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。

<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>

J. R和JZ有"特定细胞类型的适配子的siRNA的HIV - 1治疗传递系统"的未决的专利。美国专利商标局，号<strong&gt;328994分之12</strong&gt;，出版日期:2009年6月11日，日。

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。对细胞膜受体提出的适配已成为有前途的运载工具的目标鲜明的疾病或组织在一个细胞型13特有的方式，它可以提高疗效，以及减少不必要的药物的毒副作用。 到目前为止，针对特定类型的细胞或恶性细胞亚群已被成功分离，并通过使用传统的纯化膜蛋白质为基础的SELEX或完整的细胞为基础的SELEX过程的特点适配的越来越多。高效生成细胞特异性寻药的新适配仍是一个关键的挑战，表演时由于无法获得所需的受体物种，不稳定的原生和有问题的生化或物理性质，化学性质的重组蛋白的选择。因此，基于单元的SELEX提供了一个替代的一代，可以结合专为一个特定的靶细胞人口的适配。然而，它注意到死细胞产生的非特异性结合核酸，因此导致细胞SELEX失败。此外，与传统的分离纯化蛋白质为基础的SELEX，复杂性和细胞的多样性相反的表面也增加细胞SELEX实验过程的困难。到目前为止，大多数靶向给药的细胞特异性的适配已演变用纯化的蛋白为基础的SELEX方法。 硝酸纤维素膜为基础的SELEX适配隔离，那些传统的战略，是行之有效的，并在许多出版物中。在这里，我们采用此过程中，成功地分离出几个新的抗HIV gp120基因的RNA适配子，这是能具体结合gp120的细胞表达HIV包膜内在。作为这项技术的优点之一，它是没有必要的修改目标分子（例如:蛋白质），并固定在固相吸附剂（如:珠，树脂柱，板或芯片）的目标，这没有按的"影响原来目标的确认。它更可靠，更简单的选择。为了丰富与高亲和力结合的RNA种类，选择紧缩小心控制，调整的条件，如选择的缓冲系统，靶蛋白的浓度，竞争对手的tRNA和洗涤时间8。在选拔过程中，结合亲和力监测，以确保浓缩和选择的进展，从而微调在下一轮的选择条件。这些适配子，可以抑制几种不同的HIV - 1毒株，表明阻断gp120与CD4受体结合，抑制HIV - 1项的初始步骤，抗- gp120的适配可能在HIV - 1治疗中的应用非常有用。我们还利用提供抗艾滋病毒的siRNAs的净结果为艾滋病毒感染的细胞，复制和艾滋病毒的蔓延是强烈的适体的联合行动和针对达/转的siRNA抑制gp120的适体的一个精致的特异性常见的外显子的HIV - 1 7，14。因此，抗- gp120的适配功能抗病毒药物和siRNA的传递试剂。

<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 ...

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

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

19.4K 次观看.  Beckman Research Institute of City of Hope. 从RNA库是孤立的几个2' -氟RNA对HIV - 1BA - L的gp120的nanomole亲和力的适配在体外 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。

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

Development of a Negative Selectable Marker for Entamoeba histolytica

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 up- and down-regulation of gene expression rely on the transfection of stably maintained plasmids. While these have increased our understanding of Entamoeba virulence factors, the capacity to integrate exogenous DNA into genome, which would allow reverse genetics experiments, would be a significant advantage in the study of this parasite. The challenges presented by this organism include inability to select for homologous recombination events and difficulty to cure episomal plasmid DNA from transfected trophozoites. The later results in a high background of exogenous DNA, a major problem in the identification of trophozoites in which a bona fide genomic integration event has occurred. We report the development of a negative selection system based upon transgenic expression of a yeast cytosine deaminase and uracil phosphoribosyl transferase chimera (FCU1) and selection with prodrug 5-fluorocytosine (5-FC). The FCU1 enzyme converts non-toxic 5-FC into toxic 5-fluorouracil and 5-fluorouridine-5'-monophosphate. E. histolytica lines expressing FCU1 were found to be 30 fold more sensitive to the prodrug compared to the control strain.

