MOLECULAR VECTORS AND PEPTIDOMICS CORE

分子载体和肽组学核心

• 批准号: 7767527
• 负责人: NORIYUKI KASAHARA
• 金额: $ 12.12万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 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/7767527
• 关键词: Adenovirus Vector; Advisory Services; Animal Model; Arts; Biological; Biological Process; Cell Line; Cells; Chemicals; Code; Commercial Sectors; Complement; Complex; Complex Mixtures; Consultations; Core Facility; Custom; Data; Detection; Development; Digestion; Digestive System Disorders; Discipline; Disease; Disease Marker; Dose; Embryo; Endocrine; Ensure; Event; Face; Family; Figs - dietary; Functional RNA; Functional disorder; Gastrointestinal Physiology; Gastrointestinal tract structure; Gene Delivery; Gene Expression; Gene Proteins; Gene Transfer; Genes; Genetic; Growth Factor; Health; Hepatobiliary; Hormones; Human Genome Project; Immunologics; In Vitro; Individual; Investigational Therapies; Knowledge; Label; Laboratories; Libraries; Maintenance; Mammalian Cell; Mass Spectrum Analysis; Mediating; Methodology; Methods; Molecular; Molecular Weight; Morphology; Neuropeptides; Oocytes; Organogenesis; Pancreas; Pathogenesis; Pattern; Peptides; Physiological Processes; Physiology; Play; Population; Positioning Attribute; Process; Production; Productivity; Protein Analysis; Protein Isoforms; Proteins; Proteomics; Protocols documentation; RNA; RNA Interference; RNA Sequences; RNA Splicing; Radiolabeled; Reagent; Regulation; Regulator Genes; Research; Research Activity; Research Personnel; Research Project Grants; Resolution; Resources; Risk; Role; Selection Bias; Services; Signal Transduction; Somatic Cell; Source; System; Techniques; Technology; Time; Tissues; Training; Transfection; Transgenic Animals; Variant; Vendor; Viral Genes; Viral Vector; Work; absorption; base; blastocyst; cell motility; cellular imaging; cellular transduction; cost; cytokine; design; design and construction; expression vector; flexibility; functional genomics; gastrointestinal; gastrointestinal system; gene transfer vector; high throughput technology; immortalized cell; improved; in vivo; instrument; instrumentation; interest; meetings; molecular mass; molecular vector; mutant; new therapeutic target; operation; overexpression; paracrine; polyclonal antibody; protein expression; radiotracer; research study; response; small hairpin RNA; synthetic peptide; tool; transgene expression; vector

The completion of the Human Genome Project comprising approximately 30,000 genes with even more numerous splice variants generating additional protein coding sequences, and the recent identification of multiple species of RNA that are now recognized to perform critical regulatory functions through RNA interference, have provided a wealth of new genetic information. However, with this newly acquired knowledge, molecular and cellular biologists in all disciplines now face the tremendous task of understanding the functions of individual genetic sequences and protein isoforms whose significance and impact on health and disease may be unclear. Even with high-throughput technologies such as microarrays or proteomics, the gene or protein expression pattern in toto often yields a composite picture in which primary and secondary effects are difficult to unravel, and ultimately the true biological or pathogenetic significance of individual candidate cDNAs encoding proteins or non-coding RNA sequences must be confirmed directly. The field of gastrointestinal research is certainly no exception, and moreover, represents an even more complex system in which coordinately regulated genetic and protein expression patterns result in the development and maintenance of specialized physiological processes of motility, secretion, digestion, and absorption, which further coordinStely interact with pancreatic and hepatobiliary functions. In addition, the gastrointestinal system is subject to extensive paracrine, endocrine, and neurocrine regulatory mechanisms that modulate its digestive functions, as well as innate and adaptive immunologic mechanisms that mediate mucosal defense. Hence, increasing focus is aimed at elucidating molecular signaling events that mediate alterations in regulatory genes and functional protein expression, and thereby exert such modulatory influences on local physiological processes in the alimentary tract. In this context, recent developments in gene expression and protein analysis technologies indicate that the molecular research tools available are already sufficiently advanced to approach many questions relevant to normal and abnormal processes of the digestive system, including cell signaling, integrative physiology, functional disorders, diseases and its complications. The Molecular Vector and Peptidomics Core is therefore intended to serve as a comprehensive resource that will provide access to state-of-the-art molecular tools, and thereby aims to facilitate the research activities of CURE: DDRCC investigators. This Core represents the consolidation of two previous Cores (the Molecular Vector Core and the Peptidomics / RIA / Proteomics Core) into one integrated unit, intended to maximize efficiency and ease of utilization, while minimizing overiap with other existing core facilities already accessible on campus (see Fig. 1). As such, this Core is now well-positioned to offer CURE: DDRCC investigators a wide variety of unique research reagents and individually customized services, ranging from viral vector-mediated gene delivery to functional protein analysis, which cannot be readily obtained from commercial or other academic sources at comparable cost. By consolidating these services and expertise into a single integrated Core, we anticipate that users will be provided with more efficient "one-stop shopping", as well as the flexibility to offer a variety of molecular approaches that can be tailored to optimally meet the needs of individual research projects. Gene delivery and protein expression technologies: Recent advances in vector technology have made it feasible to utilize gene transfer as a methodology to elucidate the functions