GNOTOBIOTIC CORE

知生核心

• 批准号: 8374538
• 负责人: SUSAN L TONKONOGY
• 金额: $ 14.87万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8374538
• 关键词: Adherence; Administrative Supplement; Advisory Committees; Animal Experimentation; Animal Experiments; Animal Model; Animals; Antigens; Artificial Insemination; Back; Bacteria; Bacterial Genes; Biology; Biomedical Research; Biotechnology; Bone Marrow; Breeding; Cells; Chronic; Collaborations; Communities; Control Animal; Core Facility; Country; Crying; Cryopreservation; Custom; Dendritic Cells; Derivation procedure; Detection; Development; Diet; Digestive System Disorders; Direct Costs; Disease; Doctor of Medicine; Doctor of Philosophy; Embryo; Embryo Transfer; Engineering; Ensure; Environment; Epithelial; Equipment; Film; Funding; Gene Expression; Generations; Genes; Genetic; Germ-Free; Gnotobiotic; Goals; Grant; Housing; Human Resources; Immune; Immune response; Immunologist; Individual; Inflammatory; Inflammatory Bowel Diseases; Inflammatory disease of the intestine; Institution; Interleukin-10; International; Intestines; Knock-out; Knockout Mice; Laboratory Animals; Leadership; Life; Link; Location; London; Maintenance; Marines; Medicine; Mesenchymal; Methods; Microbiology; Molds; Molecular; Monitor; Mouse Strains; Mus; Mutant Strains Mice; Mutation; NIH Mouse; National Center for Research Resources; National Institute of Diabetes and Digestive and Kidney Diseases; North Carolina; Operative Surgical Procedures; Organ; Ovary; Parasites; Pathogenesis; Phenotype; Physiologic pulse; Physiological; Physiological Processes; Postdoctoral Fellow; Process; Production; Publishing; Quality Control; Rattus; Recombinants; Recovery; Regulation; Research; Research Personnel; Research Project Grants; Resources; Retirement; Retroviridae; Rodent; Role; Series; Services; Source; Specific Pathogen Frees; Sterility; Surrogate Mothers; System; T-Lymphocyte; Technical Expertise; Techniques; Time; Training; Transgenic Mice; Transgenic Organisms; Transplantation; United States National Institutes of Health; Universities; Veterinary Medicine; Virus; Washington; Wisconsin; Work; Yeasts; Zebrafish; animal resource; base; college; commensal microbes; comparative; cost; design; experience; flexibility; fungus; gastrointestinal; gene environment interaction; germ free condition; gut microbiota; in vivo; innovation; interest; male; medical schools; member; microbial; multidisciplinary; neoplastic; novel; response; sperm cell

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.

