这个项目是由塞缪尔·罗伯茨诺贝尔基金会。作者感谢MSS。珍妮Gallaway和科琳埃尔斯优良的植物护理和凯蒂·布朗女士艺术品。我们也想感谢MSS。天合光能科特雷尔，普加Uppalapati，萨哈Moumita，Swetha Vinukonda，此协议在建立技术帮助的艾萨克Greenhut先生。

1。植物生长与靶基因沉默<ol><li>植物生长的条件： <ol><li>母猪N.本生种子无土盆栽混合物，地铁混合350和发芽的种子在生长室。任何其它也可用于土壤或土壤的介质，而不是对新城混合。 </li><li>移植三周龄的苗成单个盆种植在温室中保持在21±2℃以及与其他的生长条件，详细的在以前的文献12。移栽后的两到三天，植物可以用来为TRV接种。 </li></ol></li><li>成长TRV2克隆： TRV是一个双向的病毒，其基因组由RNA1和RNA2。 RNA1编码的RNA依赖性RNA聚合酶的运动蛋白20,21。 RNA2编码外壳蛋白（CP）和两个非结构蛋白的亚基因组RNA 21。双方RNA1和RNA2需要形成成熟VIR我们粒子及其传播20,21。构建cDNA文库在TRV2载体在以前的文献22,23。简单地说，在本研究中使用的VIGS库构建从从暴露于各种生物和非生物诱导子的叶组织中提取的RNA。 <ol><li>取出根癌农杆菌 GV2260菌株含有的cDNA克隆（96孔板），从冷冻库在TRV2矢量。逐渐解冻后，培养物达到室温，接种个体的根癌农杆菌培养利福平（10微克/毫升）和卡那霉素（50微克/毫升）的Luria-Bertani（LB）琼脂平板上，用96针复制器。 </li><li>在28°C孵育板两天。我们通常生长四个复制殖民地每个克隆，以便有足够的农杆菌接种接种两种植物刺。无菌条件下执行所有步骤。 </li></ol></li><li> VIGS： <ol><li>增长农杆菌 （GV2260）携带TRV1在28°C与上述抗生素的LB液体培养基中。从过夜生长的培养物通过离心收获细胞，重新悬浮在接种缓冲液（pH为5.5的10mM MES，200μM的乙酰丁香酮），在振荡器上以50rpm在室温下孵育3小时。 </li><li>通过离心收集细胞，并重新悬浮在5mM的MES缓冲液（pH值5.5），接种量（OD 600 = 0.3）到3-4 N.背面的边叶本生使用无针注射器。详细的接种程序是体现在以前的文献24。后来，在现场的TRV1接种，接种各自TRV2菌落用牙签刺的叶子。 </li><li>保持足够的营养生长旺盛的植物是非常重要的高效沉默12。大约两个星期后接种靶基因的转录将开始减少，植物病原体接种到准备ulation TRV接种后三个星期。 </li></ol></li></ol> 2。非寄主病原文化的准备和植物接种<ol><li>在本研究中所用的病原体的详情： 野火病菌粉虱，P.番茄斑点病菌。T1，P.斑点病菌glycinea和野油菜黄单胞菌叶斑病病菌。用于此项研究。成长P.丁香株在King的B（KB）与利福平（10微克/毫升），卡那霉素（50微克/毫升）的液体培养基，在28℃12小时。成长十油菜叶斑病病菌。在LB液体培养基中16小时。所有病原体怀有质粒可以表达GFPuv的在以前的文献19。 </li><li>制备植物接种病原体培养： <ol><li>通过离心收获细菌细胞，并确认绿色荧光的存在下，使用长娃velength紫外灯在黑暗中。两次用无菌水清洗细胞，重新悬浮，用无菌水稀释至所需浓度。 </li><li>接种相应的病原体（次）目标基因沉默点（约1.5厘米直径）叶（5 日至8 日 ），背面的边。几个非寄主病原体可以同时测试它们的生长中的靶基因沉默的植物。同时接种宿主病原体，因为这可以用来作为阳性对照，用于查看在植物中的细菌生长。接种矢量控制（TRV :: 00）植物。 </li></ol></li></ol> 3。 在植物生长的细菌病原体的观察<ol><li>公开接种叶长波长的紫外线灯在黑暗中。可以被看作是在背景中的表面发出的红色荧光叶叶子的背面的边的绿色荧光点的细菌菌落。 </li><li>观察病原体生长后2天接种（dpi）的5 dpi的。在这个时间间隔期间的日常生活观察是必要的。 </li><li>后的第一个画面，短名单的克隆其沉默的一个或多个非寄主病原体的生长结果。 </li><li>重复VIGS所选克隆和再次测试非寄主病原体的基因沉默植株的响应。这种二级筛选做是为了消除过程中获得的第一个画面的误报。 </li></ol> 4。确认入围植物非寄主抗性受损的<ol><li>沉默如上所述的从屏幕上选择的cDNA克隆。接种的整个非寄主植物与病原体的浸没与相应的病原体培养的植物，具有0.01％（体积/体积）的Silwet L-77和水中植物经受真空为3 - 5分钟。通过常规的生长测定6,18,19量化细菌的生长（如下文所述）。 </li><li>在0小时接种后（HPI），3 dpi和5 dpi的两个叶片样品，收集五生物复制为每个非寄主病原体（次）接种叶用叶冲（0.5厘米2）。研磨叶样品，串行稀板块SAP KB琼脂​​培养基上辅以适当的抗生素和孵化，在28°C，2天。用菌落计数器计数的细菌菌落，并计算以前的文献6中所描述的叶片中的细菌的生长。 </li><li>植物易受非寄主病原体（次），应该表现出较高的数目相比，矢量控制病原体的生长。 </li></ol> 5。靶基因序列的插入和鉴定<ol><li>选定的克隆经重组和attB2引物的使用或使用的引物侧翼克隆网站在TRV2载体，进行菌落PCR。运行PCR产物在凝胶上，并检查是否为单一的条带的扩增。 </li><li>植物基因插入可测序在TRV2载体的使用attB位侧翼克隆位点的引物或引物。 </li><li>使用序列进行BLAST和识别基因详情。 </li><li>随着非翻译区（非编码区）获取全长基因序列。 </li><li>优先选择300-400 bp的片段，来自三个不同区域的序列和克隆到TRV2载体。独立执行VIGS使用所有三个VIGS结构和确认在所有三个基因沉默植物非寄主抗性的妥协。 </li></ol>

