我要感谢奥尔加·德索萨·席尔瓦的技术援助。这项工作是由瑞士国家科学基金会（SNSF）31003A_127029格兰特的支持。

的TransFLP方法（ 图1）通过三种不同的方法可以举出：Ⅰ）的两个相邻的基因，编码毒力决定因素五删除霍乱 （ 例如 ，ΔctxAB）;Ⅱ）去除的致病岛（ 例如 ，ΔVPI-1）和III）的T7 RNA聚合酶-依赖型启动子序列的融合利益一个基因的上游，这随后允许其人工表达的各菌株背景（ 例如，T7-tfoX）（ 图2）。 1。制备甲壳素片<ol><li>体重50-80毫克的甲壳素片在一个标准的1.5毫升的塑料管。这可以批量制备。几丁质片市售自Sigma公司（目录编号C9213）。 </li><li>保持管的盖，打开高压釜的薄片。 </li><li>高压釜冷却下来后，立即关闭盖子。 </li><li>商店蒸压甲壳素片在室温下。 </li></ol> 2。可选：人工改造五霍乱 FLP重组酶编码质粒<ol><li>准备电穿孔V。霍乱细胞使用标准的方法11。的感受态细胞的等分试样储存于-80℃。 </li><li>将1-2μl的一个普通的小编制质粒pBR-FLP 5添加到电穿孔五霍乱细胞和将混合物转移到电穿孔比色皿（0.2厘米的间隙宽度）。 </li><li>应用脉冲电压为1.6 kV。 </li><li>添加SOC培养基，0.9毫升，轻轻细胞转移到一个标准的14毫升管。孵育非移动的为2.5至3小时，在30℃下</li><li>板加入100μl和300μl的LB平板上含有100μg/ ml氨苄青霉素和孵育在30℃下保温过夜。 </li><li>净化单一的克隆和存储甘油为未来TransFLP实验。 </li></ol> 3。寡核苷酸的设计和聚合酶链反应<ol><li>需要至少六个寡核苷酸扩增的DNA区域（s）的利益，以及FRT-侧翼的抗生素盒通过PCR（ 图3）。建议还包括一个外插入的PCR片段的寡核苷酸对吸。这允许检查FRT-侧翼的抗生素抗性盒（ 例如，&#39;CHK-&#39;和&#39;CHK-向下&#39;引物，在图3至图5所示）的集成和正确的切除。 </li><li> ＃2，＃3，＃4，＃5小心设计的寡核苷酸。他们应该能够充分退火（ 例如 〜28 bp）的模板DNA。模板DNA为基因组DNA（基因组DNA）为引物，＃2和＃5和pROD17 12和PBR-FRT-KAN-FRT（本研究）为引物，＃3和＃4（FRT-阚-FRT-含有质粒DNA，如图3A）。 </li><li>设计的引物，＃2至＃5允许广泛的碱基配对分别在其5&#39;-末端＃2 /＃3和＃4 /＃5之间的，（ 图3A </strong&gt;的插图）。两轮PCR遵循。 </li><li>执行3个独立的PCR反应作为第一轮PCR。第一个，将扩增的基因/ DNA区域的感兴趣的援助的寡核苷酸＃1和＃2的上游区域。 PCR应导致在一个片段中的至少500 bp的长度，以允许在细胞内的同源重组。较短的片段（约250 bp）的就足够了8，但不建议使用。 </li><li>与此同时，进行第二次PCR，放大首次登记税两侧的抗生素磁带。