Molecular Expression and Morphology Core

点击翻译按钮获取中文摘要

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.