of specific genetic sequences by examining the phenotypic consequences of their overexpression or inhibition of specific proteins in transduced cells in vitro and in vivo; in this sense, gene transfer technology can be viewed as a highly useful tool for functional genomics and proteomics. In particular, viral vector technologies developed over the past two decades offer the advantages of consistent and reliable gene transfer that, unlike physical or chemical transfection methods, can achieve extremely high efficiencies. Depending on the vector system used, viral gene transfer can achieve longterm expression of cDNAs encoding wild type or mutant proteins (e.g., constitutively active or dominantnegative), as well as antisense and small hairpin RNA sequences, in large populations of quiescent primary cells as well as immortalized cell lines, without the need for stable selection and hence without incurring the risk of confounding clonal selection bias effects. Furthermore, stably integrating retroviral and especially lentiviral gene transfer vectors can also be introduced directly into fertilized oocytes or embryonic blastocysts to more efficiently generate transgenic animal models. If the effects of transgene expression in conventional or vector-generated transgenic animals result in embryonic lethality, viral vectors can also be used for post-natal gene transfer directly to somatic cells in target tissues. Despite the advantages cited above, use of viral vector technology requires specialized expertise and resources often not found in an individual investigator's laboratory. Easy access to these technologies can therefore facilitate and expand the scope of research activities, and will provide a means for investigators to rapidly generate preliminary data. Viral gene transfer technologies have now sufficiently matured and are robust, reliable, and useful enough to warrant offering easy access to gene expression vectors through a Core. Consolidation of these services as a Core is more cost-effective than utilizing commercially available sources, and further value is added by customized technical support available from readily accessible and knowledgeable staff who can work intensively with investigators to troubleshoot and optimize vector applications. Thus, by providing such access to these technologies, we significantly facilitate the research productivity of CURE: DDRCC investigators, and furthermore, we complement the existing strengths of other CURE: DDRCC Research Cores, including the Animal Models Core and the Morphology and Cell Imaging Core. Quantitative and functional peptide / protein analysis technologies: Proteomics has established itself as a highly valuable technology for studying complex biological problems and for the identification of disease markers, but is methodologically restricted to the analysis of proteins with higher molecular masses (>10 kDa). The development of a technology which covers peptides with low molecular weight and small proteins (0.5 to 15 kDa) has been necessary, since peptides, amongst them families of hormones, neuropeptides, cytokines and growth factors, play a central role in many biological processes, especially in the regulation of the functions of the digestive system. In many cases, particulariy for smaller peptides, direct synthesis is a simpler alternative to gene delivery-based protein expression, and for example, peptide dose-response parameters in signal transduction can be more easily controlled with use of pre-synthesized material. Improved isolation and detection technologies also permit more sensitive and quantitative analysis of peptides and small proteins in complex mixtures and cell lysates. To summarize the technologies used for this approach the term "peptidomics" is increasingly used. Recent developments in peptidomics indicate that these technologies are also already sufficiently advanced to approach many questions relevant to gastrointestinal physiology, diseases and its complications. In response to these new and evolving technologies, services previously offered by the Peptidomics, RIA and Proteomics Core, have been revised and refined, and consolidated with gene delivery and protein expression technologies previously offered by the Molecular Vector Core. As such, the current consolidated Molecular Vectors and Peptidomics Core now takes an integrated approach to the expression and characterization of the whole spectrum of gene products from peptidomics to proteomics. This integrated approach provides CURE: DDRCC investigators with a comprehensive breadth of molecular tools to study digestive functions and diseases. Most peptide- and protein-based services provided by the Core are performed on expensive instruments that require high levels of expertise for operation and maintenance. This level of expense and expertise cannot be duplicated in every CURE: DDRCC laboratory. Purification and purity analysis of synthetic peptides obtained from vendors are performed on instruments that are best utilized in core facilities. The cost of the instruments and the expertise for their optimal operation dictate their full time utilization by staff working full time on these instruments. The other benefit to users is the peptide design expertise and advisory service: investigators are not only provided with optimal peptide designs but also with detailed explanations of the reasons for design features and presented with options to ensure that the best candidate peptide is synthesized by commercial vendors in a cost-effective manner. Additional services, including facilitated access to on-campus shared facilities specializing in proteomics, and mass spectrometry, provide further benefit to CURE: DDRCC investigators. However, many such experiments and projects require careful consultation between the CURE: DDRCC investigator and knowledgeable Core staff, and frequently, resolution of obstacles can only be achieved after preliminary experiments and in-depth discussion. The need for this type of critical interaction is not met when services are sought from the commercial sector.

