GNOTOBIOTIC CORE

知生核心

• 批准号: 7764470
• 负责人: SUSAN L TONKONOGY
• 金额: $ 15.6万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-03-15 至 2014-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7764470
• 关键词: Adherence; Administrative Supplement; Advisory Committees; Animal Experimentation; Animal Experiments; Animal Model; Animals; Antigens; Artificial Insemination; Back; Bacteria; Bacterial Genes; Biomedical Research; Biotechnology; Bone Marrow; Breeding; Cells; Chronic; Collaborations; Communities; Control Animal; Core Facility; Country; 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; 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; 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; 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 foUovnng the retirement ofthe 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 ofthe 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), vnth 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 roderit 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 worldvnde. This need was precipitated by closure ofthe University of Wisconsin Gnotobiotic Unit in 2002 follovnng the retirement of Dr. Ed Balish. Our unit had operated in parallel vnth 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 ofthe Wisconsin facility for germ-free derivation of new animal strains. When the Wisconsin resource closed, we developed methods of germ-fi-ee embryo transplant and have successfully derived multiple new germfi- ee breeding colonies of vnld type and transgenic mice and cryopreserved embryos and/or sperm of each line that we established. The breeding colonies can be rederived in case of loss from contamination or other catastrophe. We have established a close working relationship vnth the UNC MMRRC, one ofthe 4 NIH NCRR-funded regional centers that maintain colonies of unique spontaneous murine mutations, transgenic and knockout lines with a broad array of phenotypes for distribution to NIH-funded investigators. The UNC MMRRC has cryopreserved embryos from 78 different murine 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 embiyo 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, r a t s and zebrafish to Center members and members of other NIDDK- supported Digestive Disease Centers, while t h e 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 ofthe intestine through innovative gene expression arrays and in vivo gene expression using NFicB^s^ 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 direcfly 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 ofthe ovary, and molecular techniques to detect and identify potential contaminants. The latter technique vnll be faciUtated by development of a new UNC Molecular Microbiology Core facility, an initiative ofthe 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。知生核心是 1985 年由 Philip Carter 博士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。