| 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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| DNA Slot Blot Repair Assay
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Author:
Date:
2015-04-20
[Abstract] Ultraviolet (UV) irradiation induces helix distorting photolesions such as cyclobutane pyrimidine dimers (CPD) and pyrimidine-pyrimidone (6-4) photoproducts (6-4PP) which threaten genomic integrity if unrepaired. In mammals, nucleotide excision repair (NER) is the only pathway that removes UV-induced DNA damages. Here we describe DNA slot blot repair assay for quantitative detection of NER activity using DNA damage specific antibodies such as anti-CPD and anti-6-4PP. Briefly, genomic DNA irradiated with UV was isolated from cells, and the genomic DNA was vacuum-transferred to a nitrocellulose ...
[摘要] 紫外线（UV）照射诱导螺旋扭曲光致损伤，例如环丁烷嘧啶二聚体（CPD）和嘧啶 - 嘧啶酮（6-4）光产物（6-4PP），如果未修复则威胁基因组完整性。 在哺乳动物中，核苷酸切除修复（NER）是去除UV诱导的DNA损伤的唯一途径。 在这里我们描述DNA狭缝印迹修复测定NER活性使用DNA损伤特异性抗体如抗CPD和抗6-4PP的定量检测。 简言之，从细胞中分离用UV照射的基因组DNA，使用Bio-Dot SF微量过滤装置（Bio-Rad）将基因组DNA真空转移到硝酸纤维素膜上。 应用识别CPD或6-4PP的单克隆抗体来检测基因组DNA中残留的光损伤量。 对于均匀负载的上样控制，可以通过SYBR金染色进一步分析DNA在膜上。
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| Preparation of Pneumococcal Proteins for Western Blot Analysis
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Author:
Date:
2013-07-05
[Abstract] This protocol was developed in a study aimed to determine the cellular localization of the lysin of pneumococcal phage SV1 (Frias et al., 2013). We obtained proteins from the surface of Streptococcus pneumoniae by elution with choline or those secreted to the medium. The analysis by Western blot of these fractions allowed us to demonstrate that the phage lysin localizes to the cell wall, associating with choline residues in the teichoic acids. Hence, protein extracts can be used to determine the localization of uncharacterized proteins and can also be useful for other ...
[摘要] 该方案是在旨在确定肺炎球菌噬菌体SV1的溶素的细胞定位的研究中开发的（Frias等人，2013）。 我们通过用胆碱或分泌到培养基的那些洗脱从肺炎链球菌的表面获得蛋白质。 通过这些级分的Western印迹分析允许我们证明噬菌体溶素定位于细胞壁，与磷壁酸中的胆碱残基相关。 因此，蛋白质提取物可用于确定未表征蛋白质的定位，并且还可用于其他生物化学分析，例如蛋白质鉴定。 该方案可以容易地适应于不同的肺炎球菌菌株和生长条件，并且其非常适合于分离其他感兴趣的蛋白质。
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