对于所提供的实例，我们在这里主要用于APH（根 ）。使用的寡核苷酸＃3和＃4用于该反应（ 图3A）。 </li><li>随之而来，扩增PCR（第三次采样）的下游DNA区域。 PCR片段的长度也应该至少500个基点。执行PCR基因组DNA为模板，寡核苷酸＃5和＃6（ 图3A）。 </li><li>所有三种PCR纯化后片段，进行第二轮的PCR。使用等量混合物为模板，在第一轮中获得的所有三个片段。扩增催化寡核苷酸＃1和＃6。将所得的PCR片段（ 图3B）在自然的转染实验中（ 图1）作为转化DNA。 </li></ol> 4。甲壳素引起的自然转化<ol><li>成长五需氧霍乱细胞在加富培养基中在30℃，直到它们到达的光密度约为0.5，在600 nm。 </li><li>通过离心收获细菌。洗沉淀一次，在规定的人工介质（DASW 6）在2倍体积的DASW前重悬细胞。加入1毫升的文化50-80毫克的无菌甲壳素片（解释第1点）。如果细菌港口质粒pBR-FLP（可选第2点），添加氨苄青霉素（50微克/毫升）的文化。在30°C孵育16-24小时不动。 </li><li>添加＆GE，200 ng的第二轮PCR衍生片段（下解释第3点）。广泛分离的几丁质表面的细菌混合，小心不要。不移动的情况下在30℃孵育24小时。 </li><li>涡广泛的文化≥30秒。 100-300微升传播选择性LB ​​培养基平板上（ 如卡那霉素或庆大霉素的LB板这里提供的例子）。使用双选择性平板怀有质粒pBR-FLP加入氨苄青霉素二者对其他抗生素的细菌。在30℃温育平板16-24小时，或直至菌落是可见的。 </li><li>从选择性平板分离单转化。这种转化已经取代了原来的染色体位点的PCR片段，由于双交叉事件。这些菌株可直接用于进一步的实验中，是没有必要的情况下，去除抗生素盒。 </li></ol> 5。去除选择性盒式（s）的FLP- 重组<ol><li>人为地改变你的转化与质粒pBR-FLP前，先自然转化试验点2下，如果不这样做。 </li><li>长出的细菌在含有氨苄青霉素的LB琼脂平板在37℃下16-24小时。选项：改变中的温度在2-3小时之间，以40°C作为flp的表达质粒pBR-flp的压制在较高温度下5,10;转印后细菌到新鲜板约。 8小时的潜伏期。 </li><li>抗生素敏感性（这里用于显示卡那霉素， 图1），通过在含抗生素和抗菌素的琼脂平板上平行restreaking克隆试验。隔离单敏感的殖民地。如果你打算进一步修改的应变与其他缺失/插入使用TransFLP，冻结氨苄青霉素抗性克隆甘油。 </li></ol> 6。质粒消除<ol><li>成长在有氧条件下培养过夜在30℃下使用加富培养基中不添加任何抗生素。 </li><li>可选：稀释过夜培养1:100，在新的抗生素的培养基，生长的新鲜文化为3-6小时。 </li><li>板或条纹稀释在普通LB琼脂平板（S）（S）的文化和孵化在30°C为8-16小时（至菌落可见）。 </li><li>试验抗生素含和不含抗生素的琼脂平板上（ 图1）上并行restreaking克隆的氨苄青霉素敏感性克隆。冻结氨苄青霉素敏感的菌株甘油。 </li></ol>

