Name: a genetic screen to isolate toxoplasma gondii host-cell egress mutants
Uploaded: 2012-02-08T13:00:00
Description: Watch this Scientific Journal Video about A Genetic Screen to Isolate Toxoplasma gondii Host-cell Egress Mutants at JoVE.com

这项工作是由美国心脏协会的科学家开发格兰特0635480N和美国国立卫生研究赠款AI081220研究院。 BIC是一个圣殿骑士眼科基金会的研究经费支持。

概观协议规定首先定义导致的寄生虫杀死70％的议定书（1）诱变剂量。下道工序提供丰富从诱变寄生虫池（协议2，图2）的诱导出口突变的。其次是协议测试中富集池出口突变的发生，或在个别突变体的出口型验证议定书（3）。最后，协议生成丰富的人口，由单一的寄生虫克隆有限稀释（协议）。 1。滴定法诱变使用双层手套，如要特别...

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<html>所描述的协议提供了一个有效的方法来分离弓形虫突变与出口缺陷。我们已经成功地分离出突变沿各个步骤的出口途径，其中一些具有双重侵袭型13。使用所谓的红绿法，区别于非侵入寄生虫入侵差抗体染色23,24，可以决定对入侵的潜在影响。对于入侵和出口检测，它可以方便表示，在寄生虫13,22细胞质萤光蛋白标记。但是，如果寄生虫诱变已经表达了荧光蛋白?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> ENU的</td><td> Sigma-Aldrich公司</td><td> N3385 </td><td> 1米股票在-20°C，储存在DMSO </td></tr><tr><td>环境管理体系</td><td> Sigma-Aldrich公司</td><td> M0880 </td><td> 1米股票在-20°C，储存在DMSO </td></tr><tr><td>硫酸葡聚糖</td><td> Sigma-Aldrich公司</td><td> D4911 </td><td></td></tr><tr><td> PDTC干预</td><td> Sigma-Aldrich公司</td><td> P8765 </td><td> 100毫米的PBS股票</td></tr><tr><td>变化快速</td><td>电解二氧化锰化学品</td><td> 65044-93 </td><td></td></tr><tr><td>过滤网架</td><td>科尔 - 帕默</td><td> 540100 </td><td></td></tr><tr><td> 3μm的聚碳酸酯过滤器</td><td>瓦特曼施莱歇尔＆Schuell </td><td> 110612 </td><td></td></tr><tr><td>血球</td><td>豪塞尔科学</td><td> 1475 </td><td></td></tr><tr><td> 二氧化碳培养箱</td><td>各个厂家</td><td></td><td>加湿，5％CO 2，35，37和40°C间</td></tr><tr><td>荧光显微镜</td><td>各个厂家</td><td></td><td>理想的反转，宽领域与63X或100X油目标</td></tr></tbody></table> HBSSc（根据黑等 11。） <ul><li> 98.0毫升汉克斯平衡盐溶液（Hyclone公司目录编号SH30588） </li><li> 100μL1M MgCl 2的 （100毫米末） </li><li> 100μL1M 氯化钙 （100毫米末） </li><li> 2.0毫升1M HEPES pH值7.3（20毫米年底） </li><li> 84毫克碳酸氢钠3（10毫米年底） </li></ul>

a genetic screen to isolate toxoplasma gondii host-cell egress mutants

广泛，专性细胞内，原生动物寄生虫弓形虫会导致免疫能力受损的患者投机性疾病和先天性感染后会导致出生缺陷。裂解复制周期的特点是通过三个阶段：1。主动入侵的核宿主细胞; 2。宿主细胞内复制; 3。从宿主细胞中活跃的出口。出口机制正在越来越多地被作为一个独特的，高度调节的过程，它是在分子水平上仍然缺乏了解表示赞赏。特点通过使用药物制剂作用于不同方面的途径1-5已基本出口的信号通路。正因为如此，出口的几个独立的触发已经确定，都收敛于细胞内钙释放的，也是一个信号，即6-8宿主细胞的入侵的关键。这种洞察力通知的候选基因的方法而导致的识别fication植物如钙依赖的蛋白激酶（CDPK）涉及出口9。此外，最近的几次突破，了解出口已用遗传方法（化学）10-12。结合起来，增加弓形虫遗传无障碍丰富的药理信息，我们最近成立了一个屏幕，允许寄生虫与宿主细胞出口13缺陷突变富集。虽然几十年来一直使用N-乙基-N-亚硝基脲（ENU的）或甲磺酸乙酯（EMS）的化学诱变的弓形虫生物学11,14,15的研究在使用，只是最近有相关基因突变的遗传图谱表型成为常规16 -18。此外，由温度敏感突变体的产生，基本过程可以解剖和底层的基因直接确定。这些突变体表现为野生型宽容的温度（35℃）下，但未能为p在严格的温度（40℃）roliferate作为一个问题的突变的结果。在这里，我们说明了一个新的表型筛选法，隔离温度 ​​敏感的出口型13的突变体。出口屏幕所面临的挑战是分开非egressed寄生虫，这是由快速重新入侵和宿主细胞寄生虫一般粘性复杂egressed的。根据先前建立的出口屏幕上的生物素化的步骤繁琐的一系列分开11个外寄生虫内。这种方法还没有产生条件突变，导致弱表型。这里介绍的方法，克服了强烈的依恋，寄生虫，包括糖的竞争对手，硫酸葡聚糖（DS），防止寄生虫宿主细胞坚持19 egressing。此外，外寄生虫特别是杀害了吡咯烷二硫代氨基甲酸（PDTC），叶细胞内寄生虫无恙20。因此，一个新的表型屏幕，专门隔离寄生虫引起的出口缺陷突变，遗传学的力量现在可以得到充分部署，以揭示宿主细胞出口的分子机制。

