E2F-6 and Repression of p19ARF Gene Expression

E2F-6 和 p19ARF 基因表达的抑制

• 批准号: 6615050
• 负责人: NICHOLAS H HEINTZ
• 金额: $ 0.06万
• 依托单位: UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-09-15 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6615050
• 关键词: DNA replication origin; apoptosis; artificial chromosomes; binding sites; cell cycle; electroporation; fluorescence microscopy; gene expression; gene induction /repression; genetic regulatory element; genetic transcription; genetically modified animals; laboratory mouse; messenger RNA; microarray technology; northern blottings; polymerase chain reaction; southern blotting; tissue /cell culture; transcription factor; transfection

DESCRIPTION (provided by applicant) The unusual organization of INK4a/ARF gene locus permits the expression of two overlapping transcripts that are translated in alternate reading frames. Recently we have developed a novel and highly efficient method for the transfer of bacterial artificial chromosomes (BACs) into cells in culture. Combined with the ability to introduce specific modifications in BACs by homologous recombination, this novel gene transfer system provides an opportunity to dissect the role of specific regulatory elements in the context of complex genetic loci, both in vitro and in BAC transgenic mice. Using this gene transfer system and modified alleles of INK4a/ARF, we propose to: 1) investigate the function of a TTTCCCGC E2F binding site in activation of pl9ARF mRNA expression in response to the transcriptional activators E2Fs 1-3, and 2) examine the specific role of E2F-6 in repression of pl9?ARF transcription. These studies will develop a new paradigm for dissecting the role of specific regulatory elements within the context of entire genetic loci in cells in culture, thereby permitting evaluation of specific BAC alleles prior to the generation of BAC transgenic mice.

描述（由申请人提供） INK4a/ARF 基因座的不寻常组织允许表达两种 在交替阅读框架中翻译的重叠转录本。 最近，我们开发了一种新颖且高效的转移方法 将细菌人工染色体（BAC）植入培养细胞中。结合 通过同源物在 BAC 中引入特定修饰的能力 重组，这种新颖的基因转移系统提供了一个机会 剖析特定监管要素在复杂环境中的作用 体外和 BAC 转基因小鼠中的遗传位点。利用这个基因 INK4a/ARF 的转移系统和修饰等位基因，我们建议：1) 研究 TTTCCCGC E2F 结合位点在 pl9ARF 激活中的功能 响应转录激活剂 E2F 1-3 和 2) 的 mRNA 表达 检查E2F-6在抑制p19？ARF转录中的具体作用。 这些研究将开发一个新的范式来剖析特定的作用 细胞中整个遗传位点范围内的调控元件 文化，从而允许在之前评估特定的 BAC 等位基因 BAC转基因小鼠的产生。