<ol>
	<li>Miller, V. L., Mekalanos, 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=A+novel+suicide+vector+and+its+use+in+construction+of+insertion+mutations:+osmoregulation+of+outer+membrane+proteins+and+virulence+determinants+in+Vibrio+cholerae+requires+toxR.">A novel suicide vector and its use in construction of insertion mutations: osmoregulation of outer membrane proteins and virulence determinants in Vibrio cholerae requires toxR.</a> J. Bacteriol. 170, 2575-2583 (1988).</li><li>Hamilton, C. M., Aldea, M., Washburn, B. K., Babitzke, P., Kushner, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+method+for+generating+deletions+and+gene+replacements+in+Escherichia+coli.">New method for generating deletions and gene replacements in Escherichia coli.</a> J. Bacteriol. 171, 4617-4622 (1989).</li><li>Kato, C., Ohmiya, R., Mizuno, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+method+for+disrupting+genes+in+the+Escherichia+coli+genome.">A rapid method for disrupting genes in the Escherichia coli genome.</a> Biosci. Biotechnol. Biochem. 62, 1826-1829 (1998).</li><li>Donnenberg, M. S., Kaper, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+of+an+eae+deletion+mutant+of+enteropathogenic+Escherichia+coli+by+using+a+positive-selection+suicide+vector.">Construction of an eae deletion mutant of enteropathogenic Escherichia coli by using a positive-selection suicide vector.</a> Infect. Immun. 59, 4310-4317 (1991).</li><li>De Souza Silva, O., Blokesch, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+manipulation+of+Vibrio+cholerae+by+combining+natural+transformation+with+FLP+recombination.">Genetic manipulation of Vibrio cholerae by combining natural transformation with FLP recombination.</a> Plasmid. 64, 186-195 (2010).</li><li>Meibom, K. L., Blokesch, M., Dolganov, N. A., Wu, C. -. Y., Schoolnik, G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chitin+induces+natural+competence+in+Vibrio+cholerae.">Chitin induces natural competence in Vibrio cholerae.</a> Science. 310, 1824-1827 (2005).</li><li>Pollack-Berti, A., Wollenberg, M. S., Ruby, E. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+transformation+of+Vibrio+fischeri+requires+tfoX+and+tfoY.">Natural transformation of Vibrio fischeri requires tfoX and tfoY.</a> Environ. Microbiol. 12, 2302-2311 (2010).</li><li>Marvig, R. L., Blokesch, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+transformation+of+Vibrio+cholerae+as+a+tool-optimizing+the+procedure.">Natural transformation of Vibrio cholerae as a tool-optimizing the procedure.</a> BMC Microbiol. 10, 155 (2010).</li><li>Blokesch, M., Schoolnik, G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serogroup+Conversion+of+Vibrio+cholerae+in+Aquatic+Reservoirs.">Serogroup Conversion of Vibrio cholerae in Aquatic Reservoirs.</a> PLoS Pathog. 3, e81 (2007).</li><li>Cherepanov, P. P., Wackernagel, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+disruption+in+Escherichia+coli:+TcR+and+KmR+cassettes+with+the+option+of+Flp-catalyzed+excision+of+the+antibiotic-resistance+determinant.">Gene disruption in Escherichia coli: TcR and KmR cassettes with the option of Flp-catalyzed excision of the antibiotic-resistance determinant.</a> Gene. 158, 9-14 (1995).</li><li>Sambrook, J., Fritsch, E. F., Maniatis, T., Ford, N., Nolan, C., Ferguson, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular Cloning: A laboratory manual. 1, (1989).</li><li>Reyes-Lamothe, R., Possoz, C., Danilova, O., Sherratt, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Independent+positioning+and+action+of+Escherichia+coli+replisomes+in+live+cells.">Independent positioning and action of Escherichia coli replisomes in live cells.</a> Cell. 133, 90-102 (2008).</li><li>Karaolis, D. K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Vibrio+cholerae+pathogenicity+island+associated+with+epidemic+and+pandemic+strains.">A Vibrio cholerae pathogenicity island associated with epidemic and pandemic strains.</a> Proc. Natl. Acad. Sci. USA. 95, 3134-3139 (1998).</li><li>Ikeda, R. A., Ligman, C. M., Warshamana, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T7+promoter+contacts+essential+for+promoter+activity+in+vivo.">T7 promoter contacts essential for promoter activity in vivo.</a> Nucleic Acids Res. 20, 2517-2524 (1992).</li><li>Pan, S. H., Malcolm, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduced+background+expression+and+improved+plasmid+stability+with+pET+vectors+in+BL21+(DE3).">Reduced background expression and improved plasmid stability with pET vectors in BL21 (DE3).</a> Biotechniques. 29, 1234-1238 (2000).</li><li>Faruque, S. M., Zhu, J., Asadulghani, M., Kamruzzaman, J. J., Mekalanos, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Examination+of+diverse+toxin-coregulated+pilus-positive+Vibrio+cholerae+strains+fails+to+demonstrate+evidence+for+Vibrio+pathogenicity+island+phage.">Examination of diverse toxin-coregulated pilus-positive Vibrio cholerae strains fails to demonstrate evidence for Vibrio pathogenicity island phage.</a> Infect. Immun. 71, 2993-2999 (2003).</li><li>Joelsson, A., Liu, Z., Zhu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+and+phenotypic+diversity+of+quorum-sensing+systems+in+clinical+and+environmental+isolates+of+Vibrio+cholerae.">Genetic and phenotypic diversity of quorum-sensing systems in clinical and environmental isolates of Vibrio cholerae.</a> Infect. Immun. 74, 1141-1147 (2006).</li><li>Heidelberg, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+sequence+of+both+chromosomes+of+the+cholera+pathogen+Vibrio+cholerae.">DNA sequence of both chromosomes of the cholera pathogen Vibrio cholerae.</a> Nature. 406, 477-483 (2000).</li></ol>