<ol>
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我们什么都没有透露。

植物免疫力限制非寄主病原体的生长，因此，很少或根本没有发出绿色荧光矢量控制植物的叶子接种非寄主病原体在长波长紫外光灯（ 图3D）。然而，当在非寄主抗性有关的基因沉默，基因沉默的植物有利于生长的非寄主的病原体和绿色荧光出现（ 图3E）。这是这个手稿中所描述的方法中所涉及的基本原理。这种方法有两个主要的优点，在人力资源为基础的检测。 在?...

非寄主抗性是对一个特定的病原体1,2种族的所有植物物种的阻力。这赋予广谱和持久抗病植物中2,3。然而，其作用机制，尤其是对细菌病原体，没有得到很好的理解。妥协非寄主抗性，高通量转录分析识别差异表达的基因在非寄主抗性5-9有两种主要方法，以前用于解剖细菌非寄主抗性的突变体或沉默植株的筛选。因为非寄主抗性是由一个复杂的机制（S）4与许多基因的参与，高通量功能基因组学方法进行基因识别控制，是为更好地理解非寄主抗性机制（S）的关键。 病毒诱导的基因沉默（VIGS）已成功地用于植物内源沉默基因在许多植物物种10,11。 烟草（Nicotiana benthamiana）是最适合的植物病毒诱导的基因沉默10,12和它的基因组序列草图，现已13。 烟草脆裂病毒 （TRV）基于VIGS技术已被广泛用作反向遗传学工具表征基因参与非寄主抗性2,4,14。这VIGS载体及衍生产品现已通过拟南芥生物资源中心（ABRC， <a href="http://www.arabidopsis.org/abrc/catalog/individ_cloned_gene_1.html" target="_blank">http://www.arabidopsis.org/abrc/catalog/individ_cloned_gene_1.html</a> ）。 VIGS也被用来确定参与植物免疫力15-17，特别是6,18非寄主抗性基因作为一个正向遗传学工具。评估过敏性反应（HR）介导的细胞死亡由一个特定的非寄主植物对病原体引起和评估疾病诱导的细胞死亡，是两个主要检测主要用于identifyin的克易感基因沉默植株。然而，HR细胞死亡不仅对II型非寄主病原体引起的，而不是针对I型非寄主病原体2。因此，人力资源分析不能被普遍用来识别非寄主抗性被植物所利用的策略，特别是针对广泛的I型非寄主病原体。此外，在基因沉默的植物非寄主抗性的部分损失并不总是导致疾病的症状，因此不能用来确定损害非寄主抗性的植物疾病的得分。相反，评估非寄主病原体的基因沉默植株的增长是一个更好的方法研究非寄主抗性基因沉默植株的损失。 以往的生长测定6,19，更快速的方法，以评估非寄 ​​主细菌生长的基因沉默的植物相比，可以缩短在正向遗传学筛选所需的时间。我们此前报道的方法观察细菌升病原体生长在紫外线（UV）光叶肉眼使用细菌表达绿色荧光蛋白（GFP）19。在这篇稿件中，我们表现出的实用性表达GFPuv的非寄主细菌病原体受到损害非寄主抗性基因沉默植株易于识别。这种方法是准确识别敏感的植物，适合高通量筛选。

