MURINE MODELS CORE

小鼠模型核心

• 批准号: 7777673
• 负责人: JEFFREY MINER
• 金额: $ 9.3万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-12-01 至 2014-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7777673
• 关键词: Ablation; Address; Adult; Affect; Air; Air Pressure; Alleles; Animal Housing; Animals; Antigens; Archaea; Area; Arts; Atomizer; Attention; Backcrossings; Bacteria; Bacterial Artificial Chromosomes; Bacteroides thetaiotaomicron; Bathing; Beds; Biological Assay; Biology; Biomedical Research; Birth; Boston; Breeding; Budgets; Caliber; Cancer Center; Cell physiology; Cells; Cesarean section; Chemicals; Chromosomes; Collection; Communication; Communities; Complex; Congenic Mice; Congenic Strain; Control Animal; Core Facility; Costs and Benefits; Coupled; Cryopreservation; Custom; DNA; Data; Derivation procedure; Development; Devices; Diarrhea; Diet; Dietary Polysaccharide; Digestive System Disorders; Disease; Disease model; Distal; Ecosystem; Education; Elbow; Embryo; Embryo Transfer; Engineering; Ensure; Environment; Equipment; Eubacterium; Evolution; Experimental Models; Extinction (Psychology); Feces; Female; Fertilization in Vitro; Film; Fogs; Food; Fostering; Founder Generation; Freezing; Future; Gene Expression; Gene Proteins; Generations; Genes; Genetic; Genetic Models; Genetic Polymorphism; Genome; Genomics; Genotype; Germ-Free; Gills; Glass; Gnotobiotic; Goals; Gossypium; Greek; Gut associated lymphoid tissue; Habitats; Harvest; Health; Horizontal Gene Transfer; Horns; Hour; Housing; Human; Human Resources; Ice; Immune system; Implant; Inbred Strain; Incubated; Individual; Induced Mutation; Infection; Inflammatory Bowel Diseases; Injection of therapeutic agent; Internet; Intervention; Intestines; Investigation; Knock-in Mouse; Knock-out; Knowledge; Laboratories; Lasers; Left; Life; Liquid substance; Maintenance; Malnutrition; Mammals; Metabolic; Metagenomics; Metals; Methanobrevibacter; Methods; Microbe; Microsatellite Repeats; Modeling; Monitor; Mothers; Mus; Mutation; Neoprene; Nitrogen; Nutrient; Obesity; Oocytes; Operative Surgical Procedures; Oral cavity; Organ; Organism; Outsourcing; Partner in relationship; Pathology; Pathway interactions; Patient currently pregnant; Pattern; Perivitelline Space; Personnel Turnover; Pharmaceutical Preparations; Phase; Phenotype; Physiological; Physiology; Plants; Plastics; Play; Polymerase; Polymorphic Microsatellite Marker; Polymorphism Analysis; Polysaccharides; Polyvinyl Chloride; Population; Povidone-Iodine; Pregnancy; Preparation; Procedures; Process; Production; Protective Devices; Protocols documentation; Puncture procedure; Quality Control; Reaction; Reagent; Reproductive Biology; Research; Research Personnel; Resources; Rewards; Risk; Rodent; Role; Running; Safety; Secure; Services; Shapes; Side; Signal Pathway; Single Nucleotide Polymorphism; Site; Solutions; Specific qualifier value; Speed; Staging; Stainless Steel; Stem cells; Sterility; Sterilization; Superovulation; Surface; Swab; Symbiosis; Synthetic Genes; System; Technical Expertise; Techniques; Technology; Temperature; Testing; Tetracyclines; Therapeutic Agents; Thick; Time; Tissues; Training; Transfection; Transgenes; Transgenic Organisms; Transplantation; Triad Acrylic Resin; Universities; Uterus; Variant; Vitamin K; Washington; Water; Work; X Chromosome; Y Chromosome; Zona Pellucida; activating transcription factor; allergic response; animal colony; assisted reproduction; base; blastocyst; chlorine dioxide; congenic; cost; cost effectiveness; density; design; embryo cell; embryo stage 2; embryo/fetus; embryonic stem cell; experience; extracellular; fiberglass; flexibility; fungus; gastrointestinal system; genetic pedigree; gut microbiota; homologous recombination; interest; laboratory facility; male; medical schools; member; microbial; microbial community; microbial genome; microbial host; microbiome; microorganism; microorganism interaction; mouse genome; mutant; mylar; novel; novel therapeutics; offspring; ovary transplantation; pathogen; pathogenic bacteria; postnatal; pregnant; prevent; processing speed; protein function; pup; quality assurance; recombinase; research study; seal; tool; trait; vector; web site

Murine models have been extensively utilized by Washington University Digestive Diseases Research Core Center investigators and are central to DDRCC biomedical research into inflammatory bowel disease and other aspects of the digestive system. The Murine Models Core has been and will continue to be dedicated to producing genetically altered animals and to providing gnotobiotic mice for DDRCC investigators in a timely and reliable manner. In addition, the Core provides ancillary sen/ices for maintaining mouse pedigrees, such as assisted reproduction and cryopreservation of embryos, as well as rapid transfer of mutations or transgenes from one genetic background to another using "speed congenics". We will continue to offer these services in the next project period. A.1 Murine Genetic Models. The mouse genome can be altered using two general approaches: (i) incorporation of synthetic