没有利益冲突的声明。

的TransFLP上述方法和其他地方已被广泛使用在我们的实验室。种遗传操作是可行的，包括其中包括：删除单个基因和基因簇的基因岛（ 例如 ，VPI-1），缺失，插入感兴趣的基因的上游侧（ 例如 ，启动子序列）的序列，并插入序列结束时的一个特定的基因（ 例如 ，编码的亲和标签）。 一个导入的先决条件TransFLP是，各自的五霍乱菌株是自然变形。?...

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>甲壳素片</td><td>西格玛</td><td> C9213 </td><td>高压灭菌要求</td></tr><tr><td>可选：Micropulser（Flp重组PBR-FLP编码的情况下，抗生素盒应该被切除） </td><td> Biorad公司</td><td> 165-2100 </td><td>或类似的electroporators </td></tr></tbody></table>

transflp &#x2014; a method to genetically modify vibrio cholerae based on natural transformation and flp-recombination

有几种方法可用来操纵细菌染色体1-3。这些协议大多依靠的条件复制型质粒插入（ 例如 PIR依赖或对温度敏感的复制窝藏1,2）。这些质粒基于同源性的介导的重组整合到细菌染色体中。这样的插入突变体往往直接用于实验的设置。可替换地，可以进行选择质粒切除后跟其损失为革兰氏阴性菌，往往依赖于计数器可选择的果聚糖蔗糖酶的酶编码的sacB基因的 4。的切除可以恢复的插入前的基因型或在经修饰的基因的染色体和质粒编码的副本之间的交换的结果。这种技术的一个缺点是，它是耗时的。首先被克隆的质粒，它需要五的水平转移到霍乱 （最多n个<html> otably通过交配与E.大肠杆菌供体菌株）或人工转变后者;和切除该质粒是随机的，可以恢复初始的基因型或创建所需的修改，如果没有施加正选择的。在这里，我们提出了一个方法，为快速操作的五霍乱弧菌染色体（）5（ 图1）。此TransFLP方法是基于最近发现的几丁质的自然能力介导的诱导在此有机体6和其他五，如弧菌属代表鲵 7。自然能力允许的游离DNA的摄取，包括PCR产生的DNA片段。一旦占用，存在一个最小的250-500的bp的侧翼同源区域8给出的染色体DNA的重组。包括选择标记这些侧翼区之间可以方便地检测经常发生转化的。 T“&gt;这种方法可以用于不同的遗传操作的霍乱弧菌 ，可能还有其他的自然胜任的细菌。我们提供了三个新的例子可以用这种方法来完成的，除了我们之前发表的研究单基因缺失和另外的亲和标签序列。优化步骤，初始协议的几丁质引起的自然转换6注册成立在此TransFLP的协议，包括（其中包括）更换螃蟹壳碎片市售甲壳素片，捐赠PCR衍生的DNA为转化材料9，和另外的FLP重组目标网站（FRT）5。FRT收网站允许位点定向切除10 Flp重组酶介导的的选择标记。

代表性的结果在图4至图6中所示的三个例子的。第一种方法的目的是在删除邻近的基因CTXA的和 ctxB。他们一起进行编码的主要致病因子V。霍乱弧菌 ，霍乱毒素。我们设计寡核苷酸为TransFLP方法，如上述那样，按照图3。中间窝藏FRT-侧翼于原始的DNA位点的ctxAB和最终删除ctxAB和释放的抗性盒的菌株的抗生素抗性盒的菌株的亲本菌株，均测试其基因型...

Watch this Scientific Journal Video about TransFLP â€” A Method to Genetically Modify Vibrio cholerae Based on Natural Transformation and FLP-recombination at JoVE.com

TransFLP &amp;#x2014; A Method to Genetically Modify &lt;em&gt;Vibrio cholerae&lt;/em&gt; Based on Natural Transformation and FLP-recombination

一个快速的方法来修改的基因组<em&gt; V.霍乱</em&gt;进行说明。这些修改包括删除单个基因，基因簇和基因组岛屿以及集成的短序列（如启动子元件或亲和标签序列）。该方法是基于对自然的改造和FLP重组。

TransFLP - A基因修改方法<em&gt;霍乱弧菌</em基于自然转化和FLP重组

Several methods are available to manipulate bacterial chromosomes1-3. Most of these protocols rely on the insertion of conditionally replicative plasmids ...

transflp-method-to-genetically-modify-vibrio-cholerae-based-on

Research

JoVE Journal

Immunology and Infection

TransFLP &#x2014; A Method to Genetically Modify Vibrio cholerae Based on Natural Transformation and FLP-recombination

14.5K 次观看.  Ecole Polytechnique Fédérale de Lausanne (EPFL). 一个快速的方法来修改的基因组 V.霍乱进行说明。这些修改包括删除单个基因，基因簇和基因组岛屿以及集成的短序列（如启动子元件或亲和标签序列）。该方法是基于对自然的改造和FLP重组。

视频: TransFLP &#x2014; A Method to Genetically Modify Vibrio cholerae Based on Natural Transformation and FLP-recombination

