ANALYSIS OF GLOBIN GENE EXPRESSION IN SINGLE CELLS

单细胞中球蛋白基因表达分析

• 批准号: 6161925
• 负责人: A N SCHECHTER
• 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/6161925
• 关键词: RNA directed DNA polymerase; blood /lymphatic pharmacology; cell line; erythroid stem cell; ethidium; fluorescent dye /probe; gel electrophoresis; gene expression; genetic models; genetic regulation; globin; human genetic material tag; messenger RNA; model design /development; natural gene amplification; polyadenylate; polymerase chain reaction; single cell analysis

Developmental and tissue specific regulation of globin gene expression has been the subject of intense investigation as part of an effort to develop a general model of eukaryotic gene regulation. In addition, pharmacologic manipulation of globin gene expression in human erythroid cells holds considerable promise as a means of controlling the clinical course of thalassemia and sickle cell disease. A system for assay of gene expression in single cells is being developed in order to elucidate the mechanism by which pharmacologic agents modulate the level of expression of globin gene. Owing to the limited quantity of mRNA contained within a single cell, the assay involves amplification of poly (A) RNA in a template independent manner while maintaining their relative abundance. This involves reverse transcription of 500-700 bases from the 3' end of each mRNA followed by poly (A) tailing of the 3' end of the new cDNA and PCR amplification using oligo(dT) based primers. Assay of specific templates by PCR coupled with real time detection of a fluorescence signal produced from a labeled gene specific probe provides the quantitation. The assay system is currently being tested using a K-562 cell line which expresses human fetal and embryonic globins. A considerable amount of heterogeneity of embryonic globin expression has already been observed between cells using agarose gel electrophoresis and ethidium bromide detection of amplified products; the implementation of real time detection will allow for rigorous quantitation of the initial amount of template. It is anticipated that such a quantitative approach will ultimately permit the identification of candidate genes as targets of drug action and contribute to a greater understanding of the globin gene switching phenomenon.

珠蛋白基因表达的发育和组织特异性调控 一直是深入调查的对象， 发展真核基因调控的一般模型。 此外，本发明还提供了一种方法， 人红系中珠蛋白基因表达的药理学操作 细胞作为控制临床疾病的一种手段， 地中海贫血和镰状细胞病的病程。 一种用于测定以下物质的系统， 单细胞中的基因表达正在发展，以阐明 药理学试剂调节 珠蛋白基因的表达。 由于mRNA的数量有限， 包含在单个细胞内，该测定涉及扩增多聚 (A)RNA在模板独立的方式，同时保持其 相对丰度 这涉及到逆转录500-700 从每个mRNA的3'端开始，然后是多聚腺苷酸加尾， 新cDNA的3'末端和使用基于寡核苷酸（dT）的PCR扩增 引物 PCR结合真实的时间检测特异性模板 检测由标记的基因特异性抗体产生的荧光信号 探针提供定量。 该分析系统目前正在 使用表达人胎儿和胚胎的K-562细胞系进行测试 球蛋白 胚胎珠蛋白的大量异质性 已经使用琼脂糖凝胶在细胞之间观察到表达 扩增产物的电泳和溴化乙锭检测; 真实的时间检测的实现将允许严格的 模板初始量的定量。 预计各国 这种定量方法最终将允许识别 作为药物作用靶点的候选基因， 对珠蛋白基因转换现象的理解。