MURINE MODELS CORE

华盛顿大学消化疾病研究中心广泛使用小鼠模型

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.