Colonization of Euprymna scolopes Squid by Vibrio fischeri

Specific bacteria are found in association with animal tissue1-5. Such host-bacterial associations (symbioses) can be detrimental (pathogenic), have no fitness consequence (commensal), or be beneficial (mutualistic). While much attention has been given to pathogenic interactions, little is known about the processes that dictate the reproducible acquisition of beneficial/commensal bacteria from the environment. The light-organ mutualism between the marine Gram-negative bacterium V. fischeri and the Hawaiian bobtail squid, E. scolopes, represents a highly specific interaction in which one host (E. scolopes) establishes a symbiotic relationship with only one bacterial species (V. fischeri) throughout the course of its lifetime6,7. Bioluminescence produced by V. fischeri during this interaction provides an anti-predatory benefit to E. scolopes during nocturnal activities8,9, while the nutrient-rich host tissue provides V. fischeri with a protected niche10. During each host generation, this relationship is recapitulated, thus representing a predictable process that can be assessed in detail at various stages of symbiotic development. In the laboratory, the juvenile squid hatch aposymbiotically (uncolonized), and, if collected within the first 30-60 minutes and transferred to symbiont-free water, cannot be colonized except by the experimental inoculum6. This interaction thus provides a useful model system in which to assess the individual steps that lead to specific acquisition of a symbiotic microbe from the environment11,12.
Here we describe a method to assess the degree of colonization that occurs when newly hatched aposymbiotic E. scolopes are exposed to (artificial) seawater containing V. fischeri. This simple assay describes inoculation, natural infection, and recovery of the bacterial symbiont from the nascent light organ of E. scolopes. Care is taken to provide a consistent environment for the animals during symbiotic development, especially with regard to water quality and light cues. Methods to characterize the symbiotic population described include (1) measurement of bacterially-derived bioluminescence, and (2) direct colony counting of recovered symbionts.

Colonization of Euprymna scolopes Squid by Vibrio fischeri

The method outlines the procedure by which the Hawaiian bobtail squid, Euprymna scolopes and its bacterial symbiont, Vibrio fischeri, are raised separately and then introduced to allow for specific colonization of the squid light organ by the bacteria. Colonization detection by bacterially-derived luminescence and by direct colony counting are described.

定植 Euprymna scolopes鱿鱼由弧菌鲵</em

Specific bacteria are found in association with animal tissue1-5. Such host-bacterial associations (symbioses) can be detrimental (pathogenic), have no ...

科研

免疫与感染

A Parasite Rescue and Transformation Assay for Antileishmanial Screening Against Intracellular Leishmania donovani Amastigotes in THP1 Human Acute Monocytic Leukemia Cell Line

Leishmaniasis is one of the world's most neglected diseases, largely affecting the poorest of the poor, mainly in developing countries. Over 350 million people are considered at risk of contracting leishmaniasis, and approximately 2 million new cases occur yearly1. Leishmania donovani is the causative agent for visceral leishmaniasis (VL), the most fatal form of the disease. The choice of drugs available to treat leishmaniasis is limited 2;current treatments provide limited efficacy and many are toxic at therapeutic doses. In addition, most of the first line treatment drugs have already lost their utility due to increasing multiple drug resistance 3. The current pipeline of anti-leishmanial drugs is also severely depleted. Sustained efforts are needed to enrich a new anti-leishmanial drug discovery pipeline, and this endeavor relies on the availability of suitable in vitro screening models.
In vitro promastigotes 4 and axenic amastigotes assays5 are primarily used for anti-leishmanial drug screening however, may not be appropriate due to significant cellular, physiological, biochemical and molecular differences in comparison to intracellular amastigotes. Assays with macrophage-amastigotes models are considered closest to the pathophysiological conditions of leishmaniasis, and are therefore the most appropriate for in vitro screening. Differentiated, non-dividing human acute monocytic leukemia cells (THP1) (make an attractive) alternative to isolated primary macrophages and can be used for assaying anti-leishmanial activity of different compounds against intracellular amastigotes.
Here, we present a parasite-rescue and transformation assay with differentiated THP1 cells infected in vitro with Leishmania donovani for screening pure compounds and natural products extracts and determining the efficacy against the intracellular Leishmania amastigotes. The assay involves the following steps: (1) differentiation of THP1 cells to non-dividing macrophages, (2) infection of macrophages with L. donovani metacyclic promastigotes, (3) treatment of infected cells with test drugs, (4) controlled lysis of infected macrophages, (5) release/rescue of amastigotes and (6) transformation of live amastigotes to promastigotes. The assay was optimized using detergent treatment for controlled lysis of Leishmania-infected THP1 cells to achieve almost complete rescue of viable intracellular amastigotes with minimal effect on their ability to transform to promastigotes. Different macrophage:promastigotes ratios were tested to achieve maximum infection. Quantification of the infection was performed through transformation of live, rescued Leishmania amastigotes to promastigotes and evaluation of their growth by an alamarBlue fluorometric assay in 96-well microplates. This assay is comparable to the currently-used microscopic, transgenic reporter gene and digital-image analysis assays. This assay is robust and measures only the live intracellular amastigotes compared to reporter gene and image analysis assays, which may not differentiate between live and dead amastigotes. Also, the assay has been validated with a current panel of anti-leishmanial drugs and has been successfully applied to large-scale screening of pure compounds and a library of natural products fractions (Tekwani et al. unpublished).

