CORE--MOLECULAR BIOLOGY FACILITY

University Of Pennsylvania, Philadelphia PA

Investigators

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Abstract

Description (provided by applicant) The purpose of the Molecular Biology Core as stated in the original Center Application in 1996 was to provide advice, equipment and technical applications for molecular cellular experimental approaches specific to digestive and liver disease. The major components of the Services of the Molecular Biology Core included: A) Digestive Disease regents, B) DNA Sequencing, C) Image analysis, D) Molecular and Cellular Biology Techniques, and E) Education. Many components of this Core have been highly successful under the directorship of Dr. Rebecca Taub. Enhancing the availability of reagents and protocols specifically targeted to digestive and liver research is of vital importance in attracting both current and potential Digestive Disease Center investigators. In this regard, components A-C have been instrument to the success of the Molecular Biology Core and the overall Center. They will be retained and expanded in the following fashion: A) Digestive Disease Reagents: We have expanded this repository of reagents to include significant additions to the stock of cDNA libraries, cDNA probes antibodies, Tab polymerase, expression vectors, and general purpose vectors already available through this core. A new component of the Molecular Biology Reagent Bank is a Human RNA Source Bank; B) DNA oligonucleotide synthesis and DNA sequencing. New components include: Direct PCR product sequencing, direct sequencing of cosmids/P1/BAC/ and PAC clones, walking strategy, and site directed mutagenesis; and c) Image Analysis: The Molecular Biology Core will continue to support the use of a Molecular Dynamics be a fully equipped workstation to analyze both custom gene arrays as well as commercially available arrays through Affymetrics (GeneChip Analysis Suite) and Genemax (Informax). The last two components of the Molecular Biology Core, D) Molecular and Cellular Biology Techniques and E) Education, have been redesigned and expanded to incorporate the analysis of gene expression at the level of mRNA through the use of array technology. At the time of the initial Center grant submission, many of the molecular techniques in item D were new to many investigators involved in digestive disease research. These include cDNA library construction, bacterial expression of proteins, and yeast two-hybrid cloning. However, with time, these techniques have become more widely used by many investigators, in part through the success of the Molecular Biology Core under Dr. Taub's direction. As a result, the need for many of the services in components D and E has diminished. In contrast, there has been considerable interest by Center Members to have services that would permit utilization of emerging technologies in the analysis of gene expression. Therefore, components D and E of the Molecular Biology Core will be replaced by a new Gene Expression Facility. This was met with great enthusiasm by the Executive Committee and the External Advisory Committee. The ability to examine and quantify alterations in gene expression is fundamental to the understanding of molecular mechanisms that determine biological processes. High density gene arrays are now a technical reality. The cost of this technology, although previous prohibitive for most investigators, has very recently become affordable for most investigators, has very recently become affordable This facility will have 2 components: 1) Digestive organ custom arrays, Affymetrix commercial arrays, and the Genomax Bioinformatics Workstation, and 2) Real-Time Quantitative PCR. The Gene Expression Facility will be highly interactive, not only with the other Cores of the Center, but also with the Clinical Investigators from both the Division of Gastroenterology and the Department of Surgery. Examples of these interactions include: 1) Interact with the transgenic/chimeric core facility by assisting in the analysis of murine tissues from wild-type and genetically engineered mice; 2) Complement the analysis of gene expression performed by the Morphology Core through in situ hybridization; 3) Assist in the analysis of in vitro cell culture model systems developed by the Cell Culture Core; and 4) Interact in a translational fashion with patient oriented research through the analysis of gene expression in human tissues. The services provided by the Gene Expression Facility are designed to provide access to several important components in the analysis of gene expression at the level of mRNA. Patterns of mRNA expression, acquired from various sources by interacting with the other Core facilities in the Digestive Disease Center, can be determined by gene array analysis either using custom gene arrays (murine) or Affymetrix arrays (murine and human). Bioinformatics and computational biology are critical components to the analysis of the data obtained from these array experiments and will be supported by the Molecular Biology Core through various Data Analysis tools provided by the Core, principally Genomax. Ultimately, with the identification of specific target genes of interest, quantification of target gene expression in a large number of samples can be performed by Quantitative Real-Time PCR and cellular localization can be determined by in situ hybridization.