Identification and Characterization of Tcf3 target genes

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中发挥关键作用。探索Tcf3在皮肤中的功能的一个重要步骤将是鉴定和表征其靶基因。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对出生后动物的影响，并评估Tcf3对体外角质形成细胞增殖特性的剂量依赖性效应。利用诱导系统，我将培养转基因小鼠的角质形成细胞，进行微阵列分析，以鉴定受Tcf3诱导影响的基因。我将使用Northern杂交和原位杂交来确认受Tcf3表达影响的基因，并分析候选基因与Tcf3结合的启动子。通过对Tcf3诱导和候选基因表达的时间过程分析，我应该可以进一步缩小候选基因的范围。同时，我将执行染色质IP结合CpG微阵列分析，以确定Tcf3直接靶基因。通过使用k14 /5启动子或敲除技术的转基因技术，我可以选择一些目标基因，通过操纵它们在皮肤中的表达来测试它们与皮肤干细胞维持的相关性。

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.