The human gut epithelium undergoes continuous rapid renewal throughout life. A major unfulfilled goal of GI research is to define the properties of the stem cells that fuel this renewal. The results will have important therapeutic and diagnostic implications for diseases as varied as GI neoplasia, the severe mucositis that accompanies radio- or chemotherapy, and the loss or disruption of intestinal absorptive function due to inflammatory bowel diseases, or surgical resection. We are proposing a gut epithelial cell progenitor GAP that uses two gnotobiotic transgenic mouse models developed in the laboratory of the Principal Investigator as part of its long-standing examination of gut stem cell biology. The representation of multipotent gastric stem cells and their immediate committed daughters is dramatically increased in one of these models through ablation of acid-producing parietal cells. In another model, the representation of intestinal epithelial progenitors is augmented markedly through removal of Paneth cells from the base of crypts of Lieberkuhn. Each model provides a unique opportunity to directly recover progenitors from anatomically distinct structures by laser capture microdissection (LCM) in sufficient numbers and purity to profile expression of their component mRNAs. Our goal is to provide the most comprehensive profile of progenitor cell gene expression possible: i.e., one that includes low abundance transcripts. To accomplish this goal, we have formed a consortium that includes members of Washington University's Genome Sequencing Center, and Michael Lovett (Professor of Genetics), an expert in the development of methods for creating normalized cDNA libraries from small numbers of cells. This GAP has 2 aims: (I) Generate and sequence normalized and subtracted cDNA libraries from LCM mouse gastric and intestinal epithelial progenitors. We will provide the public with annotated, Internet-accessible databases of expressed genes, and create tools that will allow our databases to be used as probes for examining other expression profiles. Sequenced clones will be distributed to the research community through the I.M.A.G.E. Consortium. (2) Relate what we find in mice to the human gut. Lessons learned from the adult mouse during the course of this GAP will serve as a template for a directed and focused exploration of the features of adult human gut epithelial progenitors. This will be accomplished using a 3-tier expression profiling scheme. (1) A subset of genes will be selected from our three normalized and two subtracted libraries and their expression patterns analyzed in normal adult FVBIN mouse gut using sensitive SYBR-Green based real time quantitative RT-PCR (qRT-PCR) assays of LCM epithelial cells harvested from progenitor and non-progenitor compartments within the gastric unit, the small intestinal crypt-villus unit, and the colonic crypt. (2) A subset of genes from (1) will be selected for LCM/qRT-PCR expression profiling of human gastric units, small intestinal crypt-villus units, and colonic crypts. (3) In situ hybridization studies of the adult mouse and human gut will be used to obtain a more refined view of the cellular basis of expression of genes selected from (1) and (2).
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