Name: development of cell-type specific anti-hiv gp120 aptamers for sirna delivery
Uploaded: 2011-06-23T12:00:00
Description: Watch this Scientific Journal Video about Development of Cell-type specific anti-HIV gp120 aptamers for siRNA delivery at JoVE.com

我们感谢布丽塔赫恩，孙桂花，哈里斯Soifer有益的讨论和莉萨舍雷尔。这项工作是支持通过美国国立卫生研究院艾滋病研究和参考试剂计划，艾滋病司，NIAID的，美国国立卫生研究院从国立卫生AI29329，授予JJR下列试剂HL07470研究院获得资助的CHO - EE和CHO - gp160细胞行; pNL4 - 3吕克载体; gp120的HIV - 1 BAL DAIDS，NIAID的。

1。制备的RNA库<ol><li>起始的DNA库中包含50个核苷酸的随机序列，并结合DNA技术（Coralville，衣阿华）合成。单链DNA寡核苷酸库序列为5&#39; - GGG AGG的ACG ATG CGG公司- N 50 - CAG的ACG ACT的CGC CCG一个- 3“（81新台币）。随机区域的两侧是由不断的地区， 其中包括在体外转录的T7启动子和3&#39;标签用于RT - PCR。 5&#39;和3&#39;恒定序列5&#39; - TAA交咨会GAC TCA CTA的豪GGA GGA CGA TGC GG - 3“（32 MER）和5&#39;?...

<ol>
	<li>Tuerk, C., Gold, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+evolution+of+ligands+by+exponential+enrichment:+RNA+ligands+to+bacteriophage+T4+DNA+polymerase.">Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase.</a> Science. 249, 505-510 (1990).</li><li>Ellington, A. D., Szostak, 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=In+vitro+selection+of+RNA+molecules+that+bind+specific+ligands.">In vitro selection of RNA molecules that bind specific ligands.</a> Nature. 346, 818-822 (1990).</li><li>Robertson, D. L., Joyce, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selection+in+vitro+of+an+RNA+enzyme+that+specifically+cleaves+single-stranded+DNA.">Selection in vitro of an RNA enzyme that specifically cleaves single-stranded DNA.</a> Nature. 344, 467-468 (1990).</li><li>Fitzwater, T., Polisky, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+SELEX+primer.">A SELEX primer.</a> Methods Enzymol. 267, 275-301 (1996).</li><li>Weiss, C. D., White, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+stable+Chinese+hamster+ovary+cells+expressing+wild-type,+secreted,+and+glycosylphosphatidylinositol-anchored+human+immunodeficiency+virus+type+1+envelope+glycoprotein.">Characterization of stable Chinese hamster ovary cells expressing wild-type, secreted, and glycosylphosphatidylinositol-anchored human immunodeficiency virus type 1 envelope glycoprotein.</a> J Virol. 67, 7060-706 (1993).</li><li>Vodicka, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Indicator+cell+lines+for+detection+of+primary+strains+of+human+and+simian+immunodeficiency+viruses.">Indicator cell lines for detection of primary strains of human and simian immunodeficiency viruses.</a> Virology. 233, 193-198> (1997).</li><li>Zhou, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selection,+characterization+and+application+of+new+RNA+HIV+gp+120+aptamers+for+facile+delivery+of+Dicer+substrate+siRNAs+into+HIV+infected+cells.">Selection, characterization and application of new RNA HIV gp 120 aptamers for facile delivery of Dicer substrate siRNAs into HIV infected cells.</a> Nucleic Acids Res. , (2009).</li><li>Mayer, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chemical+biology+of+aptamers.">The chemical biology of aptamers.</a> Angew Chem Int Ed Engl. 48, 2672-2689 (2009).</li><li>Famulok, M., Hartig, J. S., Mayer, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+aptamers+and+aptazymes+in+biotechnology,+diagnostics,+and+therapy.">Functional aptamers and aptazymes in biotechnology, diagnostics, and therapy.</a> Chem Rev. 107, 3715-3743 (2007).</li><li>Chu, T. C., Twu, K. Y., Ellington, A. D., Levy, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aptamer+mediated+siRNA+delivery.">Aptamer mediated siRNA delivery.</a> Nucleic Acids Res. 34, e73-e73 (2006).</li><li>McNamara, J. O., 2nd, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+type-specific+delivery+of+siRNAs+with+aptamer-siRNA+chimeras.">Cell type-specific delivery of siRNAs with aptamer-siRNA chimeras.</a> Nat Biotechnol. 24, 1005-1015 (2006).</li><li>Dassie, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systemic+administration+of+optimized+aptamer-siRNA+chimeras+promotes+regression+of+PSMA-expressing+tumors.">Systemic administration of optimized aptamer-siRNA chimeras promotes regression of PSMA-expressing tumors.</a> Nat Biotechnol. 27, 839-849 (2009).</li><li>Zhou, J., Rossi, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+therapeutic+potential+of+cell-internalizing+aptamers.">The therapeutic potential of cell-internalizing aptamers.</a> Curr Top Med Chem. 9, 1144-1157 (2009).</li><li>Zhou, J., Li, H., Li, S., Zaia, J., Rossi, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+dual+inhibitory+function+aptamer-siRNA+delivery+system+for+HIV-1+therapy.">Novel dual inhibitory function aptamer-siRNA delivery system for HIV-1 therapy.</a> Mol Ther. 16, 1481-1489 (2008).</li></ol>

