REGULATION OF SKELETAL MUSCLE DIFFERENTIATION

这个项目解决了骨骼肌的几个基本问题。 生物学：(a.)中胚层细胞是如何变得决心进入 骨骼肌细胞谱系？(B)肌肉卫星细胞是如何 派生的？(c.)骨骼肌基因是如何被激活的 生长因子剥夺？(D)基因中的数量调控是如何进行的 快肌纤维和慢肌纤维的表达受控制吗？ 具体目标是： [1.]肌酸激酶(MCK)基因调控元件的鉴定 重点将放在206 NT增强器上，它包含多个 控制和因子绑定元素。相关研究将继续进行 分析其他监管区域，并将搜索基因座控制 地区。互补性的转基因研究将评估似乎 在成年和胚胎小鼠中是更关键的控制元件。 [2.]肌纤维类型对MCK表达的调控分析。 3种MCK基因差异表达的初步研究 在快肌纤维和慢肌纤维中的转基因将在体外利用 快肌细胞系和慢肌细胞系。单元格中定义的控制元素 培养分析将在完全功能的肌肉中得到证实。 额外的转基因小鼠。 [3.]MCK结合因子的分离及功能鉴定 基因控制元件。因子结合分析将继续 AIMS 1和AIMS 2中鉴定的元件.通过cDNA文库鉴定的因子 将对他们在临终关怀期间的行为进行分析 分化，它们在发育过程中的表达，以及它们的 存在于快纤维和慢纤维类型中。以后的研究将调查 对选定的转录因子基因的控制。 [4.]骨骼肌测定的分析。细胞和分子 激活肌源性决定基因所需的环境 中胚层细胞将在培养中进行研究。 [5.]卫星细胞的形成机制。延时视频显微镜 结合单细胞检测肌肉基因激活、生长因子 将使用受体和生肌决定因子来确定 成肌细胞如何进入静止的卫星细胞状态。 这个项目的结果应该适用于骨骼肌 疾病、基因治疗和受伤肌肉的外科重建 组织。

This project addresses several basic questions of skeletal muscle biology: (a.) how do mesodermal cells become determined to enter the skeletal muscle cell lineage? (b.) how are muscle satellite cells derived? (c.) how are skeletal muscle genes activated in response to growth factor deprivation? (d.) how are quantitative modulations in gene expression controlled with respect to fast and slow muscle fiber types? The specific aims are: [1.] Identification of M-creatine kinase (MCK) gene control elements. Major emphasis will be on a 206 nt enhancer which contains multiple control and factor-binding elements. Related studies will continue analysis of other regulatory regions, and will search for locus control regions. Complementary transgenic studies will evaluate what appear to be the more critical control elements in adult and embryonic mice. [2.] Analysis of muscle fiber type control of MCK expression. Preliminary results demonstrating differential expression of 3 MCK transgenes in fast and slow muscle fibers will be pursued in vitro using fast and slow muscle cell lines. Control elements defined in cell culture assays will be confirmed in the fully functional muscles of additional transgenic mice. [3.] Isolation and functional characterization of factors that bind MCK gene control elements. Factor binding analyses will continue with elements identified in Aims 1 & 2. Factors identified via cDNA library screening will be analyzed for their behavior during terminal differentiation, for their expression during development, and for their presence in fast and slow fiber types. Later studies will investigate the control of selected transcription factor genes. [4.] Analysis of skeletal muscle determination. Cellular and molecular environments required for activating myogenic determination genes in mesodermal cells will be studied in culture. [5.] Mechanism of satellite cell formation. Time-lapse videomicroscopy coupled with single cell assays for muscle gene activation, growth factor receptors and myogenic determination factors will be used to determine how myoblasts enter a quiescent satellite cell state. Results from this project should be applicable to skeletal muscle diseases, gene therapy, and the surgical reconstruction of injured muscle tissue.