Functional Genomic Studies of Early Myogenic Differentiation

该项目的总体目标是采用一种新颖的功能基因组学方法来识别基因 其产品诱导小鼠胚胎干（ES）细胞向肌肉细胞分化。在 目标 1，将开发一种高度缺陷的、非细胞毒性的 HSV 突变体，其表达缺陷 即刻早期 (IE) 基因，能够在经过改造的细胞中复制 载体诱导型、反式互补 ICP4 基因。在目标 2 中，将创建一组小鼠 ES 细胞系 其中 EGFP 或 ICP4 基因将被重组到与 驱动肌肉分化因子 Pax7、myf5 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记物的检测相关。在 目标 3，突变病毒将被设计为包含一条细菌人工染色体和一条 重组系统“Gateway”（Invitrogen），然后将用于引入小鼠肌肉 l 将祖细胞cDNA文库导入载体中。载体库将作为单个载体在细菌中繁殖 lcopy质粒，其复杂性是通过低拷贝和中拷贝基因的定量PCR确定的 l 转染至 ICP4 互补的 Vero 细胞中用于病毒生产。在目标 4 中，汇集的文库载体将 LBE 在微量滴定孔中的补充 Vero 细胞上繁殖，并将复制品铺在三个生肌细胞上 启动子-ICP4 ES细胞系。通过诱导在表达 ICP4 的细胞中产生子代载体颗粒 通过补充 Vero 细胞的有限稀释来分离驻留的生肌启动子，并且 对 EGFP ES 细胞系进行重新筛选，以确定细胞因子是否在 激活早期和晚期生肌因子启动子。激活载体的cDNA为 测序，与已知数据库进行比较以初步评估功能并进一步 其特点是能够使用免疫学和生化测定诱导肌肉细胞标记。 最终，我们希望识别出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.