| In silico Analysis and Site-directed Mutagenesis of Promoters
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Author:
Date:
2017-03-20
[Abstract] In normal as in cancerous cells, gene expression is tightly regulated by transcription factors, which are responsible for up- or down-regulation of thousands of targets involved in different cell processes. Transcription factors can directly regulate the expression of genes by binding to specific DNA sequences known as response elements. Identification of these response elements is important to characterize targets of transcription factors in order to understand their contribution to gene regulation. Here, we describe In silico analysis coupled to selected mutagenesis and promoter gene reporter assay procedures to identify and analyze response elements in the proximal promoter sequence of genes.
[摘要] 正常情况下，在癌细胞中，基因表达受转录因子的严格调节，转录因子负责上调或下调参与不同细胞过程的成千上万个靶标。转录因子可以通过结合被称为反应元件的特定DNA序列直接调节基因的表达。识别这些响应元件对于表征转录因子的目标是重要的，以便了解它们对基因调控的作用。在这里，我们描述了与选定的诱变和启动子基因报告物测定程序相结合的电子分析，以鉴定和分析基因的近端启动子序列中的应答元件。
背景 转录因子对全球基因表达的影响可以通过其使用shRNA或CRISPR-Cas9方法的敲低来进行研究，然后进行微阵列分析，可以提供数百个失调基因的重要数据。这种分析虽然非常有用，但缺乏关于失调基因直接控制的信息。为了进一步研究这些基因，使用生物信息学的电子分析分析提供了额外的信息来鉴定研究的转录因子的直接靶标。另外，可以使用定点突变和启动子 - 基因 - 报道分析来分析这些响应元件的功能。我们最近已经表明，致癌转录因子MYC（细胞性骨髓细胞瘤癌基因）控制结肠直肠癌中ITGA1（整联蛋白α1亚基）基因的表达，并且它们的表达在72％的结肠直肠肿瘤中相关。该协议描述了用于分析ITGA1启动子的程序和用于MYC致癌基因的响应元件的鉴定。后者已知是MYC / MAX / MAD网络的成员。这些因素之间的相互作用导致基因激活或抑制，这取决于上游信号和细胞条件。
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| Cell-based Assays to Monitor AID Activity
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Author:
Date:
2016-02-05
[Abstract] The enzyme Activation induced deaminase (AID) underpins antibody affinity maturation and isotype switching through its mutagenic activity of deaminating deoxycytidine to deoxyuridine in DNA. Subsequent processing of the deoxyuridine initiates the processes of somatic hypermutation (SHM) and class switch recombination (CSR) in B cells. Structure-function analysis of AID requires sensitive and biologically relevant methods to measure its various activities. Here we describe simple but effective methods to measure 1) the ability of AID to mutate the Escherichia coli genome, which provides an indication of its catalytic activity; 2) the capacity of AID to perform SHM by complementing a derivative of the DT40 chicken B cell line; 3) the ability of AID to perform CSR by complementing ...
[摘要] 酶活化诱导的脱氨酶（AID）通过其将脱氧胞苷脱氨基到DNA中的脱氧尿苷的诱变活性来支持抗体亲和力成熟和同种型转换。脱氧尿苷的后续加工引发B细胞中体细胞超突变（SHM）和类型转换重组（CSR）的过程。 AID的结构功能分析需要灵敏和生物相关的方法来测量其各种活动。在这里我们描述简单但有效的方法来测量1）AID突变大肠杆菌基因组的能力，其提供其催化活性的指示; 2）AID通过补充DT40鸡B细胞系的衍生物来进行SHM的能力; 3）AID通过补充AID缺乏的原代小鼠B细胞来进行CSR的能力。三种方法的组合，伴随着AID亚细胞定位和蛋白质表达水平和稳定性的必要分析作为对照，允许AID的详细结构功能研究。
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| Binding to Secreted Bone Matrix in vitro
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Author:
Date:
2014-02-20
[Abstract] This method examines the bone matrix binding capacity of proteins. Using osteogenic differentiation medium, multipotent stromal cells (MSC) are induced to differentiate into osteocytes in vitro and to secrete bone matrix. The latter is confirmed using Alizarin red S staining, which detects the presence of calcific deposits (hydroxyapatite). These calcific deposits are used to test the bone binding properties of proteins. The binding to the calcific deposits is assessed by Western blot analysis.
[摘要] 这种方法检查蛋白质的骨基质结合能力。 使用成骨分化培养基，诱导多潜能基质细胞（MSC）在体外分化成骨细胞和分泌骨基质。 后者使用茜素红S染色证实，其检测钙化沉积物（羟基磷灰石）的存在。 这些钙沉积物用于测试蛋白质的骨结合性质。 通过蛋白质印迹分析评估与钙化沉积物的结合。
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