A Parasite Rescue and Transformation Assay for Antileishmanial Screening Against Intracellular Leishmania donovani Amastigotes in THP1 Human Acute Monocytic Leukemia Cell Line

A parasite-rescue and transformation assay with THP1 cells infected in vitro with Leishmania donovani has been optimized for anti-leishmanial drug screening. The assay involves differentiation of THP1 cells, infection with promastigotes, treatment with test drugs, controlled lysis of the infected macrophages, rescue of amastigotes, transformation to promastigotes and monitoring promastigote growth and proliferation with a fluorometric assay.

Antileishmanial筛选对细胞内的寄生虫救援和转化实验<em&gt;杜氏利什曼原虫</em无鞭毛体在THP1人急性单核细胞白血病细胞株

Leishmaniasis is  one of the world's most neglected diseases, largely affecting the  poorest of the poor, mainly in developing countries. Over 350 million ...

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer a biologic product with the potential for superior (i) recognition of tumor-associated antigens (TAAs), (ii) persistence after infusion, (iii) potential for migration to tumor sites, and (iv) ability to recycle effector functions within the tumor microenvironment. Most approaches to genetic manipulation of T cells engineered for human application have used retrovirus and lentivirus for the stable expression of CAR1-3. This approach, although compliant with current good manufacturing practice (GMP), can be expensive as it relies on the manufacture and release of clinical-grade recombinant virus from a limited number of production facilities. The electro-transfer of nonviral plasmids is an appealing alternative to transduction since DNA species can be produced to clinical grade at approximately 1/10th the cost of recombinant GMP-grade virus. To improve the efficiency of integration we adapted Sleeping Beauty (SB) transposon and transposase for human application4-8. Our SB system uses two DNA plasmids that consist of a transposon coding for a gene of interest (e.g. 2nd generation CD19-specific CAR transgene, designated CD19RCD28) and a transposase (e.g. SB11) which inserts the transgene into TA dinucleotide repeats9-11. To generate clinically-sufficient numbers of genetically modified T cells we use K562-derived artificial antigen presenting cells (aAPC) (clone #4) modified to express a TAA (e.g. CD19) as well as the T cell costimulatory molecules CD86, CD137L, a membrane-bound version of interleukin (IL)-15 (peptide fused to modified IgG4 Fc region) and CD64 (Fc-&gamma; receptor 1) for the loading of monoclonal antibodies (mAb)12. In this report, we demonstrate the procedures that can be undertaken in compliance with cGMP to generate CD19-specific CAR+ T cells suitable for human application. This was achieved by the synchronous electro-transfer of two DNA plasmids, a SB transposon (CD19RCD28) and a SB transposase (SB11) followed by retrieval of stable integrants by the every-7-day additions (stimulation cycle) of &gamma;-irradiated aAPC (clone #4) in the presence of soluble recombinant human IL-2 and IL-2113. Typically 4 cycles (28 days of continuous culture) are undertaken to generate clinically-appealing numbers of T cells that stably express the CAR. This methodology to manufacturing clinical-grade CD19-specific T cells can be applied to T cells derived from peripheral blood (PB) or umbilical cord blood (UCB). Furthermore, this approach can be harnessed to generate T cells to diverse tumor types by pairing the specificity of the introduced CAR with expression of the TAA, recognized by the CAR, on the aAPC.

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

T cells expressing a CD19-specific chimeric antigen receptor (CAR) are infused as investigational treatment of B-cell malignancies in our first-in-human gene therapy trials. We describe genetic modification of T cells using the Sleeping Beauty (SB) system to introduce CD19-specific CAR and selective propagation on designer CD19+ artificial antigen presenting cells.

临床应用<em&gt;睡美人</em和人工抗原呈递细胞基因修饰T细胞从外设和脐带血中

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer  a biologic product with the potential for ...

Super-resolution Imaging of the Natural Killer Cell Immunological Synapse on a Glass-supported Planar Lipid Bilayer

The glass-supported planar lipid bilayer system has been utilized in a variety of disciplines. One of the most useful applications of this technique has been in the study of immunological synapse formation, due to the ability of the glass-supported planar lipid bilayers to mimic the surface of a target cell while forming a horizontal interface. The recent advances in super-resolution imaging have further allowed scientists to better view the fine details of synapse structure. In this study, one of these advanced techniques, stimulated emission depletion (STED), is utilized to study the structure of natural killer (NK) cell synapses on the supported lipid bilayer. Provided herein is an easy-to-follow protocol detailing: how to prepare raw synthetic phospholipids for use in synthesizing glass-supported bilayers; how to determine how densely protein of a given concentration occupies the bilayer's attachment sites; how to construct a supported lipid bilayer containing antibodies against NK cell activating receptor CD16; and finally, how to image human NK cells on this bilayer using STED super-resolution microscopy, with a focus on distribution of perforin positive lytic granules and filamentous actin at NK synapses. Thus, combining the glass-supported planar lipid bilayer system with STED technique, we demonstrate the feasibility and application of this combined technique, as well as intracellular structures at NK immunological synapse with super-resolution.