适配是在体外进化承担具体的和稳定的三维形状的核酸，从而提供了非常具体的，紧密结合到目标分子 8 。低纳摩尔结合的亲和力和精致的特异性适配，他们的目标，使他们的多用途工具用于诊断，在体内成像和治疗9。随着适体进行有针对性的siRNA传递技术的到来，现在是可行的，使用的适体为受体介导的内吞作用的 siRNA 10-12的绑定功能。对细胞膜受体提出?...

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th><th>评论（可选） </th></tr><tr><td> MF微孔膜过滤器</td><td> Millipore公司</td><td> HAWP01300 </td><td>孔径0.45微米</td></tr><tr><td> Swinnex过滤持有人</td><td> Millipore公司</td><td> SX0001300 </td><td>直径13毫米</td></tr><tr><td> QIAquick凝胶提取试剂盒</td><td> QIAGEN公司</td><td> 28706 </td><td> DNA纯化</td></tr><tr><td>单片机YM - 30列</td><td> Millipore公司</td><td> 42410 </td><td> RNA浓度</td></tr><tr><td>生物旋转30列</td><td> Bio - Rad公司</td><td> 732-6250 </td><td> RNA纯化</td></tr><tr><td> Taq酶PCR扩增DNA聚合酶</td><td> Sigma - Aldrich公司</td><td> D1806 </td><td></td></tr><tr><td> ThermoScript RT - PCR检测系统</td><td> Invitrogen公司</td><td> 11146-024 </td><td></td></tr><tr><td> DuraScribe T7转录试剂盒</td><td>震中</td><td> DS010925 </td><td></td></tr><tr><td>的dNTP进行PCR </td><td>罗氏公司</td><td> 1 581 295 </td><td></td></tr><tr><td>核糖核酸，转移，从大肠杆菌</td><td> Sigma - Aldrich公司</td><td> R1753 </td><td> tRNA的竞争对手</td></tr><tr><td> HIV - 1 八- L的 gp120的蛋白质</td><td>艾滋病研究和参考试剂计划</td><td> 4961 </td><td>靶蛋白</td></tr><tr><td>消声器的siRNA标记试剂盒 - Cy3标记</td><td> Ambion公司</td><td> 1632 </td><td></td></tr><tr><td>酚/氯仿5 / 1的溶液（pH值4.5） </td><td> Ambion公司</td><td> AM9720 </td><td></td></tr><tr><td>氯仿/异丙醇24 / 1解决方案</td><td>西格玛</td><td> C0549 </td><td></td></tr><tr><td>小牛肠碱性磷酸酶（CIP） </td><td>新英格兰Biolab公司</td><td> M0290L </td><td></td></tr><tr><td> T4多聚核苷酸激酶</td><td>新英格兰Biolab公司</td><td> M0201L </td><td></td></tr><tr><td>糖原</td><td>罗氏公司</td><td> 10 901 393 001 </td><td> RNA沉淀</td></tr><tr><td> γ- P 32 + - ATP </td><td> MP生物医学</td><td> 013502002 </td><td> Radiactivity </td></tr><tr><td> 40％AccuGel 19:1 </td><td>全国诊断</td><td> EC - 850 </td><td></td></tr><tr><td> 10xTBE </td><td>全国诊断</td><td> EC - 860 </td><td></td></tr><tr><td> N，N，N&#39; -四甲基乙二胺（TMEMD） </td><td> Sigma - Aldrich公司</td><td> T9281 </td><td></td></tr><tr><td>过硫酸铵（APS） </td><td> Sigma - Aldrich公司</td><td> A3678 </td><td></td></tr><tr><td> L -蛋氨酸亚砜</td><td> Sigma - Aldrich公司</td><td> M5379 - 250毫克</td><td></td></tr><tr><td> RPMI媒体1640 </td><td> Invitrogen公司</td><td> 11835-030 </td><td></td></tr><tr><td>碳酸氢钠溶液，7.5％W / V </td><td> Invitrogen公司</td><td> 25080-094 </td><td></td></tr><tr><td>最低限度的基本培养基（MEM）（10） </td><td> Invitrogen公司</td><td> 11430-030 </td><td></td></tr><tr><td> MEM非必需氨基酸（100） </td><td> Invitrogen公司</td><td> 11140-050 </td><td></td></tr><tr><td> TA克隆试剂盒与PCR 2.1 </td><td> Invitrogen公司</td><td> K2040 - 01 </td><td></td></tr></tbody></table>

