MURINE MODELS CORE

小鼠模型核心

基本信息

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
项目类别：
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

项目摘要

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.

小鼠模型主要由华盛顿大学消化疾病研究中心使用核心中心研究人员，是 DDRCC 炎症性肠病和生物医学研究的核心小鼠模型核心已经并将继续致力于消化系统的其他方面。生产转基因动物并及时为 DDRCC 研究人员提供无菌小鼠此外，核心还提供用于维持小鼠谱系的辅助服务，例如如胚胎的辅助生殖和冷冻保存，以及突变或转基因的快速转移使用“速度遗传”从一种遗传背景到另一种遗传背景，我们将继续提供这些服务。下一个项目期。 A.1 小鼠遗传模型。可以使用两种通用方法改变小鼠基因组：（i）掺入合成基因通过直接将 DNA 注射到单细胞胚胎中，或 (ii) 创建在通过同源重组将胚胎干细胞整合到genn系中。转基因、基因敲除和基因敲入小鼠仍然是基因和蛋白质研究的支柱功能（Shastry，1998），以及疾病模型的产生（Roths 等人，1999）。改变小鼠基因组可以实现更加复杂、可操作和哺乳动物现在常见的是可诱导的、组织特异性的转基因，用于响应合成。被四环素或其他药物激活或抑制的转录因子（Saez et al. 1997）。通过在基因组中插入 Cre 和 Flp 重组酶的识别位点，可以实现条件突变感兴趣的基因座，这些技术允许组织特异性、可诱导的、有针对性的突变（Rossant 和 McMahon，1999）此外，整个基因可以作为细菌人工添加到小鼠基因组中。染色体（BAC），允许重现内源基因表达，并能够轻松地诱变基因座中的任何序列（Giraldo 和 Montoliu. 2001）。 A.2 知生动物。 “Gnotobiotics”源自希腊语“gnosis”，“知识”和“bios”，“生命”。在允许仔细定义其微生物成分的条件下饲养的那些。无菌饲养，无基因动物在完全无菌的环境中饲养。提供了研究宿主-微生物相互作用的重要工具。微生物群落）在出生后产生多谱系、空间模式微生物“器官”能够完美地适应宿主和集体“自我”的需要。有机会通过创建来检查该微生物群的特定成分对宿主生物学的影响宿主-微生物相互作用的简化实验模型。微生物和动物之间共同进化的共生关系是陆地生命的一个显着特征（Backhed 等人，2005 年；Ley 等人，2006a）。我们的微生物国家从出生时就开始传播细胞外共生体（Favier et al., 2002）。成年人中，我们的微生物总数被认为超过了我们的体细胞和生殖细胞总数至少一个数量级（Berg，1996）我们最大的微生物群存在于肠道中。（Moore 和 Holdeman，1974）。所有肠道微生物基因组的总大小可能相当于我们自己基因组的大小，这个“微生物组”中的基因数量可能超过人类的总数基因增加了 100 倍，提供了哺乳动物不需要在自己的基因组中进化的特征（Gill 等，2017）。 al.，2006；Hooper 和 Gordon，2001；Xu 和 Gordon，2003）。否则难以消化的植物多糖（Hooperetal.，2002a；Sonnenburg 等人，2005；Xu 等人，2003）。此外，出生后肠道的定植会“教育”我们的免疫系统，使我们能够耐受多种微生物免疫决定物。这种教育似乎有助于减少对微生物的过敏反应食物或环境抗原（Braun-Fahrlander et al., 2002）。肠道相关淋巴组织 (GALT) 是互惠的：例如，GALT 在塑造肠道健康方面发挥着关键作用。尽管这种相互作用背后的机制细节仍在浮现（Fagarasan 等人，2017）。等人..2002）。当前宏基因组学的革命提供了前所未有的机会来分析如何微生物群的成分调节我们产后发育的特征（Stappenbeck et al., 2002）和成人生理学的一个观点是，我们的共同进化的微生物伙伴具有这样的分析能力。