A. DEFINITION The purpose of the Molecular Biology core Is to facilitate the application of the tools of molecular genetics to the study of gastrointestinal peptides and their physiological functions. In all aspects, the Molecular Biology Core functions to provide essential services and serves as an educational resource to all Center Investigators. Although basic molecular techniques are widely available to Center investigators through commercial sources, there remains specific services that are best provided by pooled resources and by highly trained personnel such as those that can be provided by a Center Core facility. During the prior funding period, four Core programs were provided: the Transgenic Rodent Program, the Microarray Program, Viral Vector Program and the Molecular Techniques Program. The Microarray Gene Chip Program was added during the last competitive renewal and the Molecular Techniques Program was made available during the current funding cycle to provide education and training in those techniques that can be performed in the investigators own laboratory after receiving some theoretical and practical training. The latter is freely available as an educational resource for Center members interested in such techniques as chromatin structure analysis. Investigators needing assistance with basic molecular techniques, e.g., quantitative RT-PCR and more sophisticated techniques, e.g., chromatin immunoprecipitation assays (ChIP) and ChlP-Seq, can receive training from the Core Director (Dr. Merchant) and her assistant (Mr. Tessier). Fig. 1 shows the number of Center investigators for each of the Programs offered in this core. To summarize, the specific objectives of the four Programs within the Molecular Biology Core are as follows: 1. The purpose of the Transgenic Rodent Program is to facilitate the generation of mouse and rat models to study regulatory peptides in a physiologic context. Transgenic and embryonic (ES) cell technology will be available to all Center members through a centralized core facility at the University of Michigan Medical Center. The Transgenic Program's specific objectives are as follows: a. To serve as an educational resource for all aspects of transgenic and ES cell research, from the design of DNA constructs to animal husbandry and analysis of phenotypes. b. To prepare specialized BAC constructs by recombineering to generate transgenic mice, e.g., tissue specific expression of Cre recombinase or GFP. c. To prepare transgenic mice and rats from cloned DNA provided by Center investigators. d. To generate mouse ES cell clones with targeted gene mutations. e. To expand ES clones. f. To perform microinjection of ES cell clones into blastocyts. g. To provide equipment and reagents to Center investigators whose laboratory personnel wish to perform blastocyst injections or ES cell production themselves. h. To offer technologies to assist in rodent colony management, including mouse strain cryopreservation and recovery, conversion of mice to SPF status by embryo transfer, and in vitro fertilization (IVF) for genetic lines that are not reliably producing progeny. i. To monitor equitable distribution of services to center members. 2. The purpose of the Viral Vector Program is to facilitate the use of viral vectors for in vitro and in vivo gene transfer into mammalian cells. There has been a recent shift in emphasis during the prior funding period from adenoviral vectors to lentiviral vectors. The shift is related to the increased interest for gene transfer into stem cell and primary cells that divide infrequently. During the prior funding period, the services offered were expanded to include lentiviral vectors containing gene silencing technologies. The Vector Program's specific objectives are as follows: a. To serve as an educational resource for the use of viral vectors for gene transfer. b. To serve as an educational resource to Center investigators on meeting OSEH safety requirements required for use of viral vectors. c. To train Center investigators in how to construct and grow viral stocks for use in their research. d. To provide access to vectors and facilities for Center investigators to construct a variety of viral vectors. e. To provide reagent size quantities of viral vectors to Center investigators. 3. The purpose of the Microarray Gene Chip Program is to assist Center scientists in the use of highthroughput RNA profiling and genotyping methods towards delineating the role of gut hormones in health and disease. The Program comprises a separate component within the existing Affymetrix and cDNA Microarray Core, which serves all investigators affiliated with the University of Michigan Central and Medical School Campus, as well as several NIH-funded Centers. The objectives of the Microarray Gene Chip Program are as follows: a. To actively assist Center investigators with the design, performance and analysis of gene expression profiling experiments using oligonucleotide- and cDNA-based microarray technology. These services include probe preparation, labeling, hybridization, washing, and data acquisition for microarray and GeneChip experiments. b. To provide expertise and assistance in the statistical analysis of microarray-acquired data, for handling large data sets, and for data-mining large data sets; c. To provide the means for the validation of microarray-acquired data using quantitative polymerase chain reaction technology. d. To offer tools for genomic studies, including a SNP haplotyping service and Affymetrix HuSNP GeneChip services. 4. The purpose of the Molecular Technigues Training Program is to provide Center investigators with the theory behind and access to practical training in molecular techniques, e.g., protein-DNA interactions and chromatin immunoprecipitation assays (ChIP) and ChlP-Seq. The latter will be performed coordinately with the DNA Sequencing core (Robert Lyons, Director) and Bioinformatics core (James Cavacoli, Director). [See letters of support from Drs. Lyons and Cavacoli]. The Program's specific objectives are as follows: a. To serve as an educational resource for the application of gene expression and DNA-proteIn interaction techniques, e.g., electrophoretic mobility shift assays (EMSAs, gel shifts), DNA footprinting, DNA methylation, transfections, promoter construction, quantitative PCR, reporter gene assays, ChIP analysis. b. To train investigators in specialized techniques, e.g., DNA-protein binding and ChIP assays. c. To monitor the flow of applications to the Microarray Program. d. To provide and maintain the equipment to analyze reporter gene assays. e. To teach investigators on how to attach primer adaptors to generate the libraries for Next- Generation Sequencing (NGS) prior to submission to the DNA sequencing Core. f. To perform radioimmunoassays for gastrin and somatostatin,
Showing the most recent 10 out of 757 publications