genes via direct injection of DNA into single-celled embryos, or (ii) creation of targeted mutations that are induced in embryonic stem cells by homologous recombination and then are incorporated into the genn line. These transgenic, knock-out, and knock-in mice continue to be a mainstay for the investigation of gene and protein function (Shastry, 1998), and forthe production of disease models (Roths etal., 1999). Evolving technology for altering the murine genome allows for ever more sophisticated, manipulable, and informative mammalian models. Now common are inducible, tissue-specific transgenes constructed to respond to synthetic transcription factors that are activated or repressed by tetracycline or other drugs (Saez et al.. 1997). Coupled with conditional mutations made possible by insertion of recognition sites for Cre and Flp recombinases in a locus of interest, these technologies allow tissue-specific, inducible, targeted mutations (Rossant and McMahon, 1999). Furthermore, entire genes can be added to the murine genome as bacterial artificial chromosomes (BACs), allowing recapitulation of endogenous gene expression with the ability to readily mutagenize any sequence in the locus (Giraldo and Montoliu. 2001). A.2 Gnotobiotic Animals. "Gnotobiotics" derives from the Greek gnosis, 'knowledge,' and bios, 'life.' Gnotobiotic animals are those reared under conditions which allow their microbial constituents to be carefully defined. Using gnotobiotic husbandry, genn-free animals are raised in a completely sterile environment. Genn-free animals provide an essential tool for investigating host-microbia interactions. Assembly of the intestinal microbiota (the community of microorganisms) during the postnatal period produces a multi-lineage, spatially patterned microbial 'organ' that is exquisitely adapted to the needs ofthe host and collective 'self. Gnotobiotics offers an opportunity to examine the impact of defined components of this microbiota on host biology through creation of simplified experimental models of host-microbial interactions. Co-evolved symbiotic relationships between microbes and animals are a prominent feature of terrestrial life (Backhed et al.. 2005; Ley et al., 2006a). We are host to a remarkable variety and number of environmentally transmitted extracellular symbionts. Acquisition of our microbial nation begins at birth (Favier et al., 2002). As adults, our total microbial population is thought to exceed our total number of somatic and gemn cells by at least an order of magnitude (Berg, 1996). Our largest collection of microorganisms resides in the intestine (Moore and Holdeman, 1974). The aggregate size of all intestinal microbial genomes may be equivalent to the size of our own genome, and the number of genes in this 'microbiome' may exceed the total number of human genes by a factor of 100, providing traits that mammals did not need to evolve within their own genome (Gill et al., 2006; Hooper and Gordon, 2001; Xu and Gordon, 2003). These traits include the ability to break down otherwise indigestible plant polysaccharides (Hooperetal., 2002a; Sonnenburg et al., 2005; Xu et al., 2003). In addition, postnatal colonization of our intestine 'educates' our immune system so that we become tolerant of a wide variety of microbial immunodetemninants.. This education appears to help reduce allergic responses to food or environmental antigens (Braun-Fahrlander et al., 2002). The relationship between the microbiota and gut-associated lymphoid tissue (GALT) is reciprocal: for example, the GALT plays a key role in shaping the microbiota. although details ofthe mechanisms that underiie this reciprocity are still emerging (Fagarasan et al.. 2002). The current revolution in metagenomics provides an unprecedented opportunity to analyze how components of the microbiota modulate features of our postnatal development (Stappenbeck et al., 2002) and adult physiology. One notion that motivates such an analysis is that our co-evolved microbial partners have developed the capacity to synthesize novel chemical entities that help establish and sustain beneficial symbioses. Prospecting forthese chemicals (Hooperetal.. 2003), and characterizing the signaling pathways through which they operate, may provide new strategies and reagents for manipulating our biology to enforce health, and to correct, or ameliorate, certain disease states (Backhed et al., 2004; Ley et al., 2006b; Turnbaugh et al.. 2006) (e.g., infectious diarrheas, inflammatory bowel diseases, obesity/malnutrition). The potential rewards extend beyond identification of new therapeutic agents and their targets. In a dynamic, densely populated ecosystem such as the gut, horizontal gene transfer between bacterial species can have important effects on organism gene content and physiology. Thus, the intestinal ecosystem provides an opportunity to address general questions related to 'ecogenomics' (Ley et al.. 2006a; Stahl and Tiedje, 2002). For example, how do symbionts sense and respond to variations in their environments? How does a given intestinal environment