Molecular Expression and Morphology Core

分子表达和形态学核心

• 批准号: 8742146
• 负责人: Sean M Ward
• 金额: $ 23.86万
• 依托单位: UNIVERSITY OF NEVADA RENO
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至 2019-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8742146
• 关键词: Animals; Antibodies; Area; Biological; Cell Line; Cell Separation; Cells; Collaborations; Complementary DNA; Confocal Microscopy; DNA Sequence Analysis; Data; Electron Microscopy; Enteral; Epitopes; Fluorescence Microscopy; Future; Genomics; Genotype; Human; Immunohistochemistry; In Situ Hybridization; Individual; Intestinal Motility; Investigation; Ion Channel; Label; Laser Scanning Confocal Microscopy; Lasers; Maintenance; Mammalian Cell; Microscopy; Molecular; Molecular Biology; Morphology; Nerve; Population; Primates; Proteins; Protocols documentation; Reporter; Research Personnel; Reverse Transcriptase Polymerase Chain Reaction; Running; Services; Smooth Muscle Myocytes; Structure; Techniques; Technology; Testing; Time; Transcript; Transgenic Animals; Transmission Electron Microscopy; Work; cell type; cytochemistry; design; digital imaging; experience; extracellular; high standard; light microscopy; novel strategies; protein expression; receptor; research study; two-photon; vector; ward

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.

核心C设施为研究人员提供最先进的分子生物学和形态学支持 及其工作人员以及为PPG未来方向收集数据的新研究者。的 分子组分已经开发了用于许多酶促的最先进的qPCR方案， 在图尼卡肌层内有分散的细胞。这是通过使用细胞特异性荧光报告子来实现的 对于分离的ICC、平滑肌细胞、POGFRα和肠神经，以鉴定转录物表达 在这些特定的细胞类型中。核心还能够将其扩展到其他物种，包括 灵长类动物和人的抗体的细胞外表位标记。核心不断执行 RT-PCR验证已通过荧光激活细胞纯化的细胞群的身份 分选（FACS;由核心B进行）或PALM激光捕获（核心C）。这些分析是 扩展到包括对这些细胞群中特定转录本表达的实时PCR分析。 核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） 项目，设计和测试RT-PCR的引物，并为拟议的实验构建载体。的 核心还继续提供基因组克隆，cDNA和培养物的日常维护， 分子生物学研究最近，Core已将其服务扩展到单细胞 使用Fuidigm Biomark技术进行单细胞qPCR。核心提供了DNA序列分析， 克隆和扩增产物，并为表达各种 为几种离子通道和受体设计特异性cDNA。核心的形态组成部分 继续提供传统光学和荧光显微镜，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.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相结合，为定量 分析肠壁内的细胞类型。适当的细胞定量结构分析将 提供肠壁内不同细胞类型发生变化的有价值信息。