CGIBD 从一开始就有一个知生动物核心。知生核心由 Philip Carter 博士于 1985 年在北卡罗来纳州立大学兽医学院创立，他对知生研究有着长期的兴趣和丰富的专业知识。尽管服务和人员随着时间的推移而不断发展，但 CGIBD 之间在此项目上的合作 我们两个主要机构——北卡罗来纳大学教堂山分校和北卡罗来纳州立大学——的成员保持不变。随着我们强调基因与环境的相互作用并试图了解肠道微生物群在炎症性肠病发生中的作用，知生核心对该中心的重要性也日益增强。如果没有知生设施，这一极其富有成效的研究系列就不可能实现。 在两位前核心董事查尔斯·麦克弗森 (Charles McPherson) 和托马斯·哈姆 (Thomas Hamm) 退休后，卡特博士于 1998 年恢复担任董事职务。 2000 年，Carter 博士被 Sue Tonkonogy 博士取代，Sue Tonkonogy 博士是北卡罗来纳州立大学兽医学院经验丰富的细胞免疫学家，过去 15 年一直致力于慢性肠道炎症动物模型的研究。 Tonkonogy 博士与副主任 Balfour Sartor 一起领导核心，Balfour Sartor 从一开始就使用该设施。随着时间的推移，核心提供的动物和服务的范围逐渐扩大。如下文详细描述的，该核心现在提供了一系列在精心定义的微生物环境中维持的传统和基因工程啮齿动物品系和斑马鱼。根据各个实验的要求，这些动物可能是无菌的（无菌的），或者可能被一种或多种感兴趣的细菌或真菌菌株特异性定殖。 由于我们中心的成员和全国各地由 NIDDK 资助的研究人员对知生小鼠和大鼠的需求急剧增加，并且由于 UNC-CH 研究人员构成了我们用户群的绝大多数，因此我们在 2001 年通过创建平行的知生啮齿动物来扩大我们的能力北卡罗来纳大学教堂山校区的设施。此次扩建的设备由北卡罗来纳州生物技术中心提供的一次性设备补助金（直接费用 73,475 美元）提供，北卡罗来纳大学教堂山医学院提供配套资金（24,492 美元）。人员费用和一些设备费用由 NIDDK 向 CGIBD 提供的 1 年行政补助（直接费用 69,000 美元）提供，并指示该扩建设施应向 NIDDK 资助的调查人员提供无菌且选择性定殖的啮齿动物。资金也由 北卡罗来纳大学院长研究咨询委员会和北卡罗来纳大学教堂山医学系。 我们的设施扩建是由以下几个需求驱动的：1) CGIBD 成员对限生动物的需求扩大，2) 需要提供一个易于我们最大的用户群使用的设施，3) NIDDK 资助的外部研究人员的需求对于无菌啮齿动物，4）需要有效衍生新的无菌啮齿动物品系，5）需要提供支持 培育独特菌株的菌落。此外，这一扩建的设施使我们能够与 NIH 国家研究资源中心 (NCRR) 资助的位于北卡罗来纳大学教堂山分校的突变小鼠区域资源中心 (MMRRC) 建立合作伙伴关系。提供后备繁殖群体的必要性至关重要，因为我们拥有全世界唯一存在的 HLA B27 转基因大鼠和 IL-10 敲除小鼠的无菌群体，其背景为易感 129S6/SVEV。 2002 年，埃德·巴利什 (Ed Balish) 博士退休后，威斯康星大学知生部门关闭，加剧了这一需求。我们的单位自成立以来一直与巴利什博士在威斯康星大学的单位并行运作，每个单位都拥有每种独特啮齿动物品系的独立无菌繁殖群。没有任何无菌设备能够完全不受污染。公布的大鼠平均每个隔离器污染率为 5.9%，小鼠为 3.9%（Saito 和 Nomura，《无菌动物的生产》，《生物医学研究中的无菌动物》，编辑 MD Coates 和 BE） Gutaffson，实验动物有限公司，伦敦，1984 年，第 40 页）。如果一个单位受到污染，另一个设施可以为育种者提供恢复繁殖群体的服务。传统上，我们较小的 CGIBD 设施依赖于威斯康星州设施的规模和专业知识来无菌衍生新动物品系。当威斯康星州资源关闭时，我们开发了无菌胚胎移植方法，并成功衍生出多个新的野生型和转基因小鼠的无菌繁殖群体，并冷冻保存的胚胎和/或精子 我们建立的每条生产线。如果由于污染或其他灾难而损失，可以接收繁殖群体。 我们与 UNC MMRRC 建立了密切的工作关系，该中心是 NIH NCRR 资助的 4 个区域中心之一，负责维护独特的自发海洋突变、具有广泛表型的转基因和敲除品系的菌落，以分发给 NIH 资助的研究人员。北卡罗来纳大学 MMRRC 拥有 78 种不同海洋品种的冷冻胚胎，并且 在任何给定时间都维持大约 30 个繁殖群体。 Kathy Mohr 是北卡罗来纳大学 MMRRC 的技术总监，拥有 30 多年的小鼠胚胎操作经验。她完成了我们所有的胚胎移植以及胚胎和精子的冷冻保存，以便在北卡罗来纳大学的知生动物核心中衍生出新的无菌繁殖群体。 2004 年，我们在 NCRR 比较医学部门的资助下进一步扩大了 UNC 部分，该部门建立了国家知生啮齿动物资源中心 (P40 RR018603)。这种扩张是必要的，因为美国国立卫生研究院 (NIH) 资助的和对无菌和选择性定殖小鼠的需求急剧增加，导致我们的设施已达到容量极限。 国际研究人员和科学界认识到共生微生物群在基因与环境相互作用中的关键作用。 CGIBD 知生动物核心设施专注于向中心成员和其他 NIDDK 支持的消化系统疾病中心的成员提供无菌且选择性定植的知生小鼠、大鼠和斑马鱼，而国家知生啮齿动物资源中心的主要用户群是其他 NIH 资助的机构调查人员。 2007 年，CGIBD 知生动物核心的范围进一步扩大到供应无菌和单一关联的斑马鱼。将斑马鱼纳入其中的同时，北卡罗来纳大学教堂山分校招募了约翰·罗尔斯博士，他是圣路易斯华盛顿大学杰夫·戈登博士的前博士后研究员。罗尔斯博士和他的合作者，包括克里斯蒂安 乔宾博士和 Scott Plevy 医学博士利用这一新型设施，通过创新的基因表达阵列和使用 NFkB[egfp] 转基因斑马鱼的体内基因表达，探索肠道微生物群对肠道上皮、间质和先天免疫成分发育的影响，如如下所述。 为了满足对新无菌小鼠品系不断增长的需求，我们开发了创新技术，可以更快速、更有效地无菌获得更多无菌小鼠繁殖群体。在当前的融资周期中，我们定制设计了一种新型无菌衍生室/手术罩，它直接连接到用于容纳我们的无菌柔性薄膜 Trexler 隔离器 繁殖群体和实验啮齿动物。通过该系统，与已切除输精管的雄性繁殖的无菌代孕母亲可以直接转移到衍生室进行胚胎移植。在手术罩中从手术中恢复后，代孕母亲被转移回无菌的 Trexler 隔离器中。 当前资助周期中发起的其他科学创新将在所要求的新资助周期中得到优化，包括通过体内人工授精、通过输卵管内精子转移到卵巢囊来创建新的无菌育种菌株，以及检测和检测卵巢囊的分子技术。识别潜在的污染物。后一种技术将通过开发新的北卡罗来纳大学分子微生物学核心设施来促进，这是北卡罗来纳大学医学院院长办公室的一项倡议，由 CGIBD 领导层构思和组织。这些技术在本提案的计划服务和技术创新部分中有更详细的描述。