Dr. Steven Fiering who is the director of the Dartmouth Transgenic Mouse Facility will oversee the activities of Core C. Three of the five projects (projects 2,3, and 4) use transgenic (Tg) mice as central elements of their experimental approaches. The purpose of the core is to develop (where needed), maintain, and interbreed the multiple transgenic mouse strains on the appropriate genetic background and to provide these mice as required for these experiments. Part of Core C's mission is to produce unique strains of mice, specifically one strain of transgenic mice (hPSA) and two strains of speed congenic mice (TNFRI-/- and TNFRII-/- mice bred onto the Balb/c background). The production of the speed congenic mice enables very rapid (3-4 generations, as compared to the usual 10 generations) production of transgenic mice on a specific genetic background. The process entails super ovulation of females and subsequent mating, transfer of oocytes to foster mothers and screening of the offspring with multiple sets of PCR primers to identify those with the highest contribution of the desired genotype as well as the transgene. The identified and selected mice are utilized in subsequent repetitions of the process to rapidly obtain the transgenic mouse on the desired genetic background. Over the five year term of this proposal, the largest body of work to be performed by this core will be producing by interbreeding (where needed), maintaining and genotyping the following strains of transgenic mice: hCD64 transgenic, TGFbeta+/- and TGFbeta-/-, hPSA, transgenic, hCD64 transgenic, DO11.10 TCR transgenic, RAG-/-, TGFbeta+/- xRag-/-, TGFbeta-/-/Rag-/-, TNFRI-/-, TNFRII-/-, TNFRI-/-xTGFbeta+/-, TNFRI-/-xTGFbeta-/-, TNFRII-/-xTGFbeta+/-, and TNFRII-/-xTGFbeta-/-. Further efforts will include freezing of embryos from the most useful of the transgenic lines including those not produced at Dartmouth to preserve these lines so that they can be retrieved if needed without having to continually breed and genotype them. Genotyping will be a major portion of the task of Core C. TGFbeta+/-, TGFbeta-/- and hPSA mice will be genotyped by PCR of tail biopsies. Rag-/-, hCD64 transgenic, DO11.10 TCR transgenic, TNFRI-/- and TNFRII-/- mice will be genotyped by flow cytometry of peripheral blood with specific fluorescently labeled antibody reagents that are currently available commercially. The Core C director and the Dartmouth Transgenic Mouse Facility have extensive experience in the production and genotyping of transgenic mice and have all the capabilities required to perform the tasks of Core C. The Dartmouth Transgenic Facility is subsidized by the Dartmouth Medical School for production of transgenic mice, and the proposed budget reflects this subsidy for the production of the hPSA transgenic mouse, however there is no subsidy for the other tasks to be performed by Core C and the budget reflects the need to fully recover these costs.
Showing the most recent 10 out of 129 publications