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent/equipment</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>96-well U-bottom plates</td>
 <td> Becton Dickinson Labware (Franklin Lakes, NJ, USA)</td>
 <td> 35-3077</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>96-pin replicator stainless steel</td>
 <td> Nalge Nunc International (Naperville, IL, USA)</td>
 <td> 250520</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td> High Intensity UV Inspection Lamps, Model B-100ap</td>
 <td> Thomas scientific (Swedesboro, NJ, USA)</td>
 <td> 6283K50</td>
 <td> Manufacturer ID 95-0127-01</td>
 </tr>
 <tr>
 <td> Stuart SC6 colony counter</td>
 <td> Bibby Scientific Limited, Staffordshire, UK</td>
 <td> SC6PLUS</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td> Soil-less potting mixture, Metro-Mix 350</td>
 <td> SUNGRO Horticulture Distribution, Inc., (Bellevue, WA, USA)</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td> Primers: 
 attB1 (GGGGACAAGTTTGTACAAAAAAGCAGGCT) 
 attB2 (GGGGACCACTTTGTACAAGAAAGCTGGGT)</td>
 <td> Integrated DNA Technologies, Inc (Coralville, IA, USA)</td>
 <td> Custom synthesized</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td> MES, Monohydrate</td>
 <td> VWR international (Radnor, PA, USA)</td>
 <td> CAS No. 145224-94-8</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td> Acetosyringone (Dimethoxy-4'-hydroxyacetophenone)</td>
 <td> Sigma Aldrich (St. Louis, MO, USA)</td>
 <td> D134406</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td> Vac-In-Stuff (Silwet L-77)</td>
 <td> Lehle Seeds (Round Rock, TX, USA)</td>
 <td> VIS-30</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

vigs-mediated forward genetics screening for identification of genes involved in nonhost resistance

非寄主植物对细菌病原体的抗病能力控制复杂的防御途径。了解这一机制是非常重要开发耐用的抗病植物对病原体的广泛。病毒诱导的基因沉默（VIGS）基于正向遗传学筛选是用于识别赋予非寄主抗性植物防卫基因的一个有用的方法。 烟草脆裂病毒 （TRV）的沉默载体是最有效的VIGS载体和有效地使用已内源性靶基因沉默在烟草 。 在这篇稿件中，我们展示了一个正向遗传学筛选方法沉默单个克隆cDNA文库在N本生和评估对非寄主病原体损害非寄主抗性基因沉默植株的响应， 野火病菌番茄T1，P.丁香甘油 PV。成人教育学院，和X油菜叶斑病病菌。这些细菌病原体设计到快递GFPuv蛋白质和绿色荧光的菌落肉眼可以看出，如果在非寄主植物病原体接种在紫外光下沉默目标基因参与传授非寄主抗性。这有利于可靠和更快的识别基因沉默植株易受非寄主病原体。此外，有希望的候选基因的信息可以被称为TRV载体序列的植物基因插入。在这里，我们展示了VIGS介导的正向遗传学的高吞吐能力，以确定基因参与非寄主抗性。大约100个​​cDNA可以在约2至3周，对一些非寄主细菌病原体的非寄主抗性的相关个别沉默可以研究在其后的一个星期。在这篇稿件中，我们列举参与筛查的具体步骤。 VIGS介导的正向遗传学甄选NG方法不仅可以延长参与非寄主抗性基因鉴定，但也研究基因传授各种植物物种的一些生物和非生物胁迫公差。

这项研究的主要目的是要证明的方法，方便，准确识别基因沉默N. benthamiana植物非寄主抗性受到损害。这种方法有四个主要步骤。第一个步骤是个别地大量使用TRV-VIGS基因沉默。我们已经沉默了大约5000个基因6,18过了一段约1.5年的使用协议， 如图1所示。部分的基因沉默的植物表现出各种表型的改变，包括生长发育迟缓，泛黄和光漂白的表型（ 图2）。 <p c...

