The Molecular Biology and Mouse Core component of the MTCC will serve the gene transfer community by providing mouse models relevant to cystic fibrosis (CF) gene transfer and access to molecular biology equipment and expertise. Although not always predictive of results in human trials, animal models are nonetheless useful for establishing efficacy of vectors and understanding levels of gene expression necessary for functional correction. For pilot studies designed to evaluate new AAV vectors in the context of the gut, the CFTR-deficient mouse model will be provided. This model shows a highly significant gut phenotype that is very similar to human cystic fibrosis (CF). Additionally, for lung studies, the Core will provide p-ENaC over-expressing mice on an inbred background (C57BI/6N) and the same mice bred to the CFTR-deficient mice. Both of these mice provide a model for gene transfer to a mucus-filled, inflamed airway that might be typical of a human CF lung. The lack of CFTR in the (3-ENaC-CFTR model provides an opportunity to explore CFTR gene transfer in a very relevant system. To support pilot project efforts, the Core will also aid in the generation and maintenance of Dnail- and selected mucin-deficient mice. As a second major focus, the Core will assist investigators with molecular biology needs by providing access to relevant equipment and conducting specialized molecular biology services. To reach these objectives, the Core will maintain and provide access to specialized instrumentation, specifically to evaluate the quality and quantity of RNA and DMA and to conduct quantitative real-time PCR. The Core will also provide qPCR services for individuals conducting gene transfer who need to evaluate mRNA expression levels, e.g., expression of CFTR after gene transfer. In addition, the Core will be the centralized location to coordinate access to and evaluation of a library of institutionally obtained shRNA lentiviral vectors. The Core will serve to validate available shRNA vectors for functional knock-down and subsequently provide plasmids to the Vector Core for production. And finally, the Core will be the centralized location for Affymetrix gene array analysis for evaluating the disease development in the p-ENaC over-expressing mice, an effort that will help to unravel the pathogenesis in this model and provide clues to early changes in the physiology and cell biology of the lung in the context of a CF-like disease. Such information may well identify new targets for therapy. The Molecular Biology and Mouse Core has built a solid foundation of equipment and expertise. As such, use of the Core should improve efficiency and quality of the entire MTCC effort.
The Molecular Biology and Mouse Core will support faculty and staff conducting research related to gene transfer for diseases such as cystic fibrosis. Additionally, it supports research related to understanding the molecular mechanisms that lead to disease and hinder gene transfer. The results of these studies should also have broad relevance to other pulmonary diseases. Thus, the research proposed has direct relevance to public health.
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