开调节性T细胞的HIV-1感染的个体

CD4+ Regulatory T cells (Tregs) are potent immune modulators and serve an important function in human immune homeostasis. Depletion of Tregs has led to ...

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

评估的HIV-1感染的CD4的先天传感<sup&gt; +</sup&gt;性T细胞浆细胞样树突细胞使用<em&gt;体外</em&gt;共培养体系。

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

Conformational Evaluation of HIV-1 Trimeric Envelope Glycoproteins Using a Cell-based ELISA Assay

HIV-1的三聚体包膜糖蛋白的构象评估使用基于细胞的ELISA检测

HIV-1 envelope glycoproteins (Env) mediate viral entry into target cells and are essential to the infectious cycle. Understanding how those glycoproteins ...

Isolation of Exosomes from the Plasma of HIV-1 Positive Individuals

从HIV-1阳性者的血浆分离外来体的

Exosomes are small vesicles ranging in size from 30 nm to 100 nm that are released both constitutively and upon stimulation from a variety of cell types. ...

SILAC Based Proteomic Characterization of Exosomes from HIV-1 Infected Cells

从HIV-1感染细胞的外来体基于SILAC蛋白质组学表征

Proteomics is the large-scale analysis of proteins. Proteomic techniques, such as liquid chromatography tandem mass spectroscopy (LC-MS/MS), can ...

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

葡聚糖增强小鼠和人原发性 NK 细胞的慢转导效率

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...

Induction of Paralysis and Visual System Injury in Mice by T Cells Specific for Neuromyelitis Optica Autoantigen Aquaporin-4

诱导麻痹及视觉系统损伤小鼠 T 细胞特异的视神经脊髓炎抗原水通道蛋白-4

While it is recognized that aquaporin-4 (AQP4)-specific T cells and antibodies participate in the pathogenesis of neuromyelitis optica (NMO), a human ...