Molecular Expression and Morphology Core

核心C设施为研究人员提供最先进的分子生物学和形态支持 以及他们的工作人员和新的调查人员，为PPG的未来方向收集数据。这个 分子成分公司已经开发出最先进的定量聚合酶链式反应方法，用于许多酶促反应- 肌层内分散的细胞。这是通过使用细胞特异的荧光记者来实现的 对分离的血管内皮细胞、平滑肌细胞、血管内皮生长因子受体α和肠神经进行转录表达鉴定 在这些特定的细胞类型中。核心也能够将这一点扩展到其他物种，包括 通过使用抗体的胞外表位标记灵长类动物和人类。核心持续执行 RT-PCR法验证经荧光激活细胞纯化的细胞群体的身份 分类(FACS；由核心B执行)或掌上激光捕获(核心C)。这些分析已经被 扩展到包括对这些细胞群体中特定转录物表达的实时聚合酶链式反应分析。 该中心为所有适用的转基因动物提供常规基因分型(每年4000只动物) 为RT-PCR设计、设计和测试引物，并为拟议的实验构建载体。这个 CORE还继续提供基因组克隆、cDNA和培养的日常维护 分子生物学研究。最近，核心已将其服务扩展到单蜂窝 利用Fuidigm BioMark技术对单细胞进行QPCR。核心提供DNA序列分析 克隆和扩增产物，并为表达不同种类的哺乳动物细胞系提供支持 为几个离子通道和受体投射特定的cDNA。岩心的形态成分 继续提供常规光学和荧光显微镜、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、 免疫组织化学和细胞化学，以支持需要利用这些技术的个别项目 技巧。该组件具有很高的标准，并不断开发新的方法来 确定肌层内的细胞数量和体积。中的新协议和算法 与共聚焦显微镜和去卷积技术的结合为定量 肠壁内细胞类型的分析。对细胞进行适当的定量结构分析将 提供肠壁内不同细胞类型变化的有价值的信息。

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 state-of-the-art qPCR protocols for a number of enzymatically- dispersed cells within the tunica muscularis. This is achieved by using cell-specific fluorescent reporters for isolated ICC, smooth muscle cells, POGFRα and enteric nerves to identify transcript expression within these specific cell types. The core has also been able to expand this to other species including primate and human by labeling using extracellular epitopes of antibodies. The Core continually performs RT-PCR to verify the identity of cell populations that have been purified by fluorescent-activated cell sorting (FACS; conducted by Core B) or PALM laser capture (core C). These analyses have been extended to include real-time PCR analysis of specific transcript expression in these cell populations. The Core provides routine genotyping (4000 animals annually) 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. More recently the core has expanded its services to single cell qPCR for single cells using Fuidigm Biomark technology. 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 and fluorescence microscopy, laser scanning confocal microscopy, digital imaging, transmission electron microscopy, in-situ hybridization, immunohisto- and cytochemistry, 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 quantitative analysis of cells types within the gut wall. Appropriate quantitative structural analysis of cells will provide valuable information of changes which occur in different cell types within the in gut wall.