Functional Genomic Studies of Early Myogenic Differentiation

这个项目的总体目标是使用一种新的功能基因组学方法来识别基因。 其产品可诱导小鼠胚胎干细胞向肌肉细胞分化。在……里面 目的1.将开发一种高度缺陷、无细胞毒性的HSV突变体，该突变体缺乏 即刻早期(IE)基因和能够在细胞中复制的基因被设计为含有 载体可诱导、反式互补的ICP4基因。在Aim 2中，将创建一组小鼠ES细胞系 其中EGFP或ICP4基因将被重组到细胞基因组中，并与 驱动肌肉分化因子之一Pax7、myf5或myoD表达的启动子。vbl.使用 在已建立的培养条件下，EGFP细胞系将被分化为肌肉细胞和 荧光细胞的出现将与阶段特异性肌细胞标志物的检测相关。在……里面 目的3，突变病毒将被改造成包含一个细菌人工染色体和一个 重组系统，“Gateway”(Invitgen)，然后将被用来引入小鼠肌肉 将异基因细胞c DNA文库导入载体。该载体文库将以单一形式在细菌中繁殖 低拷贝和中拷贝基因的定量聚合酶链式反应确定其复杂性 将其导入ICP4互补的Vero细胞中，进行病毒生产。在AIM 4中，池化的库向量将 LBE在补充性Vero细胞上的微滴度培养和复制 启动子-ICP4 ES细胞系。诱导细胞产生表达ICP4的子代载体颗粒 通过对补充Vero细胞的有限稀释分离驻留的肌源性启动子和 对EGFP ES细胞系进行重新筛选，以确定细胞因子是否在 激活早期和晚期的生肌因子启动子。激活载体的cdna将是 已排序，与已知数据库进行比较，用于初步评估功能，并进一步 其特点是能够通过免疫学和生化分析来诱导肌肉细胞标记物。 最终，我们希望确定相互作用以控制早期肌肉的新因素或信号机制 细胞发育。

The overall goal of this project is to employ a novel functional genomics approach to identify genes whose products induce the differentiation of mouse embryonic stem (ES) cells towards muscle cells. In Aim 1, a highly defective, non-cytotoxic HSV mutant will be developed that is deficient for expression of the immediate early (IE) genes and capable of replication in cells engineered to contain a vector-inducible, trans-complementing ICP4 gene. In Aim 2, a panel of mouse ES cell lines will be created in which either the EGFP or ICP4 gene will be recombined into the cellular genome juxtaposed to the promoter that drives expression of one of the muscle differentiation factors Pax7, myf5, or myoD. Using established culture conditions, the EGFP cell lines will be differentiated towards muscle cells and the appearance of fluorescing cells will be correlated with detection of stage specific myocytic markers. In Aim 3, the mutant virus will be engineered to contain a bacterial artificial chromosome and a recombination system, "Gateway" (Invitrogen), which will then be used to introduce a mouse muscle lprogenitor cell cDNA library into the vector. The vector library will be propagated in bacteria as single lcopy plasmids, its complexity established by quantitative PCR for low and medium copy genes and ltransfected into ICP4-complementing Vero cells for virus production. In Aim 4, pooled library vectors will lbe propagated on complementing Vero cells in microtiter wells and replica plated onto the three myogenic promoter-ICP4 ES cell lines. Progeny vector particles generated in cells expressing ICP4 by induction of the resident myogenic promoter will be isolated by limiting dilution on complementing Vero cells and rescreened on the panel of EGFP ES cell lines to determine whether the cellular factor is functional in activating early and late stage myogenic factor promoters. The cDNA of activating vectors will be sequenced, compared with known data bases for preliminary assessment of function and further characterized for their ability to induce muscle cell markers using immunological and biochemical assays. Ultimately, we hope to identify novel factors or signaling mechanisms that interact to control early muscle cell development.