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.）中胚层细胞是如何决定进入 骨骼肌细胞系？（B.）肌肉卫星细胞 衍生？（c.）骨骼肌基因是如何被激活的 生长因子缺乏（d.）基因的数量调节 表达控制相对于快和慢肌纤维类型？ 具体目标是： 【一、】M-肌酸激酶（MCK）基因控制元件的鉴定。 主要重点将是206 nt增强子，其含有多个 控制和因子结合元件。 相关研究将继续进行 分析其他调控区域，并将寻找基因座控制 地区 互补的转基因研究将评估似乎 是成年和胚胎小鼠中更关键的控制元件。 【2.】肌纤维类型控制MCK表达的分析。 初步结果表明3 MCK的差异表达 在快肌和慢肌纤维中的转基因将在体外进行， 快肌和慢肌细胞系。 单元格中定义的控制元素 培养试验将在功能齐全的肌肉中得到证实， 其他转基因小鼠。 【三、】MCK结合因子的分离和功能表征 基因控制元件 将继续进行因素约束分析， 目标1和2中确定的要素。通过cDNA文库鉴定的因子 将分析其在终末期间的行为 分化，在发育过程中表达， 存在于快速和慢速纤维类型中。 以后的研究将调查 控制选定的转录因子基因。 【4.】骨骼肌测定分析。 细胞和分子 激活成肌决定基因所需的环境， 将在培养物中研究中胚层细胞。 【五、】卫星细胞的形成机制。 延时视频显微镜 再加上单细胞测定肌肉基因激活，生长因子 受体和生肌决定因子将用于确定 成肌细胞如何进入静止的卫星细胞状态 本项目的结果应适用于骨骼肌 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.