Development of a Negative Selectable Marker for Entamoeba histolytica

We report development of a negative selection system in E. histolytica based upon transgenic expression of a chimeric protein (FCU1) and selection with the prodrug 5-fluorocytosine. The FCU1 protein is a fusion of yeast cytosine deaminase and uracil phosphoribosyltransferase. Expression of FCU1 resulted in increased E. histolytica sensitivity towards 5-fluorocytosine.

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

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 antigen receptor (TCR) specificity. Many indirect methods are available in which a bulk population of T cells is stimulated in vitro with a specific antigen and epitope-specific T cells are identified through the measurement of a functional response such as proliferation, cytokine production, or expression of activation markers1. However, these methods only identify epitope-specific T cells exhibiting one of many possible functions, and they are not sensitive enough to detect epitope-specific T cells at naive precursor frequencies. A popular alternative is the TCR transgenic adoptive transfer model, in which monoclonal T cells from a TCR transgenic mouse are seeded into histocompatible hosts to create a large precursor population of epitope-specific T cells that can be easily tracked with the use of a congenic marker antibody2,3. While powerful, this method suffers from experimental artifacts associated with the unphysiological frequency of T cells with specificity for a single epitope4,5. Moreover, this system cannot be used to investigate the functional heterogeneity of epitope-specific T cell clones within a polyclonal population.
	The ideal way to study adaptive immunity should involve the direct detection of epitope-specific T cells from the endogenous T cell repertoire using a method that distinguishes TCR specificity solely by its binding to cognate peptide:MHC (pMHC) complexes. The use of pMHC tetramers and flow cytometry accomplishes this6, but is limited to the detection of high frequency populations of epitope-specific T cells only found following antigen-induced clonal expansion. In this protocol, we describe a method that coordinates the use of pMHC tetramers and magnetic cell enrichment technology to enable detection of extremely low frequency epitope-specific T cells from mouse lymphoid tissues3,7. With this technique, one can comprehensively track entire epitope-specific populations of endogenous T cells in mice at all stages of the immune response.

This protocol describes the use of peptide:MHC tetramers and magnetic microbeads to isolate low frequency populations of epitope-specific T cells and analyze them by flow cytometry. This method enables the direct study of endogenous T cell populations of interest from in vivo experimental systems.

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

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, it is therefore important to have a quick and reliable way of measuring transcriptional activity in infected cells. Traditionally, transcription has been measured either by incorporation of radioactive nucleosides such as 3H-uridine followed by detection via autoradiography or scintillation counting, or incorporation of halogenated uridine analogs such as 5-bromouridine (BrU) followed by detection via immunostaining. The use of radioactive isotopes, however, requires specialized equipment and is not feasible in a number of laboratory settings, while the detection of BrU can be cumbersome and may suffer from low sensitivity.
The recently developed click chemistry, which involves a copper-catalyzed triazole formation from an azide and an alkyne, now provides a rapid and highly sensitive alternative to these two methods. Click chemistry is a two step process in which nascent RNA is first labeled by incorporation of the uridine analog 5-ethynyluridine (EU), followed by detection of the label with a fluorescent azide. These azides are available as several different fluorophores, allowing for a wide range of options for visualization.
This protocol describes a method to measure transcriptional suppression in cells infected with the Rift Valley fever virus (RVFV) strain MP-12 using click chemistry. Concurrently, expression of viral proteins in these cells is determined by classical intracellular immunostaining. Steps 1 through 4 detail a method to visualize transcriptional suppression via fluorescence microscopy, while steps 5 through 8 detail a method to quantify transcriptional suppression via flow cytometry. This protocol is easily adaptable for use with other viruses.

