Functional Genomic Studies of Early Myogenic Differentiation

该项目的总体目标是采用一种新的功能基因组学方法来鉴定基因 其产物诱导小鼠胚胎干（ES）细胞向肌细胞分化。在 目的1，将开发一种高度缺陷的、无细胞毒性的HSV突变体，该突变体缺乏 立即早期（IE）基因，并能够在细胞中复制，工程改造含有一个 载体可诱导的反式互补ICP 4基因。在目标2中，将创建一组小鼠ES细胞系 其中EGFP或ICP 4基因将被重组到细胞基因组中， 驱动肌肉分化因子Pax 7、myf 5或myoD之一表达的启动子。使用 在建立的培养条件下，EGFP细胞系将向肌细胞分化， 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 目标3，突变病毒将被改造成含有细菌人工染色体和 重组系统，“Gateway”（Invitrogen），然后将其用于引入小鼠肌肉 将祖细胞cDNA文库插入载体中。载体文库将在细菌中作为单一载体进行繁殖。 lcopy质粒，其复杂性通过定量PCR建立低和中拷贝基因， 转染到ICP 4-互补Vero细胞中用于病毒生产。在目标4中，合并的文库载体将 l在微量滴定威尔斯孔中的补充Vero细胞上增殖，并将复制品接种到三个肌源性细胞上。 启动子-ICP 4 ES细胞系。在表达ICP 4的细胞中通过诱导 通过在互补的Vero细胞上有限稀释来分离固有的生肌启动子， 在一组EGFP ES细胞系上重新筛选，以确定细胞因子是否在 激活早期和晚期生肌因子启动子。激活载体的cDNA将被 测序，与已知的数据库进行比较，以初步评估功能，并进一步 使用免疫学和生物化学测定表征它们诱导肌细胞标志物的能力。 最终，我们希望确定新的因素或信号机制，相互作用，以控制早期肌肉 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.