Watch this Scientific Journal Video about A Genetic Screen to Isolate Toxoplasma gondii Host-cell Egress Mutants at JoVE.com

A Genetic Screen to Isolate Toxoplasma gondii Host-cell Egress Mutants

正向遗传学是一个功能强大的方法来解开如何在分子水平<em&gt;弓形虫</em从它的宿主细胞外出。协议提供化学诱变寄生虫，丰富与缺陷突变引起的出口，并验证克隆突变体的表型。

遗传筛选隔离弓形虫宿主细胞的出口突变

The widespread, obligate intracellular, protozoan parasite Toxoplasma gondii causes opportunistic disease in immuno-compromised patients and causes birth ...

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A Genetic Screen to Isolate Toxoplasma gondii Host-cell Egress Mutants

Obtaining Highly Purified Toxoplasma gondii Oocysts by a Discontinuous Cesium Chloride Gradient

Obtaining Highly Purified Toxoplasma gondii Oocysts by a Discontinuous Cesium Chloride Gradient

取得高纯度弓形虫</em一个不连续的氯化铯梯度>卵囊

Toxoplasma gondii is an obligate intracellular protozoan pathogen that commonly infects humans. It is a well characterized apicomplexan associated with ...

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Toxoplasma gondii Cyst Wall Formation in Activated Bone Marrow-derived Macrophages and Bradyzoite Conditions

弓形虫囊肿壁形成激活骨髓衍生的巨噬细胞和Bradyzoite条件

Toxoplasma gondii is an obligate intracellular parasite that can invade any nucleated cell of warm-blooded animals. During infection, T. gondii ...

Transnuclear Mice with Pre-defined T Cell Receptor Specificities Against Toxoplasma gondii Obtained Via SCNT

Transnuclear Mice with Pre-defined T Cell Receptor Specificities Against Toxoplasma gondii Obtained Via SCNT

与预先定义的T细胞受体特异性Transnuclear小鼠对弓形虫获得通过体细胞核移植

Lymphocytes, such as T cells, undergo genetic V(D)J recombination, to generate a receptor with a certain specificity1. Mice transgenic for a rearranged ...

Using a Pan-Viral Microarray Assay (Virochip) to Screen Clinical Samples for Viral Pathogens

Using a Pan-Viral Microarray Assay Virochip to Screen Clinical Samples for Viral Pathogens

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The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence ...

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一种简单而有效的隔离混合原代培养成人肝细胞巨噬细胞的方法

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although ...

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Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

建立的液体覆盖的文化偏光人类呼吸道上皮的CALU-3细胞研究呼吸系统病原宿主细胞的反应<em在体外</em

The  apical and basolateral surfaces of airway epithelial cells demonstrate  directional responses to pathogen exposure in  vivo. Thus, ideal in vitro ...

Genetic Manipulation in &Delta;ku80 Strains for Functional Genomic Analysis of Toxoplasma gondii

Genetic Manipulation in ÃƒÅ½Ã¢â‚¬Âku80 Strains for Functional Genomic Analysis of Toxoplasma gondii

遗传操纵<em&gt;Δku80</em&gt;株功能基因组分析<em&gt;弓形虫</em

Targeted genetic manipulation using homologous recombination is the method of choice for functional genomic analysis to obtain a detailed view of gene ...

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

使用点击化学测量的影响，病毒感染宿主细胞RNA合成

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

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

Forward Genetics Screens Using Macrophages to Identify Toxoplasma gondii Genes Important for Resistance to IFN-ÃƒÅ½Ã‚Â³-Dependent Cell Autonomous Immunity

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 ...

Use of the Soft-agar Overlay Technique to Screen for Bacterially Produced Inhibitory Compounds

软琼脂覆盖技术使用筛选细菌产生的抑制化合物

The soft-agar overlay technique was originally developed over 70 years ago and has been widely used in several areas of microbiological research, ...