FACTOR(S) CONTROLLING GLOBIN GENE EXPRESSION IN K562 CELLS

K562 细胞中控制球蛋白基因表达的因子

• 批准号: 3964304
• 负责人: J ELION
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3964304
• 关键词: autoradiography; azacitidine; developmental genetics; gel electrophoresis; gel filtration chromatography; gene expression; genes; genetic manipulation; genetic transcription; human tissue; ion exchange chromatography; messenger RNA; nucleic acid sequence; plasmids; radiotracer; tissue /cell culture

K562 cells are used as a model to study the control of human globin gene expression. They express embryonic and fetal but not adult globin genes. Results suggest that the lack of transcription of the adult beta-globin gene results from an alteration of a regulating trans-acting factors(s). Our objectives are to provide evidence for the existence of such a factor, to elucidate its nature as an activator or a repressor and to set up an in vitro assay for its measurement. Globin promoters activity is deduced from the transient expression of the bacterial CAT enzyme in K562 cells transfected with plasmids in which transcription of the CAT gene is driven by various globin gene promoters. In competition experiments, cells have been cotransfected with these plasmids and an excess of the corresponding globin promoter. However, low efficiency and poor reproducibility of transfection impaired analysis of the results. Therefore, details of the transfection procedure have been thoroughly worked out to optimize reproducibility and efficiency. Since competition experiments required final DNA concentrations found to be inhibitory of CAT expression in control experiments, we are now constructing competitor plasmids containing multiple copies of the globin promoter sequences. Experiments conducted with K562 cells grown in the presence of 5-azacytidine, did not provide evidence for expression of the CAT plasmid containing the beta-promoter, but showed non-specific enhancement of expression of the other CAT plasmids. Finally, we are attempting to develop an assay for trans-acting factors that would allow use to track them in a purification scheme. We constructed SP6 promoter-containing plasmids in which the globin promoters are used as templates for in vitro transcription. We hope to be able to set up conditions in which binding of proteins to the promoter will result in premature transcription termination. Labelled transcripts could then be analyzed by polyacrylamide gel electrophoresis and autoradiography.

以K562细胞为模型研究人珠蛋白基因的调控 表达。 它们表达胚胎和胎儿的珠蛋白基因，但不表达成人的珠蛋白基因。 结果表明，成人β-珠蛋白转录的缺乏 基因是调节反式作用因子改变的结果。 我们的目标是为这种因素的存在提供证据， 阐明其作为激活剂或阻遏剂的性质，并建立一个in 其测量的体外测定。 球蛋白启动子活性是从 K562 细胞中细菌 CAT 酶的瞬时表达 用驱动 CAT 基因转录的质粒转染 由各种珠蛋白基因启动子。 在竞争实验中，细胞 用这些质粒和过量的相应质粒共转染 珠蛋白启动子。 但效率低、重复性差 转染损害了结果的分析。 因此，详细信息 转染程序已彻底制定以优化 再现性和效率。 由于需要进行竞争实验 发现最终 DNA 浓度抑制 CAT 表达 对照实验，我们现在正在构建含有 球蛋白启动子序列的多个拷贝。 进行的实验 在 5-氮杂胞苷存在下生长的 K562 细胞，没有提供 含有 β 启动子的 CAT 质粒表达的证据， 但显示出其他 CAT 表达的非特异性增强 质粒。 最后，我们正在尝试开发一种反式作用检测方法 允许在纯化方案中跟踪它们的因素。 我们 构建了含有 SP6 启动子的质粒，其中珠蛋白启动子 用作体外转录的模板。 我们希望能够 设置蛋白质与启动子结合的条件 转录过早终止。 标记的转录本可以是 通过聚丙烯酰胺凝胶电泳和放射自显影进行分析。