shape the evolution of its component microbial species? If there is significant microevolution of a given species and re-distribution of genetic traits to other members of the consortium, what are genome-based definitions of speciation and extinction? What is the genomic basis for nutrient cycling and syntrophy? The intestinal microbiota operates through a complex network of interspecies communications and an elaborate web of nutrient sharing/cycling. The complexity of the system presents a seemingly overwhelming experimental challenge when envisioning how to (i) identify the principles that govern establishment of these environmentally transmitted communities, (ii) characterize the spectrum of contributions that community members make to postnatal gut development and adult physiology, (iii) dissect microbial-host and microbialmicrobial- communications pathways, (iv) understand the forces that direct co-evolution and co-adaptation of symbiotic bacteria and their host in specified intestinal habitats, and/or (v) decipher host-pathogen interactions. A key experimental strategy for defining the impact of microorganisms on host physiology is to first examine cellular function in the absence of bacteria (i.e., under gemi-free conditions) and then to evaluate the effects of adding a single species, or a defined number of species of bacteria. Gem-free mice can be viewed as having a complete ablation of their multi-lineage microbial 'organ'. They can be colonized with a recognized or candidate intestinal symbiont (or pathogen), during or after completion of postnatal gut development (Hooper et al., 2002a; Hooper et al.. 2002b; Xu et al., 2003). The impact of colonization with one species can be compared and contrasted to another species, or to defined collections of species, or to an unfractionated microbiota harvested from a region of the intestines of mice that have acquired a microbiota from birth (conventionally raised' animals). The Murine Models Core will continue to provide the key investigational tools for exploration of host-microbial interactions to DDRCC members as services: (i) Provision of unmanipulated germ-free mice and genotypically matched conventionally-raised control animals, (ii) Colonization of germ-free mice with bacteria, (iii) Rederivation of germ-free mice from existing conventionally-raised strains of genetically modified mice. A.3. Cost Effectiveness Creation of genetically altered and gnotobiotic mice requires expensive equipment, specialized technical expertise, and long experience¿requirements that put their production beyond the resources of most individual laboratories. The Murine Models Core provides timely access to these murine models at an affordable cost to DDRCC investigators, and also provides advice to investigators who need guidance in project design or technical implementation of these models. In addition, the Core will also provide services for maintaining murine pedigrees, including cryopreservation, assisted reproduction, and rapid transfer of mutations or transgenes into inbred strain backgrounds. These techniques are invaluable to laboratories without proficiency in murine reproductive biology, and their availability allows investigators to focus on the relevant physiology or pathology that is their area of expertise. The DDRCC will achieve a significant cost benefit by utilizing two existing facilities to provide all Murine Models Core services: ¿Gnotobiotic animals will continue to be derived and maintained in a facility operated by Jeffrey Gordon's laboratory. Creation of an independent gnotobiotic facility would be prohibitively difficult and expensive for the DDRCC, involving purchase of equipment and hiring and training of personnel for mouse work and microbiological assays. Sen/ice is most economically and reliably provided through incurring the additional variable costs of DDRCC usage in the current Gordon Laboratory facility. ¿All no n-g no to biotic services will be provided by the Mouse Genetics Core (MGC). The Mouse Genetics Core (http://mgc.wustl.edu/) is a large facility dedicated to producing genetically altered mice for all Washington University investigators, and is also co-directed by the Murine Models Core director. Dr. Miner. Services provided by the MGC to DDRCC investigators are also perfomned more economically than could be accomplished by a dedicated DDRCC facility; the size of the MGC allows an economy of scale so that the DDRCCs overall costs are lower. In addition, use of the MGC ensures reliable and timely provision of a wide variety of services and spares the DDRCC from problems associated with employee turnover. The DDRCC subsidizes MGC services to DDRCC investigators by covering ~75% ofthe cost, thus removing a significant barrier to murine model production and facilitating the initiation of new mouse projects. Removing a cost barrier for cryopreservation of digestive diseases-relevant mouse lines encourages DDRCC investigators to utilize this service, thus ensuring that valuable lines are safely maintained and presen/ed for many years to come.