开发了合成新型化学实体的能力，有助于建立和维持有益的探索这些化学物质（Hooperetal.. 2003），并表征信号传导途径。通过它们的运作，可能会提供新的策略和试剂来操纵我们的生物学以强制执行健康，并纠正或改善某些疾病状态（Backhed 等人，2004 年；Ley 等人，2006b；Turnbaugh et al.. 2006）（例如，感染性腹泻、炎症性肠病、肥胖/营养不良）。奖励不仅仅限于动态、密集地识别新的治疗药物及其靶标。在肠道等人口密集的生态系统中，细菌物种之间的水平基因转移可能具有重要意义因此，肠道生态系统提供了一个机会解决与“生态基因组学”相关的一般问题（Ley 等人，2006a；Stahl 和 Tiedje，2002 年）。共生体如何感知和应对环境的变化？环境是否影响其组成微生物物种的进化？给定物种并将遗传特征重新分配给联盟的其他成员，什么是基于基因组的物种形成和灭绝的定义是什么？营养循环和综合症的基因组基础是什么？肠道微生物群通过一个复杂的种间通讯网络和复杂的养分共享/循环网络看似令人难以承受。设想如何（i）确定管理这些建立的原则时的实验挑战环境传播的社区，(ii) 描述社区的贡献范围成员对产后肠道发育和成人生理学进行研究，（iii）解剖微生物宿主和微生物微生物- 沟通、途径 (iv) 了解指导共同进化和共同适应的力量特定肠道栖息地中的共生细菌及其宿主，和/或 (v) 破译宿主与病原体的相互作用。定义微生物对宿主生理学影响的一个关键实验策略是首先在没有细菌的情况下（即在无双生条件下）检查细胞功能，然后评估可以查看添加单一物种或指定数量的无宝石小鼠的效果。因为它们的多谱系微生物“器官”被完全消融，它们可以被一种公认的微生物定殖。或候选肠道共生体（或病原体），在产后肠道发育期间或完成后（Hooper 等人，2002a；Hooper 等人，2002b；Xu 等人，2003）。与另一个物种、或特定的物种集合、或未分级的物种进行比较和对比从出生时就获得微生物群的小鼠肠道区域采集微生物群（传统饲养的动物）。小鼠模型核心将继续提供关键的研究。向 DDRCC 成员提供探索宿主-微生物相互作用的工具作为服务：(i) 提供未经操作的无菌小鼠和基因型匹配的常规饲养的对照动物， (ii) 用细菌定植无菌小鼠，(iii) 从现有的无菌小鼠中重新衍生出无菌小鼠传统饲养的转基因小鼠品系。 A.3. 成本效益培育遗传性和知生性小鼠需要昂贵的设备和专门的设备技术专长和长期经验¿其生产超出了大多数人的资源范围小鼠模型核心可以及时访问这些小鼠模型。 DDRCC 调查人员负担得起的费用，并为需要指导的调查人员提供建议此外，Core还将为这些模型提供项目设计或技术实现服务。维持小鼠血统，包括冷冻保存、辅助生殖和快速转移这些技术对于实验室来说是无价的。不精通小鼠生殖生物学，并且它们的可用性使研究人员能够专注于 DDRCC 的专业领域是相关的生理学或病理学，这将带来巨大的成本。利用两个现有设施提供所有小鼠模型核心服务，从而受益： ¿知生动物将继续在杰弗里运营的设施中衍生和维持戈登的实验室的建立将是极其困难和令人望而却步的。对于 DDRCC 来说成本高昂，涉及购买设备以及雇用和培训鼠标人员 Sen/ice 的提供最经济、最可靠。当前戈登实验室设施中 DDRCC 使用的额外可变成本。 ¿所有禁止生物服务将由小鼠遗传学核心 (MGC) 提供。 Genetics Core (http://mgc.wustl.edu/) 是一个大型设施，致力于为所有人生产转基因小鼠华盛顿大学的研究人员，也是小鼠模型核心主任 Miner 博士的共同指导者。 MGC 向 DDRCC 调查人员提供的服务也比实际提供的更经济由专门的 DDRCC 设施完成；MGC 的规模允许规模经济，以便 DDRCC 的总体成本较低，此外，MGC 的使用确保了广泛的可靠和及时的提供。提供各种服务，并使 DDRCC 免受与员工流动相关的问题。通过覆盖约 75% 的成本来补贴 MGC 向 DDRCC 调查人员提供的服务，从而消除了显着的小鼠模型生产的障碍并促进新小鼠项目的启动。用于冷冻保存消化系统疾病相关小鼠品系，鼓励 DDRCC 研究人员利用此技术服务，从而确保有价值的线路在未来许多年得到安全维护和展示。