Watch this Scientific Journal Video about VIGS-Mediated Forward Genetics Screening for Identification of Genes Involved in Nonhost Resistance at JoVE.com

VIGS-Mediated Forward Genetics Screening for Identification of Genes Involved in Nonhost Resistance

病毒诱导的基因沉默是一个有用的工具，用于识别植物非寄主抗性基因参与。我们演示了如何使用中表达GFPuv的识别基因沉默植株易受非寄主病原体的细菌病原体。这种做法是方便，快捷，有利于大规模筛选和类似协议可以应用到其他各种植物与微生物相互作用研究。

VIGS介导的正向遗传学筛选参与非寄主抗性基因鉴定


	Nonhost disease resistance of plants against bacterial pathogens is controlled by complex defense pathways. Understanding this mechanism is important ...

vigs-mediated-forward-genetics-screening-for-identification-genes

Research

JoVE Journal

Immunology and Infection

16.9K 次观看.  The Samuel Roberts Noble Foundation. 病毒诱导的基因沉默是一个有用的工具，用于识别植物非寄主抗性基因参与。我们演示了如何使用中表达GFPuv的识别基因沉默植株易受非寄主病原体的细菌病原体。这种做法是方便，快捷，有利于大规模筛选和类似协议可以应用到其他各种植物与微生物相互作用研究。 

视频: VIGS-Mediated Forward Genetics Screening for Identification of Genes Involved in Nonhost Resistance

RNAi Screening for Host Factors Involved in Vaccinia Virus Infection using Drosophila Cells

Viral pathogens represent a significant public health threat; not only can viruses cause natural epidemics of human disease, but their potential use in bioterrorism is also a concern. A better understanding of the cellular factors that impact infection would facilitate the development of much-needed therapeutics. Recent advances in RNA interference (RNAi) technology coupled with complete genome sequencing of several organisms has led to the optimization of genome-wide, cell-based loss-of-function screens. Drosophila cells are particularly amenable to genome-scale screens because of the ease and efficiency of RNAi in this system 1. Importantly, a wide variety of viruses can infect Drosophila cells, including a number of mammalian viruses of medical and agricultural importance 2,3,4. Previous RNAi screens in Drosophila have identified host factors that are required for various steps in virus infection including entry, translation and RNA replication 5. Moreover, many of the cellular factors required for viral replication in Drosophila cell culture are also limiting in human cells infected with these viruses 4,6,7,8, 9. Therefore, the identification of host factors co-opted during viral infection presents novel targets for antiviral therapeutics. Here we present a generalized protocol for a high-throughput RNAi screen to identify cellular factors involved in viral infection, using vaccinia virus as an example.

RNAi Screening for Host Factors Involved in Vaccinia Virus Infection using Drosophila Cells

Novel host factors involved in viral infection can be identified through cell-based genome-wide loss of function RNAi screening. A Drosophila cell culture model is particularly amenable to this approach due to the ease and efficiency of RNAi. Here we demonstrate this technique using vaccinia virus as an example.

RNAi筛选参与宿主因素牛痘病毒感染使用果蝇细胞

Viral pathogens represent a significant public health threat; not only can viruses cause natural epidemics of human disease, but their potential use in ...

科研

免疫与感染

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response following an infection. Despite efforts to identify unique surface marker on Tregs, the only unique feature is their ability to suppress the proliferation and function of effector T cells. While it is clear that only in vitro assays can be used in assessing human Treg function, this becomes problematic when assessing the results from cross-sectional studies where healthy cells and cells isolated from subjects with autoimmune diseases (like Type 1 Diabetes-T1D) need to be compared. There is a great variability among laboratories in the number and type of responder T cells, nature and strength of stimulation, Treg:responder ratios and the number and type of antigen-presenting cells (APC) used in human in vitro suppression assays. This variability makes comparison between studies measuring Treg function difficult. The Treg field needs a standardized suppression assay that will work well with both healthy subjects and those with autoimmune diseases. We have developed an in vitro suppression assay that shows very little intra-assay variability in the stimulation of T cells isolated from healthy volunteers compared to subjects with underlying autoimmune destruction of pancreatic &beta;-cells. The main goal of this piece is to describe an in vitro human suppression assay that allows comparison between different subject groups. Additionally, this assay has the potential to delineate a small loss in nTreg function and anticipate further loss in the future, thus identifying subjects who could benefit from preventive immunomodulatory therapy1. Below, we provide thorough description of the steps involved in this procedure. We hope to contribute to the standardization of the in vitro suppression assay used to measure Treg function. In addition, we offer this assay as a tool to recognize an early state of immune imbalance and a potential functional biomarker for T1D.