华盛顿大学消化疾病研究已广泛使用鼠模型 核心中心研究人员是DDRCC生物医学研究的核心 消化系统的其他方面。鼠模型核心已经并且将继续致力于 及时生产一般改变的动物，并为DDRCC研究人员提供gnotobiotic小鼠 和可靠的方式。此外，核心提供了维持小鼠血统的辅助剂量，这样 作为胚胎的辅助生殖和冷冻保存，以及突变或翻译的快速转移 从一个遗传背景到另一个遗传背景，使用“速度友善”。我们将继续提供这些服务 下一个项目期。 A.1鼠遗传模型。 可以使用两种一般方法来改变小鼠基因组：（i）合成基因的合并 通过将DNA直接注射到单细胞胚胎中，或（ii）产生诱导的靶向突变 通过同源重组，然后将胚胎干细胞掺入Genn系。这些 转基因，敲除和敲除小鼠仍然是研究基因和蛋白质的主要支柱 功能（Shastry，1998），以及用于疾病模型的生产（Roths等，1999）。不断发展的技术 改变鼠基因组可以使疗程更复杂，可操作和信息丰富的哺乳动物 型号。现在常见的诱导性，组织特异性翻译构建以响应合成 被四环素或其他药物激活或抑制的转录因子（Saez等。1997）。耦合 随着条件突变通过插入CRE和FLP重组酶的识别位点而成为可能 这些技术允许组织特异性，可诱导，靶向突变（rossant和rossant和 麦克马洪，1999年）。此外，可以将整个基因添加到鼠基因组中作为细菌艺术 染色体（BAC），允许对内源基因表达概括，并能轻松地 诱变的基因序列（Giraldo andMontoliu。2001）。 A.2 gnotobiotic动物。 “ gnotobiotics”源自希腊的gnosis，'知识，和bios，'生活。 gnotobiotic动物是 在允许其微生物构成的条件下饲养的人仔细定义。使用 在完全无菌的环境中饲养了gnotobiotic饲养，无genn的动物。无genn动物 提供了研究宿主微生物相互作用的重要工具。肠道菌群的组装（ 微生物社区）在产后时期产生多条形，空间图案的 微生物“器官”完全适应了宿主和集体自我的需求。 Gnotobiotics提供 通过创建该微生物群的定义组成部分对宿主生物学的影响的机会 简化的宿主 - 微生物相互作用的实验模型。 微生物与动物之间的共同发展的共生关系是陆生生物的重要特征 （Backhed等人2005； Ley等，2006a）。我们在环境方面拥有非凡的种类和数量 传输细胞外符号。我们的微生物国家的获取始于出生（Favier等，2002）。作为 成年人，我们的总微生物种群被认为超过了我们的体细胞和gemn细胞总数 至少一个数量级（Berg，1996）。我们在肠道中最大的微生物住宅集合 （Moore and Holdeman，1974）。所有肠道微生物基因组的骨料大小可能等效于 我们自己的基因组的大小以及此“微生物组”中的基因数可能会超过人类的总数 基因的100倍，提供了哺乳动物不需要在自己的基因组中发展的特征（Gill et Al。，2006年； Hooper和Gordon，2001年； Xu和Gordon，2003年）。这些特征包括分解的能力 否则，不可消化的植物多糖（Hooperetal。，2002a; Sonnenburg等，2005; Xu等，2003）。 此外，我们的肠道“教育”免疫系统的产后殖民化，以便我们宽容 多种微生物免疫季节性。该教育似乎有助于减少对 食品或环境抗原（Braun-Fahrlander等，2002）。微生物群和 肠道相关的淋巴组织（GALT）是相互的：例如，galt在塑造塑造中起关键作用 尽管互惠互惠的机制的细节仍在出现（Fagarasan et Al .. 2002）。 当前的宏基因组革命提供了一个前所未有的机会，可以分析如何 我们产后发育的微生物群调节特征（Stappenbeck等，2002）和 成人生理学。激励这种分析的一个观念是我们共同发展的微生物伴侣有 发展了合成新型化学实体的能力，以帮助建立和维持有益 象征。探测化学物质（Hooperetal .. 