This method describes the use of click chemistry to measure changes in host cell transcription after infection with the Rift Valley fever virus (RVFV) strain MP-12. Results can be visualized qualitatively via fluorescence microscopy or obtained quantitatively through flow cytometry. This method is adaptable for use with other viruses.

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, ...

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

The intestinal lymphatic system plays key roles in fluid transport, lipid absorption and immune function. Lymph flows directly from the small intestine via a series of lymphatic vessels and nodes that converge at the superior mesenteric lymph duct. Cannulation of the mesenteric lymph duct thus enables the collection of mesenteric lymph flowing from the intestine. Mesenteric lymph consists of a cellular fraction of immune cells (99% lymphocytes), aqueous fraction (fluid, peptides and proteins such as cytokines and gut hormones) and lipoprotein fraction (lipids, lipophilic molecules and apo-proteins). The mesenteric lymph duct cannulation model can therefore be used to measure the concentration and rate of transport of a range of factors from the intestine via the lymphatic system. Changes to these factors in response to different challenges (e.g., diets, antigens, drugs) and in disease (e.g., inflammatory bowel disease, HIV, diabetes) can also be determined. An area of expanding interest is the role of lymphatic transport in the absorption of orally administered lipophilic drugs and prodrugs that associate with intestinal lipid absorption pathways. Here we describe, in detail, a mesenteric lymph duct cannulated rat model which enables evaluation of the rate and extent of lipid and drug transport via the lymphatic system for several hours following intestinal delivery. The method is easily adaptable to the measurement of other parameters in lymph. We provide detailed descriptions of the difficulties that may be encountered when establishing this complex surgical method, as well as representative data from failed and successful experiments to provide instruction on how to confirm experimental success and interpret the data obtained.

Here we describe a technique to cannulate the mesenteric lymph duct in rats which enables quantification of lipid and drug transport via the lymphatic system following intestinal delivery. The technique can be adapted to assess mesenteric lymph concentrations and/or transport of fluid, immune cells, peptides, proteins and lipophilic molecules.

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

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 replicative niche. A cohort of effectors are translocated through this system into the host cell to manipulate host processes and allow the establishment of a unique lysosome-derived vacuole for replication. The method presented here involves the combination of two well-established techniques: specific gene silencing using siRNA and measurement of effector translocation using a FRET-based substrate that relies on &#946;-lactamase activity. Applying these two approaches, we can begin to understand the role of host factors in bacterial secretion system function and effector translocation. In this study we examined the role of Rab5A and Rab7A, both important regulators of the endocytic trafficking pathway. We demonstrate that silencing the expression of either protein results in a decrease in effector translocation efficiency. These methods can be easily modified to examine other intracellular and extracellular pathogens that also utilize secretion systems. In this way, a global picture of host factors involved in bacterial effector translocation may be revealed.

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

Investigating the interactions between bacterial pathogens and their hosts is an important area of biological research. Here, we describe the necessary techniques to measure effector translocation by Coxiella burnetii during siRNA gene silencing using BlaM substrate.

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

Foodborne infections in the US caused by Vibrio species have shown an upward trend. In the genus Vibrio, V. parahaemolyticus is responsible for the majority of Vibrio-associated infections. Thus, accurate differentiation among Vibrio spp. and detection of V. parahaemolyticus is critically important to ensure the safety of our food supply. Although molecular techniques are increasingly common, culture-depending methods are still routinely done and they are considered standard methods in certain circumstances. Hence, a novel chromogenic agar medium was tested with the goal of providing a better method for isolation and differentiation of clinically relevant Vibrio spp. The protocol compared the sensitivity, specificity and detection limit for the detection of V. parahaemolyticus between the new chromogenic medium and a conventional medium. Various V. parahaemolyticus strains (n=22) representing diverse serotypes and source of origins were used. They were previously identified by Food and Drug Administration (FDA) and Centers for Disease Control and Prevention (CDC), and further verified in our laboratory by tlh-PCR. In at least four separate trials, these strains were inoculated on the chromogenic agar and thiosulfate-citrate-bile salts-sucrose (TCBS) agar, which is the recommended medium for culturing this species, followed by incubation at 35-37 &#176;C for 24-96 hr. Three V. parahaemolyticus strains (13.6%) did not grow optimally on TCBS, nonetheless exhibited green colonies if there was growth. Two strains (9.1%) did not yield the expected cyan colonies on the chromogenic agar. Non-V. parahaemolyticus strains (n=32) were also tested to determine the specificity of the chromogenic agar. Among these strains, 31 did not grow or exhibited other colony morphologies. The mean recovery of V. parahaemolyticus on the chromogenic agar was ~96.4% relative to tryptic soy agar supplemented with 2% NaCl. In conclusion, the new chromogenic agar is an effective medium to detect V. parahaemolyticus and to differentiate it from other vibrios.