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Tregs are potent suppressors of the immune system. There is a lack of unique surface markers to define them, hence, definitions of Tregs are primarily functional. Here we describe an optimized in vitro assay capable of identifying immune imbalance in subjects at risk to develop T1D.

人类<em在体外承认免疫失衡的早期国家禁止作为筛检工具

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response ...

Reverse Genetics Mediated Recovery of Infectious Murine Norovirus

Human noroviruses are responsible for most cases of human gastroenteritis (GE) worldwide and are recurrent problem in environments where close person-to-person contact cannot be avoided 1, 2. During the last few years an increase in the incidence of outbreaks in hospitals has been reported, causing significant disruptions to their operational capacity as well as large economic losses. The identification of new antiviral approaches has been limited due to the inability of human noroviruses to complete a productive infection in cell culture 3. The recent isolation of a murine norovirus (MNV), closely related to human norovirus 4 but which can be propagated in cells 5 has opened new avenues for the investigation of these pathogens 6, 7. 
MNV replication results in the synthesis of new positive sense genomic and subgenomic RNA molecules, the latter of which corresponds to the last third of the viral genome (Figure 1). MNV contains four different open reading frames (ORFs), of which ORF1 occupies most of the genome and encodes seven non-structural proteins (NS1-7) released from a polyprotein precursor. ORF2 and ORF3 are contained within the subgenomic RNA region and encode the capsid proteins (VP1 and VP2, respectively) (Figure 1). Recently, we have identified that additional ORF4 overlapping ORF2 but in a different reading frame is functional and encodes for a mitochondrial localised virulence factor (VF1) 8.
Replication for positive sense RNA viruses, including noroviruses, takes place in the cytoplasm resulting in the synthesis of new uncapped RNA genomes. To promote viral translation, viruses exploit different strategies aimed at recruiting the cellular protein synthesis machinery 9-11. Interestingly, norovirus translation is driven by the multifunctional viral protein-primer VPg covalently linked to the 5' end of both genomic and subgenomic RNAs 12-14. This sophisticated mechanism of translation is likely to be a major factor in the limited efficiency of viral recovery by conventional reverse genetics approaches. 
Here we report two different strategies based on the generation of murine norovirus-1 (referred to as MNV herewith) transcripts capped at the 5' end. One of the methods involves both in vitro synthesis and capping of viral RNA, whereas the second approach entails the transcription of MNV cDNA in cells expressing T7 RNA polymerase. The availability of these reverse genetics systems for the study of MNV and a small animal model has provided an unprecedented ability to dissect the role of viral sequences in replication and pathogenesis 15-17.

Noroviruses are a major cause of gastroenteritis yet molecular techniques for their characterisation are still relatively new. Here we report two different reverse genetics approaches for the efficient recovery of murine norovirus (MNV), the only member of this genus which can be propagated in cell culture.

反向遗传学感染小鼠诺沃克病毒介导的恢复

Human noroviruses are responsible for most cases of human gastroenteritis (GE) worldwide and are recurrent problem in environments where close ...

Forward Genetic Approaches in Chlamydia trachomatis

Chlamydia trachomatis, the etiological agent of sexually transmitted diseases and ocular infections, remains poorly characterized due to its intractability to experimental transformation with recombinant DNA. We developed an approach to perform genetic analysis in C. trachomatis&#160;despite the lack of molecular genetic tools. Our method involves: i.) chemical mutagenesis to rapidly generate comprehensive libraries of genetically-defined mutants with distinct phenotypes; ii.) whole-genome sequencing (WGS) to map the underlying genetic lesions and to find associations between mutated gene(s) and a common phenotype; iii.) generation of recombinant strains through co-infection of mammalian cells with mutant and wild type bacteria. Accordingly, we were able to establish causal relationships between genotypes and phenotypes. The coupling of chemically-induced gene variation and WGS to establish correlative genotype&#8211;phenotype associations should be broadly applicable to the large list of medically and environmentally important microorganisms currently intractable to genetic analysis.

