Identification and Characterization of Tcf3 target genes

Tcf3 靶基因的鉴定和表征

• 批准号: 6648110
• 负责人: HOANG NGUYEN
• 金额: $ 4.16万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-05-01 至 2006-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6648110
• 关键词: cell growth regulation; cell proliferation; cytogenetics; gene expression; genetic promoter element; genetically modified animals; immunoprecipitation; in situ hybridization; keratinocyte; laboratory mouse; microarray technology; northern blottings; postdoctoral investigator; skin; stem cells; terminal nick end labeling; tissue /cell culture; transcription factor; western blottings

DESCRIPTION (provided by applicant): Several evidence suggest that the transcription factor Tcf3 might play a key role in maintaining skin stem cell properties. An important step in exploring Tcf3's functions in skins will be to identify and characterize its target genes. I will first generate inducible Tcf3 transgenic mice using the tetracycline inducible system. This will allow me to characterize Tcf3's effect on postnatal animals and assess the dose dependent effect of Tcf3 on keratinocytes proliferative characteristics in vitro. With the inducible system, I will culture keratinocytes from the transgenic mice to perform microarray analyses to identify genes that are affected by Tcf3 induction. I will use Northern and in situ hybridization to confirm for genes that are affected by Tcf3 expression, and analyze promoters of the candidate genes for binding to Tcf3. By time course analyses of Tcf3 induction and the candidate genes' expression, I should be able to further narrow down the list of the candidate genes. In parallel, I will perform chromatin IP coupled with CpG microarray analyses to identify Tcf3 direct target genes. The combined approaches will allow me to select a few target genes to test for their relevance in skin stem cell maintenance by manipulating their expression in the skin, by either transgenic technology using K 14/5 promoter or knockout technology.

描述(由申请人提供)：一些证据表明，转录因子Tcf3可能在维持皮肤干细胞特性方面发挥关键作用。鉴定和鉴定Tcf3的靶基因是探索Tcf3‘S在皮肤中功能的重要一步。我将首先使用四环素诱导系统产生可诱导的TCF3转基因小鼠。这将使我能够表征TCF3对出生后动物的S效应，并评估TCF3对体外培养的角质形成细胞增殖特性的剂量依赖性影响。利用诱导系统，我将从转基因小鼠中培养角质形成细胞，进行微阵列分析，以确定受Tcf3诱导影响的基因。我将使用Northern和原位杂交来确认受Tcf3表达影响的基因，并分析候选基因的启动子与Tcf3结合的情况。通过对Tcf3诱导和候选基因表达的时间进程分析，我应该能够进一步缩小候选基因的范围。同时，我将进行染色质IP和CpG微阵列分析，以确定Tcf3直接靶基因。这种结合的方法将使我能够选择一些目标基因来测试它们在皮肤干细胞维持中的相关性，方法是通过使用K14/5启动子的转基因技术或基因敲除技术来操纵它们在皮肤中的表达。