GNOTOBIOTIC CORE

The CGIBD has had a gnotobiotic animal core from its inception. The gnotobiotic core was founded in 1985 at the College of Veterinary Medicine at NC State University, by Philip Carter, PhD, who had a long-standing interest and considerable expertise in gnotobiotic research. Although the services and personnel have evolved over time, the collaboration on this project between CGIBD members at our two principle institutions - UNC Chapel Hill and North Carolina State University - has remained constant. The Gnotobiotic Core has also grown in importance to the Center as we have emphasized gene-environmental interactions and sought to understand the role of intestinal microbiota in the genesis of inflammatory bowel diseases. Without a gnotobiotic facility this extremely productive line of research would be impossible. Dr. Carter resumed the directorship in 1998 following the retirement of the two prior core directors, Charles McPherson and Thomas Hamm. Dr. Carter was replaced in 2000 by Sue Tonkonogy, Ph.D., an experienced cellular immunologist at NC State University, College of Veterinary medicine who has worked with animal models of chronic intestinal inflammation for the past 15 years. Dr. Tonkonogy leads the core in concert with Balfour Sartor, Associate Director, who has used this facility from its inception. The range of animals and services provided by the core has expanded progressively over time. As described in detail below, the core now provides a range of both conventional and genetically engineered rodent strains and zebrafish maintained in carefully defined microbial environments. Depending on the requirements of the individual experiment, these animals maybe axenic (germ-free), or maybe specifically colonized with one or a combination of bacterial or fungal strains of interest. Because demand by members of our Center and NIDDK-funded investigators around the country for gnotobiotic mice and rats dramatically increased, and because UNC-CH investigators comprised the vast majority of our user base, we expanded our capacity in 2001 by creating a parallel Gnotobiotic Rodent Facility on the UNC-Chapel Hill campus. Equipment for this expansion was provided by a one-time equipment grant from the North Carolina Biotechnology Center ($73,475 direct costs), with matching funds from the UNC-Chapel Hill School of Medicine ($24,492). Personnel costs and some equipment expenses were provided by a 1 year administrative supplement by the NIDDK to the CGIBD ($69,000 direct costs), with the directive that this expanded facility should provide germfree and selectively colonized rodents to NIDDK-funded investigators. Funds were also provided by the UNC Dean's Research Advisory Committee and UNC-Chapel Hill Department of Medicine. Expansion of our facility was driven by several needs: 1) the expanded needs of CGIBD members for gnotobiotic animals, 2) the need to provide a facility that was easily accessible to our largest user base, 3) the needs of external NIDDK-funded investigators for gnotobiotic rodents, 4) the need for efficient derivation of new germ-free rodent strains, and 5) the need to provide back up breeding colonies for unique strains. In addition, this expanded facility permitted us to establish a partnership with the NIH National Center for Research Resources (NCRR)-funded Mutant Mouse Regional Resource Center (MMRRC) at UNC-Chapel Hill. The need to provide back up breeding colonies was essential because we had the only existing germ-free colonies of HLA B27 transgenic rats and IL-10 knockout mice on a susceptible 129S6/SVEV background worldwide. This need was precipitated by closure of the University of Wisconsin Gnotobiotic Unit in 2002 following the retirement of Dr. Ed Balish. Our unit had operated in parallel with Dr. Balish's unit at the University of Wisconsin since its inception, with each unit having independent germ-free breeding colonies of each unique rodent strain. No germ-free unit has ever been maintained entirely free of contamination. The average published contamination rate is 5.9% per isolator per month for rats and 3.9% for mice, (Saito and Nomura, Production of Germ-free Animals, in The Germ-free Animal in Biomedical Research, Eds. MD Coates and BE Gutaffson, Laboratory Animals Ltd., London, 1984, p 40). In case of contamination in one unit, the other facility could supply breeders to restore breeding colonies. Traditionally, our smaller CGIBD facility had depended on the size and expertise of the Wisconsin facility for germ-free derivation of new animal strains. When the Wisconsin resource closed, we developed methods of germ-free embryo transplant and have successfully derived multiple new germ-free breeding colonies of wild type and transgenic mice and cry preserved embryos and/or sperm of each line that we established. The breeding colonies can be received in case of loss from contamination or other catastrophe. We have established a close working relationship with the UNC MMRRC, one of the 4 NIH NCRR-funded regional centers that maintain colonies of unique spontaneous marine mutations, transgenic and knockout lines with a broad array of phenotypes for distribution to NIH-funded investigators. The UNC MMRRC has cry preserved embryos from 78 different marine strains, and maintains approximately 30 breeding colonies at any given time. Kathy Mohr, Technical Director of the UNC MMRRC, has over 30 years experience with mouse embryo manipulation. She has performed all of our embryo transfers and cryopreservation of embryos and sperm for derivation of new germ-free breeding colonies in the UNC Gnotobiotic Animal Core. In 2004, we further expanded the UNC component under funding from the NCRR Division of Comparative Medicine that established the National Gnotobiotic Rodent Resource Center (P40 RR018603). This expansion was necessary because our facilities were at the limits of their capacity due to the dramatic increase in requests for germ-free and selectively colonized mice by NIH-funded and international investigators as the scientific community recognized the key role of commensal microbiota in gene-environment interactions. The CGIBD Gnotobiotic Animal Core facility concentrates on supplying germ-free and selectively colonized gnotobiotic mice, rats and zebrafish to Center members and members of other NIDDK- supported Digestive Disease Centers, while the National Gnotobiotic Rodent Resource Center's primary user base is other NIH- funded investigators. In 2007 the scope of the CGIBD Gnotobiotic Animal Core further expanded to supply germ-free and monoassociated zebrafish. The expansion to include zebrafish occurred in conjunction with the recruitment to UNC-Chapel Hill of J o h n Rawls, Ph.D., a former postdoctoral fellow of Dr. Jeff Gordon at Washington University in St. Louis. Dr. Rawls and his collaborators, including Christian Jobin, Ph.D. and Scott Plevy, M.D., have used this novel facility to explore the influence of intestinal microbiota on development of epithelial, mesenchymal and innate immune components of the intestine through innovative gene expression arrays and in vivo gene expression using NFkB[egfp] transgenic zebrafish, as described below. To accommodate increased requests for new axenic mouse strains, we have developed innovative techniques to more rapidly and efficiently sterilely derive additional breeding colonies of germ-free mice. In the current funding cycle, we have custom designed a novel sterile derivation chamber/surgical hood that directly links to the sterile flexible film Trexler isolators used to house our breeding colonies and experimental rodents. With this system, a germ-free surrogate mother bred to a vasectomized male can be directly transferred to the derivation chamber for embryo transfer. After recovery from surgery in the surgical hood, the surrogate mother is transferred back into the sterile Trexler isolator. Additional scientific innovations initiated in the current funding cycle that will be optimized in the requested new funding cycle include creation of new germ-free breeding strains by in vivo artificial insemination by intratubal sperm transfer to the bursa of the ovary, and molecular techniques to detect and identify potential contaminants. The latter technique will be facilitated by development of a new UNC Molecular Microbiology Core facility, an initiative of the UNC School of Medicine Dean's office that was conceived and organized by the CGIBD leadership. These techniques are described in greater detail in the Planned Services and Technical Innovations section of this proposal.