development of cell-type specific anti-hiv gp120 aptamers for sirna delivery

全球艾滋病毒感染疫情已创建了一个新类抗逆转录病毒药物的迫切需要。很强的能力，抑制小分子干扰（SI）的RNA互补的RNA转录表达是被人利用作为一个新类的疗法，为各种疾病，包括艾滋病毒。许多以前的报告表明，新型RNAi为基础的治疗策略anti-HIV/AIDS有相当大的承诺，然而，成功的siRNAs的临床治疗中的应用和翻译的一个主要障碍是有效地提供。特别是，考虑RNAi为基础的疗法的安全性和有效性，它是非常可取的，制定有针对性的细胞内的siRNA传递到特定的细胞群体或组织的方法。 HIV - 1的gp120的蛋白质，对HIV - 1病毒表面的糖蛋白的信封，到病毒进入CD4细胞起着重要作用。已通过验证的触发HIV - 1项，并启动细胞融合的gp120与CD4的相互作用作为临床相关的抗病毒的药物发现策略。 在此，我们首先讨论2&#39; - F改性抗- HIV的gp120基因的RNA适配子的选择和鉴定。使用传统的硝化纤维过滤器SELEX方法，几个新的适配纳摩尔亲和力，从50个随机NT RNA文库中分离。为了成功地获得绑定的物种具有较高的亲和力，选择紧缩小心控制调整的条件。选定适配特异性结合，并迅速进入细胞表达的HIV - 1包膜蛋白的内化。此外，单独的适配，可中和HIV - 1病毒的传染性。根据最好的适体A - 1中，我们还创建了一种新型的双抑菌功能抗- gp120的适配子的siRNA的嵌合体适体和siRNA的部分具有强大的抗艾滋病毒的活动。此外，我们利用gp120的适体的siRNA嵌合体细胞类型特定的siRNA传递到HIV - 1感染细胞。这种双重功能的嵌合体，显示了相当大的潜力相结合的各种核酸治疗剂（适体和siRNA）抑制HIV - 1感染，使适配子的siRNA嵌合体，没有高活性抗逆转录病毒疗法（HAART）的患者有吸引力的治疗候选人。

Watch this Scientific Journal Video about Development of Cell-type specific anti-HIV gp120 aptamers for siRNA delivery at JoVE.com

Development of Cell-type specific anti-HIV gp120 aptamers for siRNA delivery

从RNA库是孤立的几个2&#39; -氟RNA对HIV - 1BA - L的gp120的nanomole亲和力的适配<em&gt;在体外</em&gt; SELEX程序。创建一种新的双抑菌功能抗- gp120的适配子的siRNA的嵌合体，显示了相当的全身抗艾滋病毒治疗的承诺。

发展的细胞类型特异性的抗艾滋病毒gp120基因的siRNA传递适配

The global epidemic of infection by HIV has created an urgent need for new classes of antiretroviral agents. The potent ability of small interfering ...