2003），并表征信号通路 通过它们的操作，可以提供新的策略和试剂来操纵我们的生物学来执行 健康，纠正或改善某些疾病状态（Backhed等，2004； Ley等，2006b； Turnbaugh 等。2006）（例如，感染性腹泻，炎症性肠病，肥胖/营养不良）。潜力 奖励超出了新的治疗剂及其目标的识别。在动态，密集的 人口稠密的生态系统，例如肠道，细菌之间的水平基因转移可以具有重要的 对生物基因含量和生理学的影响。这，肠生态系统为 解决与“生态学学”有关的一般问题（Ley等人2006a； Stahl和Tiedje，2002）。例如， 符号如何感知并响应其环境中的变化？给定的肠道如何 环境塑造其成分微生物物种的演变？如果有显着的微发展 给定物种并将遗传特征重新分配给财团的其他成员，什么是基于基因组的 规范和扩展的定义？营养循环和综合体的基因组基础是什么？ 肠道菌群通过复杂的种间通信网络运行 精心制作的营养共享/骑自行车网。系统的复杂性表现出似乎压倒性的 实验性挑战在设想如何（i）确定统治这些建立的原则 环境传播的社区（ii）表征了社区的贡献范围 成员提高产后肠道发展和成人生理学，（iii）剖析微生物宿主和微生物 - 通信途径，（iv）了解直接共同进化和共同适应的力 共生细菌及其宿主在指定的肠道栖息地和/或（v）破译的宿主 - 病原体相互作用。 定义微生物对宿主生理的影响的关键实验策略是首先 在没有细菌的情况下检查细胞功能（即在无宝石条件下），然后评估 添加一种物种或定义的细菌数量的影响。可以查看无宝石的小鼠 因为它们完全消融了他们的多部件微生物“器官”。他们可以通过公认的 或候选肠道符号（或病原体），在产后肠道发育完成期间或之后 （Hooper等，2002a; Hooper等，2002b; Xu等，2003）。与一个物种殖民化的影响可以 比较并与其他物种或定义的物种收集或与未分离的物种进行比较并形成鲜明对比 从一只从出生开始获得菌群的小鼠肠道区域收获的菌群 （常规饲养的动物）。鼠模型核心将继续提供关键的研究 用于向DDRCC成员探索宿主 - 微生物互动的工具：（i）提供 无操纵的无菌小鼠和基因型匹配的传统饲养的对照动物， （ii）用细菌定植的无细菌小鼠，（iii）从现有 常规修饰的小鼠的常规饲养菌株。 A.3。成本效益 创建一般改变和gnotobiotic小鼠需要昂贵的设备，专门的设备 技术专长和长期的经验要求，其生产超出了大多数资源 个别实验室。鼠模型核心在 向DDRCC调查人员负担得起的成本，还向需要指导的调查人员提供建议 这些模型的项目设计或技术实施。此外，核心还将为 维持鼠类血统，包括冷冻保存，辅助繁殖和快速转移 突变或转化为近交性菌株背景。这些技术对实验室非常宝贵 没有熟悉鼠的生殖生物学，它们的可用性使研究人员可以专注于 相关的生理或病理学是其专家领域。 DDRCC将实现巨大的成本 通过使用两个现有设施提供所有鼠模型核心服务的好处： „将继续在Jeffrey经营的设施中得出和维护Gnotobiotic动物 戈登的实验室。建立独立的gnotobiotic设施将被禁止难以 DDRCC的昂贵，涉及购买设备，雇用和培训鼠标的人员 工作和微生物学测定。森/冰是经济和可靠的 当前戈登实验室设施中DDRCC使用的额外可变成本。 „将由小鼠遗传学核心（MGC）提供所有NO N-G NO。鼠标 遗传学核心（http://mgc.wustl.edu/）是一个大型设施，致力于为所有人生成遗传改变的小鼠 华盛顿大学的调查人员，也由Murine Model Model Core主管共同执导。矿工博士。 MGC向DDRCC调查人员提供的服务在经济上也比可能更经济 由专门的DDRCC设施完成； MGC的规模允许规模经济，因此 DDRCCS的总成本较低。此外，使用MGC确保可靠及时提供广泛的 与员工流动相关的问题中的多种服务和DDRCC的多种多样。 DDRCC 通过支付约75％的成本，为DDRCC调查人员提供了MGC服务，从而消除了大量费用 鼠模型生产的障碍和支持新鼠标项目的倡议。消除成本障碍 为了冷冻保存消化疾病，相关的鼠标线鼓励DDRCC调查人员利用这一点 服务，从而确保在未来的许多年中安全地维护和呈现有价值的线条。