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

Detection and isolation of clinically relevant Vibrio species require selective and differential culture media. This study evaluated the ability of a new chromogenic medium to detect and identify V. parahaemolyticus and other related species. The new medium was found to have better sensitivity and specificity than the conventional medium.

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

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic self-tolerance. Tregs represent about 5 - 10% of the mature CD4+ T cell subpopulation in mice and humans, with about 1 - 2% of those Tregs circulating in the peripheral blood. Induced pluripotent stem cells (iPSCs) can be differentiated into functional Tregs, which have a potential to be used for cell-based therapies of autoimmune diseases. Here, we present a method to develop antigen (Ag)-specific Tregs from iPSCs (i.e., iPSC-Tregs). The method is based on incorporating the transcription factor FoxP3 and an Ag-specific T cell receptor (TCR) into iPSCs and then differentiating on OP9 stromal cells expressing Notch ligands delta-like (DL) 1 and DL4. Following in vitro differentiation, the iPSC-Tregs express CD4, CD8, CD3, CD25, FoxP3, and Ag-specific TCR and are able to respond to Ag stimulation. This method has been successfully applied to cell-based therapy of autoimmune arthritis in a murine model. Adoptive transfer of these Ag-specific iPSC-Tregs into Ag-induced arthritis (AIA)-bearing mice has the ability to reduce joint inflammation and swelling and to prevent bone loss.

We present here a method to develop functional antigen (Ag)-specific regulatory T cells (Tregs) from induced pluripotent stem cells (iPSCs) for immunotherapy of autoimmune arthritis in a murine model.

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

Dysregulation of immune response in skin is associated with numerous human skin disorders. Direct transfer of immune-related genes into skin tissue is a fascinating approach to investigate immune modulation of cutaneous inflammation in mouse models of human diseases. Here we present a cost-effective protocol that delivered naked DNA in mouse skin and leads to transgene expression. The method is coined &#34;acufection&#34;, denoting acupuncture-mediated DNA transfection. To perform acufection, mouse skin was first infused with DNA in phosphate-buffered saline (PBS) and then pricked lightly with a bundle of acupuncture needles to facilitate the absorption of DNA and transfection into cells. The plasmid DNA is presumably taken up by the keratinocyte and dendritic cells (DCs) in the skin and expressed into protein. Mechanical prick with the needles per se did not cause skin damage or induce keratinocyte activation. The expression of the transfected genes was detected in the skin at both transcriptional and translational levels following acufection for 2 days and maintained up to 7 days. The primary goal for the development of this acufection method was to investigate a previously undefined isoform of IL-15. Using this method, an alternatively spliced IL-15 isoform with partially deleted exon 7 (IL-15&#916;E7) was expressed in the skin and subsequently treated with a Toll-like receptor 7 (TLR7) agonist, imiquimod (IMQ), to induce inflammation. Acufection-delivered IL-15&#916;E7 in skin suppressed keratinocyte proliferation, epidermal thickness and neutrophil recruitment in IMQ-induced cutaneous inflammation. With increasing interest in identifying the regulatory mechanisms of cutaneous inflammation, the protocol described here provides a cost effective and versatile alternative to the gene gun system or microseeding for DNA delivery in vivo. It may potentially allow discovery of the function of a novel gene in the skin or for investigating new treatment for cutaneous diseases.