Research

JoVE Journal

Immunology and Infection

Development of a Negative Selectable Marker for Entamoeba histolytica

Development of a Negative Selectable Marker for Entamoeba histolytica

一个负选择标记的发展阿米巴</em

Entamoeba histolytica is the causative agent of amebiasis and infects up to 10% of the world's population. The molecular techniques that have enabled the ...

科研

免疫与感染

Peptide:MHC Tetramer-based Enrichment of Epitope-specific T cells

肽：MHC四聚体为基础的抗原表位特异性的T细胞的富集

A  basic necessity for researchers studying adaptive immunity with in vivo experimental models is an  ability to identify T cells based on their T cell ...

Using Click Chemistry to Measure the Effect of Viral Infection on Host-Cell RNA Synthesis

使用点击化学测量的影响，病毒感染宿主细胞RNA合成

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, ...

Development of an IFN-&#947; ELISpot Assay to Assess Varicella-Zoster Virus-specific Cell-mediated Immunity Following Umbilical Cord Blood Transplantation

Development of an IFN-ÃƒÅ½Ã‚Â³ ELISpot Assay to Assess Varicella-Zoster Virus-specific Cell-mediated Immunity Following Umbilical Cord Blood Transplantation

的IFN-γ酶联免疫斑点检测，以评估水痘 - 带状疱疹病毒特异性细胞免疫继脐血移植的发展

Varicella zoster virus (VZV) is a significant cause of morbidity and mortality following umbilical cord blood transplantation (UCBT). For this reason, ...

The Mesenteric Lymph Duct Cannulated Rat Model: Application to the Assessment of Intestinal Lymphatic Drug Transport

在肠系膜淋巴管空心​​大鼠模型：应用肠淋巴药物运输的评估

The intestinal lymphatic system plays key roles in fluid transport, lipid absorption and immune function. Lymph flows directly from the small intestine ...

Applying Fluorescence Resonance Energy Transfer (FRET) to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing

Applying Fluorescence Resonance Energy Transfer FRET to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing

应用荧光共振能量转移（FRET）审查效应转运效率由<em&gt;贝氏柯克斯体</em&gt; siRNA沉默中

Coxiella burnetii, the causative agent of Q fever, is an intracellular pathogen that relies on a Type IV Dot/Icm Secretion System to establish a ...

Development of a More Sensitive and Specific Chromogenic Agar Medium for the Detection of Vibrio parahaemolyticus and Other Vibrio Species

Development of a More Sensitive and Specific Chromogenic Agar Medium for the Detection of Vibrio parahaemolyticus and Other Vibrio Species

对于检测到的敏感性和特异性显色琼脂培养基发展<em&gt;副溶血性弧菌</em&gt;及其他<em&gt;弧菌</em&gt;物种

Foodborne infections in the US caused by Vibrio species have shown an upward trend. In the genus Vibrio, V. parahaemolyticus is responsible for the ...

Development of Stem Cell-derived Antigen-specific Regulatory T Cells Against Autoimmunity

干细胞衍生的抗原特异性调节性T细胞对自身免疫的发展

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic ...

Development of an Economical DNA Delivery System by "Acufection" and its Application to Skin Research

通过“Acufection”和它的皮肤研究中的应用的经济型DNA递送系统的开发

Dysregulation of immune response in skin is associated with numerous human skin disorders. Direct transfer of immune-related genes into skin tissue is a ...

Zika Virus Specific Diagnostic Epitope Discovery

Zika 病毒特定诊断表位发现

High-density peptide microarrays allow screening of more than six thousand peptides on a single standard microscopy slide. This method can be applied for ...