虽然 服务和人员随着时间的推移而发展，CGIBD 之间在该项目上的合作 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。知生核心对中心的重要性也随着我们的发展而增长 强调基因与环境的相互作用，并试图了解肠道的作用 微生物群在炎症性肠病发生中的作用。如果没有一个无菌设施，这极其 富有成效的研究将是不可能的。 卡特博士于 1998 年在两位前核心退休后重新担任董事职务 导演查尔斯·麦克弗森和托马斯·哈姆。卡特博士于 2000 年被苏取代 Tonkonogy 博士，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，他使用了这个 设施自成立以来。核心提供的动物和服务范围扩大了 随着时间的推移逐渐。如下文详细描述，该核心现在提供一系列 传统的和基因工程的啮齿动物品系和斑马鱼保存在精心定义的环境中 微生物环境。根据个别实验的要求，这些动物 可能是无菌的（无菌的），或者可能被细菌或细菌中的一种或多种特异性定植。 感兴趣的真菌菌株。 因为我们中心的成员和 NIDDK 资助的调查人员要求 国家的限生小鼠和大鼠急剧增加，并且因为 UNC-CH 研究人员 由于我们的用户群占绝大多数，我们在 2001 年通过创建并行系统来扩大我们的容量 北卡罗来纳大学教堂山校区的知生啮齿动物设施。此次扩建的设备是 由北卡罗来纳州生物技术中心提供的一次性设备补助金提供（直接73,475美元） 费用），以及北卡罗来纳大学教堂山医学院的配套资金（24,492 美元）。人员费用 一些设备费用由 NIDDK 提供为期 1 年的行政补助 CGIBD（直接成本 69,000 美元），并指示该扩建设施应提供无菌设施 并选择性地向 NIDDK 资助的研究人员定殖啮齿类动物。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。 我们设施的扩建是由以下几个需求驱动的：1) CGIBD 扩大的需求 限生动物成员，2) 需要提供一个易于我们使用的设施 最大的用户群，3) NIDDK 资助的外部研究人员对限生啮齿动物的需求，4) 需要有效衍生新的无菌 Roderit 菌株，以及 5) 需要提供支持 培育独特菌株的菌落。此外，这一扩大的设施使我们能够建立一个 与 NIH 国家研究资源中心 (NCRR) 资助的突变小鼠合作 北卡罗来纳大学教堂山分校区域资源中心 (MMRRC)。需要提供后备育种 菌落至关重要，因为我们拥有唯一现存的 HLA B27 转基因大鼠无菌菌落 和易感 129S6/SVEV 背景世界的 IL-10 敲除小鼠。这个需求是 2002 年，威斯康星大学知生单位关闭，引发了 埃德·巴利什博士退休。我们的单位与巴利什博士在巴利什大学的单位同时运作。 威斯康星州自成立以来，每个单位都有独立的无菌繁殖群体 独特的啮齿动物品系。没有任何无菌设备能够完全不受污染。这 公布的大鼠平均每个隔离器污染率为 5.9%，小鼠为 3.9%（Saito 和野村，无菌动物的生产，在“生物医学研究中的无菌动物”中， 编辑。 MD Coates 和 BE Gutaffson，实验动物有限公司，伦敦，1984 年，第 40 页）。如果出现以下情况 一个单位受到污染，另一单位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的规模和专业知识 无菌衍生新动物品系的设施。当威斯康星州资源关闭时，我们 开发了胚芽移植方法并成功衍生出多种新胚芽移植方法 vnld型和转基因小鼠的ee繁殖群体以及冷冻保存的胚胎和/或精子 我们建立的每条生产线。繁殖群落一旦丧失，可以重新获得 污染或其他灾难。 我们与 UNC MMRRC 建立了密切的工作关系，该中心是 4 个 NIH 之一 NCRR 资助的区域中心维持独特的自发鼠突变群体， 具有广泛表型的转基因和敲除品系，分发给 NIH 资助的机构 调查人员。北卡罗来纳大学 MMRRC 冷冻保存了 78 种不同小鼠品系的胚胎，并且 在任何给定时间都维持大约 30 个繁殖群体。凯西·莫尔，技术总监 北卡罗来纳大学 MMRRC 在小鼠胚胎操作方面拥有 30 多年的经验。她曾表演过 我们所有的胚胎移植以及胚胎和精子的冷冻保存，用于衍生新的无菌胚胎 北卡罗来纳大学知生动物核心的繁殖群体。 2004 年，我们在 NCRR 部门的资助下进一步扩大了 UNC 部分。 建立国家知生啮齿动物资源中心的比较医学（P40 RR018603）。这次扩建是必要的，因为我们的设施已达到容量极限 NIH 资助的和对无菌和选择性定植小鼠的需求急剧增加 随着科学界认识到共生的关键作用，国际调查人员 基因与环境相互作用中的微生物群。 CGIBD 知生动物核心设施 专注于提供无菌且选择性定植的无菌小鼠、大鼠和 斑马鱼向中心成员和其他 NIDDK 支持的消化中心成员 疾病中心，而国家知生啮齿动物资源中心的主要用户 基地是其他 NIH 资助的研究人员。 2007年，CGIBD Gnotobiotic Animal Core的范围进一步扩大到供应无菌产品 和单关联斑马鱼。斑马鱼的扩展与 北卡罗来纳大学教堂山分校招聘 J o h n Rawls 博士，他是 Jeff 博士的前博士后研究员 戈登在圣路易斯华盛顿大学。罗尔斯博士和他的合作者，包括克里斯蒂安 乔宾博士和斯科特·普莱维 (Scott Plevy) 医学博士使用这种新颖的设备来探索肠道的影响 微生物群对肠道上皮、间质和先天免疫成分发育的影响 通过创新的基因表达阵列和使用 NFicB^s^ 转基因的体内基因表达 斑马鱼，如下所述。 为了满足对新无菌小鼠品系的日益增长的需求，我们开发了 创新技术，更快速、更有效地无菌地获得更多的繁殖群体 无菌小鼠。在当前的融资周期中，我们定制设计了一种新颖的无菌衍生 腔室/手术罩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。通过这个系统，无菌代孕母亲可以培育出 切除输精管的雄性可直接转移至衍生室进行胚胎移植。后 在手术罩中从手术中恢复后，代孕母亲被转移回无菌室 Trexler 隔离器。 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包括通过体内人工培育新的无菌育种菌株 通过输卵管内精子转移至卵巢囊进行授精，并通过分子技术进行检测 并识别潜在的污染物。后一种技术将通过开发一种新的技术来促进 北卡罗来纳大学分子微生物学核心设施，北卡罗来纳大学医学院院长的倡议 该办公室由 CGIBD 领导层构思和组织。这些技术描述于 本提案的计划服务和技术创新部分中有更多详细信息。