Forward Genetic Approaches in Chlamydia trachomatis

We describe a methodology to perform genetic analysis in Chlamydia based on chemical mutagenesis and whole genome sequencing. In addition, a system for DNA exchange within infected cells is described that can be used for genetic mapping. This method may be broadly applicable to microbial systems lacking transformation systems and molecular genetic tools.

正向遗传途径<em&gt;沙眼衣原体</em

Chlamydia trachomatis, the etiological agent of sexually transmitted diseases and ocular infections, remains poorly characterized due to its ...

Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

Macrophages are key phagocytic innate immune cells. When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the pathogen, produce various cytokines and chemokines to recruit and stimulate other immune cells, and present antigens to stimulate the adaptive immune response. Thus, being able to efficiently manipulate macrophages with techniques such as RNA-interference (RNAi) is critical to our ability to investigate this important innate immune cell. However, macrophages can be technically challenging to transfect and can exhibit inefficient RNAi-induced gene knockdown. In this protocol, we describe methods to efficiently transfect two mouse macrophage cell lines (RAW264.7 and J774A.1) with siRNA using the Amaxa Nucleofector 96-well Shuttle System and describe procedures to maximize the effect of siRNA on gene knockdown. Moreover, the described methods are adapted to work in 96-well format, allowing for medium and high-throughput studies. To demonstrate the utility of this approach, we describe experiments that utilize RNAi to inhibit genes that regulate lipopolysaccharide (LPS)-induced cytokine production.

In this protocol, we describe methods to efficiently transfect murine macrophage cell lines with siRNAs using the Amaxa Nucleofector 96-well Shuttle System, stimulate the macrophages with lipopolysaccharide, and monitor the effects on inflammatory cytokine production.

利用RNA干扰调查的先天免疫反应的小鼠巨噬细胞

Macrophages are key phagocytic innate immune cells.  When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the ...

Identification of Novel Genes Associated with Alginate Production in Pseudomonas aeruginosa Using Mini-himar1 Mariner Transposon-mediated Mutagenesis

Pseudomonas aeruginosa is a Gram-negative, environmental bacterium with versatile metabolic capabilities. P. aeruginosa is an opportunistic bacterial pathogen which establishes chronic pulmonary infections in patients with cystic fibrosis (CF). The overproduction of a capsular polysaccharide called alginate, also known as mucoidy, promotes the formation of mucoid biofilms which are more resistant than planktonic cells to antibiotic chemotherapy and host defenses. Additionally, the conversion from the nonmucoid to mucoid phenotype is a clinical marker for the onset of chronic infection in CF. Alginate overproduction by P. aeruginosa is an endergonic process which heavily taxes cellular energy. Therefore, alginate production is highly regulated in P. aeruginosa. To better understand alginate regulation, we describe a protocol using the mini-himar1 transposon mutagenesis for the identification of novel alginate regulators in a prototypic strain PAO1. The procedure consists of two basic steps. First, we transferred the mini-himar1 transposon (pFAC) from host E. coli SM10/&#955;pir into recipient P. aeruginosa PAO1 via biparental conjugation to create a high-density insertion mutant library, which were selected on Pseudomonas isolation agar plates supplemented with gentamycin. Secondly, we screened and isolated the mucoid colonies to map the insertion site through inverse PCR using DNA primers pointing outward from the gentamycin cassette and DNA sequencing. Using this protocol, we have identified two novel alginate regulators, mucE (PA4033) and kinB (PA5484), in strain PAO1 with a wild-type mucA encoding the anti-sigma factor MucA for the master alginate regulator AlgU (AlgT, &#963;22). This high-throughput mutagenesis protocol can be modified for the identification of other virulence-related genes causing change in colony morphology.

Identification of Novel Genes Associated with Alginate Production in Pseudomonas aeruginosa Using Mini-himar1 Mariner Transposon-mediated Mutagenesis

Here we describe a protocol using the mini-himar1 mariner transposon-mediated mutagenesis for generating a high-density insertion mutant library to screen, isolate and identify novel alginate regulators in the prototypic Pseudomonas aeruginosa strain PAO1.

