Molecular biology, which in a broad sense covers the manipulation of DMA and RNA, has become an integral part of most biological research programs, including the UNC Program Project Grant (PPG). Research areas that utilize molecular biology techniques include cloning genes for expression of proteins in cell model systems, site-directed mutagenesis and tagging for cellular localization and functional studies, polymerase chain reaction for cloning and genotyping, RNA isolation and expression analysis, and the development of transgenic mice. The Molecular Biology Core was established at the University of North Carolina (UNC) Cystic Fibrosis Center in 1998 to provide molecular biology services and expertise to faculty who are often primarily trained in cell biology and/or physiology. The Core is currently an integral part of the ongoing PPG to provide primarily cloning and antibody production services. The functions of the Core have been expanded in the latest proposal to suit new project needs, and now include the development and genotyping of transgenic mouse models and RNA expression analyses services. Specifically, the PPG investigators will require the construction of adenoviral, retroviral, and Xenopus expression vectors for expression of a variety of tagged and/or modified proteins, such as CFTR, ENaC subunits, proteases, and nucleotide transporters, in a variety of cell models including well-differentiated human bronchial epithelial (HBE) cell cultures (provided by Core C. Cell Culture). In addition, the Molecular Biology/Mouse Genotyping Core will provide for the generation of new transgenic mouse models (both knock-out and overexpressing models) to study the role of nucleotides and nucleosides release and metabolism in the airway surface liquid regulation and to study the role of proteases in the regulation of ENaC function. Along with this, genotyping of transgenic models for generation of experimental and control animals will also be provided by the Molecular Biology/Mouse Genotyping Core. And finally, RNA isolation and expression analysis services, including access to quantitative real-time PCR equipment and expertise, will be provided. In addition, the Core is continuously striving to provide access to new research tools, including the development of siRNA technologies, and we will seek to make these technologies available for PPG investigators as protocols and strategies are developed. The Molecular Biology/Mouse Genotyping Core will be utilized by all four proposed PPG projects, and thus is an integral component of the efforts outlined in this application.
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