We describe here a combination of the glass-supported lipid bilayer technique of forming immunological synapses with the super-resolution imaging technique of stimulated emission depletion (STED) microscopy. The goal of this protocol is to provide users with the instructions necessary to successfully carry out these two techniques.

的自然杀伤细胞免疫突触上的玻璃支承平面脂双层超分辨率成像

The glass-supported planar lipid bilayer system has been utilized in a variety of disciplines. One of the most useful applications of this technique has ...

Implementation of a Permeable Membrane Insert-based Infection System to Study the Effects of Secreted Bacterial Toxins on Mammalian Host Cells

Many bacterial pathogens secrete potent toxins to aid in the destruction of host tissue, to initiate signaling changes in host cells or to manipulate immune system responses during the course of infection. Though methods have been developed to successfully purify and produce many of these important virulence factors, there are still many bacterial toxins whose unique structure or extensive post-translational modifications make them difficult to purify and study in in vitro systems. Furthermore, even when pure toxin can be obtained, there are many challenges associated with studying the specific effects of a toxin under relevant physiological conditions. Most in vitro cell culture models designed to assess the effects of secreted bacterial toxins on host cells involve incubating host cells with a one-time dose of toxin. Such methods poorly approximate what host cells actually experience during an infection, where toxin is continually produced by bacterial cells and allowed to accumulate gradually during the course of infection. This protocol describes the design of a permeable membrane insert-based bacterial infection system to study the effects of Streptolysin S, a potent toxin produced by Group A Streptococcus, on human epithelial keratinocytes. This system more closely mimics the natural physiological environment during an infection than methods where pure toxin or bacterial supernatants are directly applied to host cells. Importantly, this method also eliminates the bias of host responses that are due to direct contact between the bacteria and host cells. This system has been utilized to effectively assess the effects of Streptolysin S (SLS) on host membrane integrity, cellular viability, and cellular signaling responses. This technique can be readily applied to the study of other secreted virulence factors on a variety of mammalian host cell types to investigate the specific role of a secreted bacterial factor during the course of infection.

Here, a method using a permeable membrane insert-based infection system to study the effects of Streptolysin S, a secreted toxin produced by Group A Streptococcus, on keratinocytes is described. This system can be readily applied to the study of other secreted bacterial proteins on various host cell types during infection.

基于插入渗透膜感染系统的实现研究分泌细菌病毒对哺乳动物宿主细胞的作用

Many bacterial pathogens secrete potent toxins to aid in the destruction of host tissue, to initiate signaling changes in host cells or to manipulate ...

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 ...

A Platform of Anti-biofilm Assays Suited to the Exploration of Natural Compound Libraries

Biofilms are regarded as one of the most challenging topics of modern biomedicine, and they are potentially responsible for over 80% of antibiotic-tolerant infections. Biofilms have displayed an exceptionally high tolerance for chemotherapy, which is thought to be multifactorial. For instance, the matrix provides a physical barrier that decreases the penetration of antibiotics into the biofilm. Also, cells within the biofilms are phenotypically diverse. Likely, biofilm resilience arises from a combination of these and other, yet unknown, mechanisms. All of the currently existing antibiotics have been developed against single-cells (planktonic) bacteria. Therefore, so far, a very limited repertoire of molecules exists that can selectively act on mature biofilms. This situation has driven a progressive paradigm shift in drug discovery, in which searching for anti-biofilms has been urged to occupy a more prominent place. An additional challenge is that there are a very limited number of standardized methods for biofilm research, especially those that can be used for large-throughput screening of chemical libraries. Here, an experimental anti-biofilm platform for chemical screening is presented. It uses three assays to measure biofilm viability (with resazurin staining), total biomass (with crystal violet staining), and biofilm matrix (using a wheat germ agglutinin, WGA-fluorescence-based staining of the poly-N-acetyl-glucosamine, PNAG, fraction). All the assays were developed using Staphylococcus aureus as the model bacteria. Examples of how the platform can be used for primary screening as well as for functional characterization of identified anti-biofilm hits are presented. This experimental sequence further allows for the classification of the hits based upon the measured end-points. It also provides information on their mode of action, especially on long-term versus short-term chemotherapeutic effects. Thus, it is very advantageous for the quick identification of high-quality hit compounds that can serve as starting points for various biomedical applications.

Biofilm infections show high tolerance towards chemotherapy. No single assay captures the complexity of biofilms. Instead, complementary assays are needed. We present a screening platform (developed for S. aureus) that combines assays for viability, biomass, and biofilm matrix. It allows anti-biofilm drug discovery, including the assessment of long-term chemotherapeutic effects.