在伴有海藻酸钠生产新基因的鉴定<em&gt;铜绿假单胞菌</em&gt;使用小型<em&gt; himar1</em&gt;水手转座子介导的诱变

Pseudomonas aeruginosa is a Gram-negative, environmental bacterium with versatile metabolic capabilities. P. aeruginosa is an opportunistic bacterial ...

Rapid Screening of HIV Reverse Transcriptase and Integrase Inhibitors

Although a number of anti HIV drugs have been approved, there are still problems with toxicity and drug resistance. This demonstrates a need to identify new compounds that can inhibit infection by the common drug resistant HIV-1 strains with minimal toxicity. Here we describe an efficient assay that can be used to rapidly determine the cellular cytotoxicity and efficacy of a compound against WT and mutant viral strains.
The desired target cell line is seeded in a 96-well plate and, after a 24 hr incubation, serially dilutions of the compounds to be tested are added. No further manipulations are necessary for cellular cytotoxicity assays; for anti HIV assays a predetermined amount of either a WT or drug resistant HIV-1 vector that expresses luciferase is added to the cells. Cytotoxicity is measured by using an ATP dependent luminescence assay and the impact of the compounds on infectivity is measured by determining the amount of luciferase in the presence or the absence of the putative inhibitors.
This screening assay takes 4 days to complete and multiple compounds can be screened in parallel. Compounds are screened in triplicate and the data are normalized to the infectivity/ATP levels in absence of target compounds. This technique provides a quick and accurate measurement of the efficacy and toxicity of potential anti HIV compounds.

Here we describe cellular cytotoxicity and single round infectivity assays that allow for the rapid and accurate screening of compounds to determine their cellular cytotoxicity (CC50) and IC50 values against WT and drug resistant HIV-1.

HIV逆转录酶和整合酶抑制剂的快速筛选

Although a number of anti HIV drugs have been approved, there are still problems with toxicity and drug resistance. This demonstrates a need to identify ...

Forward Genetics Screens Using Macrophages to Identify Toxoplasma gondii Genes Important for Resistance to IFN-&#947;-Dependent Cell Autonomous Immunity

Toxoplasma gondii, the causative agent of toxoplasmosis, is an obligate intracellular protozoan pathogen. The parasite invades and replicates within virtually any warm blooded vertebrate cell type. During parasite invasion of a host cell, the parasite creates a parasitophorous vacuole (PV) that originates from the host cell membrane independent of phagocytosis within which the parasite replicates. While IFN-dependent-innate and cell mediated immunity is important for eventual control of infection, innate immune cells, including neutrophils, monocytes and dendritic cells, can also serve as vehicles for systemic dissemination of the parasite early in infection. An approach is described that utilizes the host innate immune response, in this case macrophages, in a forward genetic screen to identify parasite mutants with a fitness defect in infected macrophages following activation but normal invasion and replication in na&#239;ve macrophages. Thus, the screen isolates parasite mutants that have a specific defect in their ability to resist the effects of macrophage activation. The paper describes two broad phenotypes of mutant parasites following activation of infected macrophages: parasite stasis versus parasite degradation, often in amorphous vacuoles. The parasite mutants are then analyzed to identify the responsible parasite genes specifically important for resistance to induced mediators of cell autonomous immunity. The paper presents a general approach for the forward genetics screen that, in theory, can be modified to target parasite genes important for resistance to specific antimicrobial mediators. It also describes an approach to evaluate the specific macrophage antimicrobial mediators to which the parasite mutant is susceptible. Activation of infected macrophages can also promote parasite differentiation from the tachyzoite to bradyzoite stage that maintains chronic infection. Therefore, methodology is presented to evaluate the importance of the identified parasite gene to establishment of chronic infection.

Forward Genetics Screens Using Macrophages to Identify Toxoplasma gondii Genes Important for Resistance to IFN-&amp;#947;-Dependent Cell Autonomous Immunity

Forward genetics is a powerful approach to identify genes in intracellular pathogens important for resistance to cell autonomous immunity. The current approach uses innate immune cells, specifically macrophages, to identify novel Toxoplasma gondii genes important for immune evasion.

正向遗传学屏风使用巨噬细胞识别<em&gt;弓形虫</em&gt;基因的重要的抗IFN-γ依赖性细胞免疫自主

Toxoplasma gondii, the causative agent of toxoplasmosis, is an obligate intracellular protozoan pathogen. The parasite invades and replicates within ...