人类基因组计划完成，包含约 30,000 个基因，甚至更多 许多剪接变体产生额外的蛋白质编码序列，并且最近鉴定 多种 RNA 现已被认为通过 RNA 执行关键的调节功能 干扰，提供了丰富的新遗传信息。然而，随着这个新收购的 所有学科的分子和细胞生物学家现在都面临着巨大的任务 了解个体基因序列和蛋白质亚型的功能，其重要性和 对健康和疾病的影响可能尚不清楚。即使采用微阵列等高通量技术 或蛋白质组学，基因或蛋白质表达模式的整体通常会产生一幅合成图，其中 主要和次要影响很难阐明，最终真正的生物学或致病性 编码蛋白质或非编码RNA序列的各个候选cDNA的重要性必须是 直接确认。 胃肠道研究领域当然也不例外，而且代表着更广泛的领域。 协调调节遗传和蛋白质表达模式的复杂系统 运动、分泌、消化和消化等特殊生理过程的发展和维持 吸收，进一步协调胰腺和肝胆功能。此外， 胃肠系统受到广泛的旁分泌、内分泌和神经分泌调节机制的影响 调节其消化功能，以及介导先天性和适应性免疫机制 粘膜防御。因此，越来越多的关注旨在阐明介导的分子信号传导事件 调节基因和功能蛋白表达的改变，从而发挥这种调节作用 对消化道局部生理过程的影响。在此背景下，近期事态发展 基因表达和蛋白质分析技术表明可用的分子研究工具是 已经足够先进来解决与正常和异常过程相关的许多问题 消化系统，包括细胞信号传导、综合生理学、功能障碍、疾病及其 并发症。 因此，分子载体和肽组学核心旨在作为综合资源 这将提供最先进的分子工具，从而促进研究 CURE 的活动：DDRCC 调查员。该核心代表了之前两个核心的整合 （分子载体核心和肽组学/RIA/蛋白质组学核心）整合为一个集成单元，旨在 最大限度地提高效率和易用性，同时最大限度地减少与其他现有核心设施的重叠 校园内已经可以使用（见图 1）。 因此，该核心现在处于有利地位，可以为 CURE：DDRCC 研究人员提供各种独特的 研究试剂和个性化定制服务，范围从病毒载体介导的基因传递到 功能性蛋白质分析，不能轻易地从商业或其他学术来源获得 可比成本。通过将这些服务和专业知识整合到一个集成核心中，我们预计 将为用户提供更高效的“一站式购物”，以及提供多种选择的灵活性 可以定制的分子方法，以最佳地满足各个研究项目的需求。 基因递送和蛋白质表达技术：载体技术的最新进展 使得利用基因转移作为一种方法来阐明特定遗传功能成为可能 通过检查特定序列的过度表达或抑制的表型后果来分析序列 体外和体内转导细胞中的蛋白质；从这个意义上说，基因转移技术可以被视为一种 功能基因组学和蛋白质组学非常有用的工具。 特别是，过去二十年发展起来的病毒载体技术具有以下优点： 与物理或化学转染方法不同，一致且可靠的基因转移可以实现 效率极高。根据所使用的载体系统，病毒基因转移可以实现长期 编码野生型或突变蛋白（例如，组成型活性或显性失活）的cDNA的表达， 以及反义和小发夹 RNA 序列，在大量静止初级群体中 细胞以及永生化细胞系，无需稳定选择，因此不会产生 混淆克隆选择偏差效应的风险。此外，稳定整合逆转录病毒，尤其是 慢病毒基因转移载体也可以直接导入受精卵母细胞或胚胎中 囊胚以更有效地产生转基因动物模型。如果转基因表达的影响 传统或载体产生的转基因动物会导致胚胎致死，病毒载体也可以 用于产后基因直接转移至目标组织的体细胞。 尽管具有上述优点，但使用病毒载体技术需要专业知识和 个别研究者的实验室中通常找不到资源。轻松获取这些技术可以 因此，促进和扩大研究活动的范围，并将为研究人员提供一种手段 快速生成初步数据。病毒基因转移技术现已足够成熟， 强大、可靠且有用，足以保证通过 核。将这些服务整合为核心比利用商业服务更具成本效益 来源，并通过可轻松访问和获得的定制技术支持增加了进一步的价值 知识渊博的工作人员可以与调查人员密切合作，排除故障并优化载体 应用程序。因此，通过提供对这些技术的访问，我们极大地促进了研究 CURE：DDRCC 研究人员的生产力，此外，我们补充了 其他 CURE：DDRCC 研究核心，包括动物模型核心以及形态和细胞 成像核心。 定量和功能性肽/蛋白质分析技术：蛋白质组学已建立 它本身是一种非常有价值的技术，用于研究复杂的生物问题和识别 疾病标志物，但方法论仅限于分析具有较高分子量的蛋白质 (>10 kDa)。开发涵盖低分子量、小分子肽的技术 蛋白质（0.5 至 15 kDa）是必需的，因为肽，其中包括激素家族， 神经肽、细胞因子和生长因子在许多生物过程中发挥着核心作用，特别是在 消化系统功能的调节。在许多情况下，特别是对于较小的肽， 直接合成是基于基因传递的蛋白质表达的更简单的替代方案，例如，肽 使用预合成的药物可以更容易地控制信号转导中的剂量反应参数 材料。改进的分离和检测技术还可以提高灵敏度和定量性 分析复杂混合物和细胞裂解物中的肽和小蛋白质。总结一下 用于这种方法的技术越来越多地使用术语“肽组学”。最近的发展 肽组学表明这些技术也已经足够先进，可以接近许多 与胃肠道生理学、疾病及其并发症相关的问题。 为了应对这些不断发展的新技术，Peptidomics、RIA 之前提供的服务 和蛋白质组学核心，已被修订和完善，并与基因传递和蛋白质相整合 之前由 Molecular Vector Core 提供的表达技术。因此，当前 整合的分子载体和肽组学核心现在采用综合方法来表达 从肽组学到蛋白质组学的整个基因产物谱的表征。这 综合方法为 CURE：DDRCC 研究人员提供了全面的分子生物学知识 研究消化功能和疾病的工具。 核心提供的大多数基于肽和蛋白质的服务都是在昂贵的仪器上进行的 需要高水平的操作和维护专业知识。这种水平的费用和专业知识 无法在每个 CURE: DDRCC 实验室中重复。合成物的纯化和纯度分析 从供应商处获得的肽是在核心设施中最适合使用的仪器上进行的。这 仪器的成本及其最佳操作的专业知识决定了工作人员的全职使用 全职从事这些仪器工作。用户的另一个好处是肽设计专业知识和 咨询服务：不仅为研究人员提供最佳的肽设计，还为研究人员提供详细的肽设计 解释设计特征的原因并提供选项以确保最佳效果 候选肽由商业供应商以具有成本效益的方式合成。 其他服务，包括方便使用专门从事蛋白质组学的校园共享设施， 和质谱分析，为 CURE：DDRCC 研究人员提供进一步的好处。然而，许多这样的 实验和项目需要 CURE：DDRCC 研究者和 知识渊博的核心人员，并且通常只有在经过初步准备后才能解决障碍 实验和深入讨论。当服务无法满足这种类型的关键交互的需求时 是从商业部门寻求的。