Molecular Expression and Morphology

核心C设施为研究者提供最先进的分子生物学和形态学支持， 他们的工作人员以及新的研究人员为PPG的未来方向收集数据。分子 一个研究小组已经开发出了用于酶促分散、分离平滑肌和ICC的RT-PCR方案 用绿色报告基因鉴定图尼卡内这些特定细胞类型内的转录本表达 包括人类在内的几种动物的肌层。核心不断进行RT-PCR，以验证 已通过荧光激活细胞纯化的图尼卡肌层内细胞群的同一性 分选（FACS;由核心B进行）。这些分析已经扩展到包括实时PCR分析 在这些细胞群中的特定转录表达。核心提供常规的转基因 为所有适用的项目设计和测试RT-PCR引物，并构建载体， 实验核心还继续提供基因组克隆，cDNA和 用于分子生物学研究的培养物。核心提供克隆的DNA序列分析， 扩增产物，并为表达各种项目特异性的哺乳动物细胞系提供支持 几种离子通道和受体的cDNA。核心的形态部分继续提供 在传统光学显微镜，相差显微镜，荧光显微镜， 激光扫描共聚焦显微镜，数字成像，透射电子显微镜，原位杂交， 免疫组织化学和免疫细胞化学，以支持有需要利用的个别项目， 这些技术。该组件具有很高的标准，并不断开发新的方法， 确定图尼卡肌层内的细胞数量和体积。新的协议和算法 与共聚焦显微镜和去卷积的结合为定量分析建立了新的标准 肠壁内的细胞类型，包括肠神经和ICC群体。适当的定量 细胞的结构和超显微分析将提供有价值的信息， 健康和疾病时肠道壁内的不同细胞类型。

Core C facility provides state of the art molecular biological and morphological support to investigators and their staff as well as new investigators collecting data for future directions of the PPG. The molecular component has developed RT-PCR protocols for enzymatically dispersed, isolated smooth muscle and ICCs with green reporters to identify transcript expression within these specific cell types within the tunica muscularis of several animal species including humans. The Core continually performs RT-PCR to verify the identity of cell populations within the tunica muscularis that have been purified by fluorescent-activated cell sorting (FACS; conducted by Core B). These analyses have been extended to include real-time PCR analysis of specific transcript expression in these cell populations. The Core provides routine genotyping of transgenic animals for all applicable projects, designs and tests primers for RT-PCR and constructs vectors for proposed experiments. The Core also continues to provide day to day maintenance of genomic clones, cDNAs and cultures for molecular biological investigations. The core provides DNA sequence analysis of clones and amplification products and provides support with mammalian cell lines expressing various project specific cDNAs for several ion channels and receptors. The morphology component of the Core continues to provide expertise in the areas of conventional light microscopy, phase contrast microscopy, fluorescence microscopy, laser scanning confocal microscopy, digital imaging, transmission electron microscopy, in-situ hybridization, immunohistochemistry and immunocytochemistry, to support individual projects that have the need to utilize these techniques. This component holds a high standard and is continually developing novel approaches to determine cellular numbers and volumes within the tunica muscularis. New protocols and alogrithims in combination with confocal microscopy and deconvolution have set new standards for the quantitiative analysis of cells types within the gut wall including enteric nerves and ICC populations. Appropriate quantatitive structural and ultrascructural analysis of cells will provide valuable information of changes which occur in different cell types within the in gut wall in health and disease.