Microscopy-based Assays for High-throughput Screening of Host Factors Involved in Brucella Infection of Hela Cells

Brucella species are facultative intracellular pathogens that infect animals as their natural hosts. Transmission to humans is most commonly caused by direct contact with infected animals or by ingestion of contaminated food and can lead to severe chronic infections.
Brucella can invade professional and non-professional phagocytic cells and replicates within endoplasmic reticulum (ER)-derived vacuoles. The host factors required for Brucella entry into host cells, avoidance of lysosomal degradation, and replication in the ER-like compartment remain largely unknown. Here we describe two assays to identify host factors involved in Brucella entry and replication in HeLa cells. The protocols describe the use of RNA interference, while alternative screening methods could be applied. The assays are based on the detection of fluorescently labeled bacteria in fluorescently labeled host cells using automated wide-field microscopy. The fluorescent images are analyzed using a standardized image analysis pipeline in CellProfiler which allows single cell-based infection scoring.
In the endpoint assay, intracellular replication is measured two days after infection. This allows bacteria to traffic to their replicative niche where proliferation is initiated around 12 hr after bacterial entry. Brucella which have successfully established an intracellular niche will thus have strongly proliferated inside host cells. Since intracellular bacteria will greatly outnumber individual extracellular or intracellular non-replicative bacteria, a strain constitutively expressing GFP can be used. The strong GFP signal is then used to identify infected cells.
In contrast, for the entry assay it is essential to differentiate between intracellular and extracellular bacteria. Here, a strain encoding for a tetracycline-inducible GFP is used. Induction of GFP with simultaneous inactivation of extracellular bacteria by gentamicin enables the differentiation between intracellular and extracellular bacteria based on the GFP signal, with only intracellular bacteria being able to express GFP. This allows the robust detection of single intracellular bacteria before intracellular proliferation is initiated.

Microscopy-based Assays for High-throughput Screening of Host Factors Involved in Brucella Infection of Hela Cells

Two assays for microscopy-based high-throughput screening of host factors involved in Brucella infection are described. The entry assay detects host factors required for Brucella entry and the endpoint assay those required for intracellular replication. While applicable for alternative approaches, siRNA screening in HeLa cells is used to illustrate the protocols.

在参与宿主因素的高通量筛选基于显微镜的检测方法<em&gt;布鲁氏菌</em&gt; Hela细胞感染

Brucella species are facultative intracellular pathogens that infect animals as their natural hosts. Transmission to humans is most commonly caused by ...

Screening Assays to Characterize Novel Endothelial Regulators Involved in the Inflammatory Response

The endothelial layer is essential for maintaining homeostasis in the body by controlling many different functions. Regulation of the inflammatory response by the endothelial layer is crucial to efficiently fight against harmful inputs and aid in the recovery of damaged areas. When the endothelial cells are exposed to an inflammatory environment, such as the outer component of gram-negative bacteria membrane, lipopolysaccharide (LPS), they express soluble pro-inflammatory cytokines, such as Ccl5, Cxcl1 and Cxcl10, and trigger the activation of circulating leukocytes. In addition, the expression of adhesion molecules E-selectin, VCAM-1 and ICAM-1 on the endothelial surface enables the interaction and adhesion of the activated leukocytes to the endothelial layer, and eventually the extravasation towards the inflamed tissue. In this scenario, the endothelial function must be tightly regulated because excessive or defective activation in the leukocyte recruitment could lead to inflammatory-related disorders. Since many of these disorders do not have an effective treatment, novel strategies with a focus on the vascular layer must be investigated. We propose comprehensive assays that are useful to the search of novel endothelial regulators that modify leukocyte function. We analyze endothelial activation by using specific expression targets involved in leukocyte recruitment (such as, cytokines, chemokines, and adhesion molecules) with several techniques, including: real-time quantitative polymerase chain reaction (RT-qPCR), western-blot, flow cytometry and adhesion assays. These approaches determine endothelial function in the inflammatory context and are very useful to perform screening assays to characterize novel endothelial inflammatory regulators that are potentially valuable for designing new therapeutic strategies.

Vascular endothelium tightly controls leukocyte recruitment. Inadequate leukocyte extravasation contributes to human inflammatory diseases. Therefore, searching for novel regulatory elements of endothelial activation is necessary to design improved therapies for inflammatory disorders. Here, we describe a comprehensive methodology to characterize novel endothelial regulators that can modify leukocyte trafficking during inflammation.