Genetics, Genomics and Molecular Biology Core
|Hans-Christian Reinecker, MD
|Mark Daly, PhD
|Aylwin Ng, PhD
|Brian Seed, PhD
Manager of CSIBD Genetics and Microbiome Studies
The major roles of the Genetics, Genomics and Molecular Biology (GGMB) Core are 1) to provide services in the utilization of advanced genetics and molecular biological techniques and 2) to facilitate the implementation of advances in genomics and molecular biology to IBD research. Application of genetics and molecular biological approaches is central to the evaluation of processes underlying the development of IBD according to the hypotheses that serve as a foundation for this center. As genetics play a significant role in determining the risk of developing IBD, identifying specific risk-associated genes is a priority for CSIBD researchers who are seeking to determine underlying disease processes. This core principally provides access to high-demand, mission-enabling tools for the investigator cohort participating in the CSIBD. The core also provides access to capital or resource-intensive methodologies, such as laboratory automation cDNA and siRNA libraries, and BAC-centered recombinant DNA techniques. The importance of molecular methods for the understanding of mechanisms of immunity and generating transgenic/knockout mice is undeniable, and their impact and dissemination throughout biological disciplines grow every year. These techniques permit the identification and characterization of genes regulating epithelial and immune cell function, analysis of the expression of these genes, determination of the functions and interactions of the encoded proteins, and expression of reporter genes and proteins to allow cellular localization and physiological analysis. For those laboratories with limited expertise in molecular biology, the core has developed a major educational effort consisting of the annual current techniques course in molecular genetics and training in genetic epidemiology, quantitative biology, and genomic medicine.
The objectives of the GGMB Core are to provide:
- Facilitation of the application of advanced experimental platforms for genetics, genomics and high throughput data analysis to discovery efforts relevant to inflammatory bowel diseases. The GGMB Core will provide access to and training in the use of bioinformatics analysis and software, including the large variety of databases and software tools available via the internet, as well as molecular biology and statistical applications.
A centralized facility and personnel for performing state-of-the-art recombinant and PCR-based DNA procedures and RNA interference.
Cost-effective and high-quality supplies of molecular biology reagents and services including nucleic acid probes, expression plasmids, reporter gene constructs, viral vectors, real-time PCR, mutagenesis, expertise for differential mRNA display analysis, RNA purification for DNA microarray analysis and facilitated access to bioinformatics support.
A resource for disseminating a wide range of molecular biology, genetic and bioinformatics technologies. Services are initially provided by the core as part of hands-on training for center personnel, allowing transfer of the technology to the laboratories of individual CSIBD investigators. Training opportunities include the formal molecular biology laboratory course, (directed by Dr. Reinecker) as well as ad hoc focused training experiences within the GGMB Core laboratories. Techniques covered have included methods to examine gene expression and transcription regulation, library preparation, several types of expression screening and differential display protocols, competitive and real-time PCR, microarray analysis, protein expression in prokaryotes, yeast, insect cells and mammalian cells, site-directed mutagenesis, two-hybrid screening, recombination engineering of gene targeting constructs, and methods to examine signal transduction, cell-cell, protein-protein and DNA-protein interactions.
Gene Profiling Services
- Real-time qPCR validation of profiling data
- DNA Sequencing
- High Throughput Oligonucleotide Synthesis
- Research Laboratory Automation for siRNA and cDNA library screening
- Plasmid Repository
- Technology Transfer Services
Research techniques currently available for transfer from the core include the following:
- Preparation and analysis of genomic DNA, including DNA isolation from tissues, cell lines, and paraffin-embedded specimens, PCR amplification of genomic DNA, and Southern and dot blot analyses
- mRNA preparation and analysis, including extraction from cells and tissues, RNA purification, Northern blot and in situ hybridization analysis, S1 nuclease and RNase protection mapping, and primer extension analysis
- General PCR applications, including anchored PCR, quantitative, real-time PCR, in situ PCR, inverse PCR and single-stranded conformation polymorphism analysis for mutation detection
- Subcloning and sequencing of DNA fragments, including single- and double-stranded sequencing protocols, and direct sequencing of PCR products
- Transfection of DNA into eukaryotic cells by chemical and electrical protocols, and generation of stably transfected eukaryotic cells
- Generation of targeting constructs by recombination engineering in bacterial artificial chromosomes, as well as by standard cloning techniques
- Use and assay of reporter gene constructs to examine gene transcription and protein synthesis
- Nuclear run-on assays, intron-specific hybridization and mRNA half-life analysis to examine the molecular basis of gene regulation
- Assay of DNA-protein interactions, including avidin-biotin DNA complexes, gel mobility shift, DNA footprinting and methylation interference, UV crosslinking and southwestern blotting
- Analysis of protein-protein interactions by precipitation-based techniques and yeast two-hybrid and related technologies
- Chromosomal gene localization by fluorescent in situ hybridization (FISH)
- Protein expression in bacteria, yeast, insect cells, mammalian cells, and eukaryotic virus systems, including His6 and glutathione-S-transferase fusion proteins
- Site-directed DNA mutagenesis, including use of PCR, oligonucleotide hybridization, exonuclease III digestion and insertion of oligonucleotide cassettes
- Immunoassays, including immunoprecipitation, Western blots, enzyme-linked and radioimmunoassays
- Measurement of signal transduction, including protein phosphorylation and phosphatase activities, cAMP and IP3 generation, and protein association/dissociation
- Gene expression profiling by DNA microarray technology, including statistical analysis of results
- Use of bioinformatics tools for molecular biology and genetic analysis