MOUSE DIHYDROFOLATE REDUCTASE GENE EXPRESSION

小鼠二氢叶酸还原酶基因表达

• 批准号: 2085180
• 负责人: DAVID G COLLART
• 金额: $ 2.86万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-02-11 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/2085180
• 关键词: 3T3 cells; artificial chromosomes; cell cycle; cell growth regulation; clone cells; dihydrofolate reductase; gene expression; gene mutation; genetic regulation; laboratory mouse; messenger RNA; nucleic acid sequence; polymerase chain reaction; posttranscriptional RNA processing

The primary emphasis of this proposal is to study the regulation of dihydrofolate reductase (Dhfr) gene expression during changes in the proliferative state of the cell. The steady state levels and stability of various mouse Dhfr mRNA species will be analyzed during growth stimulation and quiescence in both the amplified and un-amplified states. Using cell lines with amplified copies of the Dhfr gene has several advantages: i)Dhfr MRNA can easily be detected due to the elevated level of expression in amplified cells and ii)Dhfr expression in the amplified state extends our understanding of its regulation in tumor cells which have used gene amplification as a method of resistance to folate antagonists such as methotrexate. However, Dhfr gene expression in an unamplified cell may be different than in the amplified state. There- fore, Dhfr gene expression will also be studied in the unamplified state. It has previously been difficult to study Dhfr expression in unamplified cells, but the recent development of quantitative PCR to detect mRNA has made this feasible. The sensitivity of the PCR assay will allow the analysis of thiouridine pulsed labeled RNA separated on organomercurial columns, thereby enabling us to study both stable and newly made Dhfr RNA. Secondly, various changes will be made in the primary structure of the Dhfr gene. Yeast genetics will be employed to make changes in the mouse Dhfr gene contained in a YAC clone. These YAC constructs will be transferred to Dhfr-deficient CHO cells and Dhfr expression in normal and tumorigenic cells, and additional insight into the role of Dhfr gene expression in the development of drug resistance in neoplastic cell.

该提案的主要重点是研究 二氢叶酸还原酶（DHFR）基因表达在变化中 细胞的增殖状态。 稳态水平和稳定性 在生长过程中将分析各种小鼠DHFR mRNA物种 放大和未扩增状态下的刺激和静止。 使用与DHFR基因放大副本的细胞系具有多个 优点：i）由于水平升高，很容易检测到DHFR mRNA 放大细胞中的表达和II）DHFR表达在扩增的 状态扩展了我们对其在肿瘤细胞中调节的理解 已经使用基因扩增作为一种抗叶酸的方法 甲氨蝶呤等拮抗剂。 但是，DHFR基因表达 未放大细胞可能与放大状态不同。 那里- 最重要的是，DHFR基因表达也将在未扩增的状态下进行研究。 以前很难研究未扩大的DHFR表达 细胞，但最近的定量PCR检测mRNA的发展已 使这个可行。 PCR分析的敏感性将允许 分析在有机尾部分离的标记RNA的硫氨醇脉冲分析 列，从而使我们能够研究稳定和新制作的DHFR RNA。 其次，将在主要结构中进行各种更改 DHFR基因。 酵母遗传学将被用来改变 小鼠DHFR基因包含在YAC克隆中。 这些YAC结构将是 在正常和 肿瘤细胞，以及对DHFR基因作用的额外洞察力 在肿瘤细胞中耐药性发展中的表达。