适合与天然化合物库的探索抗生物试验的平台

Biofilms are regarded as one of the most challenging topics of modern biomedicine, and they are potentially responsible for over 80% of ...

Fluorescence-based Neuraminidase Inhibition Assay to Assess the Susceptibility of Influenza Viruses to The Neuraminidase Inhibitor Class of Antivirals

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against currently circulating strains. In addition to their use in treating seasonal influenza, the NA inhibitors have been stockpiled by a number of countries for use in the event of a pandemic. It is therefore important to monitor the susceptibility of circulating influenza viruses to this class of antivirals. There are different types of assays that can be used to assess the susceptibility of influenza viruses to the NA inhibitors, but the enzyme inhibition assays using either a fluorescent substrate or a chemiluminescent substrate are the most widely used and recommended. This protocol describes the use of a fluorescence-based assay to assess influenza virus susceptibility to NA inhibitors. The assay is based on the NA enzyme cleaving the 2&#8242;-(4-Methylumbelliferyl)-&#945;-D-N-acetylneuraminic acid (MUNANA) substrate to release the fluorescent product 4-methylumbelliferone (4-MU). Therefore, the inhibitory effect of an NA inhibitor on the influenza virus NA is determined based on the concentration of the NA inhibitor that is required to reduce 50% of the NA activity, given as an IC50 value.

We describe the use of a phenotypic fluorescence-based neuraminidase inhibition assay to assess the susceptibility of influenza A and B viruses to the neuraminidase inhibitor class of antivirals.

荧光为基础的神经氨酸酶抑制测定流感病毒的易感性评估抗病毒药的神经氨酸酶抑制剂类

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against ...

Laboratory Techniques Used to Maintain and Differentiate Biotypes of Vibrio cholerae Clinical and Environmental Isolates

The aquatic Gram-negative bacterium Vibrio cholerae is the etiological agent of the infectious gastrointestinal disease cholera. Due to the global prevalence and severity of this disease, V. cholerae has been extensively studied in both environmental and laboratory settings, requiring proper maintenance and culturing techniques. Classical and El Tor are two main biotypes that compose the V. cholerae O1 serogroup, each displaying unique genotypic and phenotypic characteristics that provide reliable mechanisms for biotype characterization, and require distinct virulence inducing culturing conditions. Regardless of the biotype of the causative strain for any given infection or outbreak, the standard treatment for the disease involves rehydration therapy supplemented with a regimen of antibiotics. However, biotype classification may be necessary for laboratory studies and may have broader impacts in the biomedical field. In the early 2000&#39;s clinical isolates were identified which exhibit genotypic and phenotypic traits from both classical and El Tor biotypes. The newly identified hybrids, termed El Tor variants, have caused clinical and environmental isolate biotype identification to become more complex than previous traditional single assay identification protocols. In addition to describing V. cholerae general maintenance and culturing techniques, this manuscript describes a series of gene specific (ctxB and tcpA) PCR-based genetic screens and phenotypic assays (polymyxin B resistance, citrate metabolism, proteolytic activity, hemolytic activity, motility, and glucose metabolism via Voges-Proskauer assay) collectively used to characterize and/or distinguish between classical and El Tor biotypes. Together, these assays provide an efficient systematic approach to be used as an alternative, or in addition, to costly, labor-intensive experiments in the characterization of V. cholerae clinical (and environmental) isolates.

Laboratory Techniques Used to Maintain and Differentiate Biotypes of Vibrio cholerae Clinical and Environmental Isolates

This manuscript describes proper Vibrio cholerae maintenance techniques in addition to a series of biochemical assays, collectively utilized for quick and reliable differentiation between clinical and environmental V. cholerae biotypes in a laboratory setting.

用于维持和区分生物型的实验室技术<em&gt;霍乱弧菌</em&gt;临床和环境隔离

The aquatic Gram-negative bacterium Vibrio cholerae is the etiological agent of the infectious gastrointestinal disease cholera. Due to the global ...

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor microenvironment and the underlying mechanism is still largely unknown. The lack of a consistent and reliable source of NK cells limits the research progress of NK cell immunity. Here, we report an in vitro system that can provide high quality and quantity of bone marrow-derived murine NK cells under a feeder-free condition. More importantly, we also demonstrate that siRNA-mediated gene silencing successfully inhibits the E4bp4-dependent NK cell maturation by using this system. Thus, this novel in vitro NK cell differentiating system is a biomaterial solution for immunity research.

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Here, we present a protocol to mass-produce gene-silencing murine NK cells by using a feeder-free differentiation system for mechanistic study in vitro and in vivo.

一种新型大鼠自然杀伤细胞无饲养系统的研究体外

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor ...