MOLECULAR VECTORS AND PEPTIDOMICS CORE

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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.