This protocol is a cost-effective alternative for expressing naked plasmid DNA in mouse skin. The overall goal of the protocol is to deliver immune-related genes into skin tissue to delineate the functional role of a specific gene in cutaneous inflammation.

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

High-density peptide microarrays allow screening of more than six thousand peptides on a single standard microscopy slide. This method can be applied for drug discovery, therapeutic target identification, and developing of diagnostics. Here, we present a protocol to discover specific Zika virus (ZIKV) diagnostic peptides using a high-density peptide microarray. A human serum sample validated for ZIKV infection was incubated with a high-density peptide microarray containing the entire ZIKV protein translated into 3,423 unique 15 linear amino acid (aa) residues with a 14-aa residue overlap printed in duplicate. Staining with different secondary antibodies within the same array, we detected peptides that bind to Immunoglobulin M (IgM) and Immunoglobulin G (IgG) antibodies present in serum. These peptides were selected for further validation experiments. In this protocol, we describe the strategy followed to design, process, and analyze a high-density peptide microarray.

In this protocol, we describe a technique to discover Zika virus specific diagnostic peptides using a high-density peptide microarray. This protocol can readly be adapted for other emerging infectious diseases.

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 ...

Quantitative PCR of T7 Bacteriophage from Biopanning

This protocol describes the use of quantitative PCR (qPCR) to enumerate T7 phages from phage selection experiments (i.e., &#34;biopanning&#34;). qPCR is a fluorescence-based approach to quantify DNA, and here, it is adapted to quantify phage genomes as a proxy for phage particles. In this protocol, a facile phage DNA preparation method is described using high-temperature heating without additional DNA purification. The method only needs small volumes of heat-treated phages and small volumes of the qPCR reaction. qPCR is high-throughput and fast, able to process and obtain data from a 96-well plate of reactions in 2&#8211;4 h. Compared to other phage enumeration approaches, qPCR is more time-efficient. Here, qPCR is used to enumerate T7 phages identified from biopanning against in vitro cystic fibrosis-like mucus model. The qPCR method can be extended to quantify T7 phages from other experiments, including other types of biopanning (e.g.,&#160;immobilized protein binding, in vivo phage screening) and other sources (e.g., water systems or body fluids). In summary, this protocol can be modified to quantify any DNA-encapsulated viruses.

A reproducible, accurate, and time efficient quantitative PCR (qPCR) method to enumerate T7 bacteriophage is described here. The protocol clearly describes phage genomic DNA preparation, PCR reaction preparation, qPCR cycling conditions, and qPCR data analysis.

以此 T7 噬菌体的定量 PCR 研究

This protocol describes the use of quantitative PCR (qPCR) to enumerate T7 phages from phage selection experiments (i.e., &#34;biopanning&#34;). qPCR is a ...

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

摘要

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此视频中的章节

一个负选择标记的发展阿米巴

肽:MHC四聚体为基础的抗原表位特异性的T细胞的富集

使用点击化学测量的影响，病毒感染宿主细胞RNA合成

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

应用荧光共振能量转移（FRET）审查效应转运效率由

对于检测到的敏感性和特异性显色琼脂培养基发展

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

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

Zika 病毒特定诊断表位发现

以此 T7 噬菌体的定量 PCR 研究

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

摘要

探索更多视频

此视频中的章节

一个负选择标记的发展阿米巴

肽:MHC四聚体为基础的抗原表位特异性的T细胞的富集

使用点击化学测量的影响，病毒感染宿主细胞RNA合成

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

应用荧光共振能量转移（FRET）审查效应转运效率由

对于检测到的敏感性和特异性显色琼脂培养基发展

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

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

Zika 病毒特定诊断表位发现

以此 T7 噬菌体的定量 PCR 研究

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