| A Simple Method to Generate Gene Knockout Clones in Human Cells Using Transcription Activator-Like Effector Nuclease (TALEN)
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Author:
Date:
2015-07-20
[Abstract] Transcription activator-like effectors (TALEs) are naturally occurring proteins secreted by the plant pathogen, Xanthomonas, and fused to the Fok1 endonuclease to generate TALE nucleases (TALENs). TALEN pairs bind to specific DNA sequences initiating Fok1 dimerization and double-stand cleavage of DNA within the TALEN target site. This cleavage event triggers cellular repair mechanisms that result in insertions and/or deletions (indels), which enable gene knockout. The high specificity and efficiency of TALENs makes them important tools for genome editing. Here, we describe a method ...
[摘要] 转录激活子样效应器（TALE）是由植物病原体黄单胞菌分泌的天然存在的蛋白质，并且与Fok1内切核酸酶融合以产生TALE核酸酶（TALEN）。 TALEN对与特异性DNA序列结合，启动FAL1二聚化和在TALEN靶位点内双链切割DNA。 该切割事件触发导致插入和/或缺失（插入缺失）的细胞修复机制，其使得能够进行基因敲除。 TALEN的高特异性和高效性使其成为基因组编辑的重要工具。 在这里，我们描述了使用共转染和FACS与荧光报告基因通过TALEN产生具有靶向基因敲除的单细胞克隆的方法。 该方案设计为在Huh7.5细胞中敲除诱导细胞死亡的DFFA样效应子b，CIDEB; 然而，该协议可以应用于广泛的细胞类型和感兴趣的基因。
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| Murine in vivo CD8+ T Cell Killing Assay
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Author:
Date:
2014-07-05
[Abstract] Antigen-specific killing ability of effector CD8+ T cells is critical for protective immunity against infection. Here, we describe in vivo cytotoxic T cell assay to examine effector function of antigen-specific CD8+ T cells. Mice infected with Listeria monocytogenes (L. monocytogenes) expressing chicken ovalbumin as a model antigen mount ovalbumin-specific CD8+ T cell responses. Effector CD8+ T cell function in vivo is determined by mixed transfer of OVA peptide-pulsed target cells with control target cells into ...
[摘要] 效应CD8 + T细胞的抗原特异性杀伤能力对于感染的保护性免疫是关键的。 在这里，我们描述了在体内细胞毒性T细胞测定来检查抗原特异性CD8 + T细胞的效应子功能。 用表达鸡卵白蛋白作为模型抗原的单核细胞增生性李斯特菌（单核细胞增生李斯特氏菌）感染的小鼠装载卵清蛋白特异性CD8 + T细胞应答。 通过将OVA肽脉冲的靶细胞与对照靶细胞混合转移到先前免疫的小鼠中来测定体内效应物CD8 + T细胞功能。 CFSE表达水平的差异清楚地标记两种不同的群体：抗原脉冲的靶细胞-CFSE 低与未突发的靶细胞-FSE hi 。 抗原脉冲的靶细胞和对照靶细胞之间的频率用作抗原特异性杀伤的读数。
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| Shigella IpaD and IpaB Surface Localizations
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Author:
Date:
2013-11-20
[Abstract] Shigella uses a type III secretion system to invade host cell and to cause disease. Secretion control and insertion of a translocation pore into cell membrane are critical steps for pathogenesis and are tightly linked to the formation of the needle tip complex formed by the IpaB and IpaD proteins (Veenendaal et al., 2007). Surface localizations of IpaD and IpaB were monitored by FACS analysis according to the localization protocol for Pseudomonas aeruginosa homolog PcrV (Lee et al., 2010).
[摘要] 志贺氏菌使用III型分泌系统侵入宿主细胞并引起疾病。 分泌控制和易位孔插入细胞膜是发病的关键步骤，并且与由IpaB和IpaD蛋白形成的针尖复合物的形成紧密相关（Veenendaal等人，2007） 。 根据铜绿假单胞菌同源物PcrV的定位方案通过FACS分析监测IpaD和IpaB的表面定位（Lee等人，2010）。
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