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MOLECULAR BIOLOGY CORE

分子生物学核心

基本信息

批准号：
8788007
负责人：
JUANITA L. MERCHANT
金额：
$ 17.14万
依托单位：
UNIVERSITY OF MICHIGAN AT ANN ARBOR
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至 2017-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8788007
关键词：
Address Adenovirus Vector Adopted Agreement Aliquot Alleles Animal Experiments Animal Husbandry Animal Model Animals Antibodies Bacterial Artificial Chromosomes Bioconductor Bioinformatics Biological Biological Assay Biological Models Biometry Breeding Caenorhabditis elegans Candidate Disease Gene Cataloging Catalogs Cell Culture Techniques Cell Line Cell Separation Cells Chimera organism Chromatin Chromatin Structure Chromosomes Clinical Cobalt Complement Complementary DNA Consultations Core Facility Cre-LoxP Cryopreservation Custom DNA DNA Footprint DNA Methylation DNA Sequence DNA Sequencing Facility DNA analysis DNA-Binding Proteins DNA-Protein Interaction Data Data Set Databases Disease Doctor of Medicine Drosophila melanogaster EMSA ES Cell Line Education Educational process of instructing Effectiveness Electrophoretic Mobility Shift Assay Electroporation Embryo Embryo Transfer Equipment Evaluation Experimental Designs Feline Immunodeficiency Virus Fellowship Fertilization in Vitro Firefly Luciferases Funding Gastrins Gel Gene Chips Gene Expression Gene Expression Profiling Gene Mutation Gene Silencing Gene Targeting Gene Transfer Generations Genes Genetic Genetic Engineering Genetic Recombination Genetic screening method Genome Genomic DNA Genotype Germ-Free Goals HIV Health Hormones Human Human Genome Human Resources Hypersensitivity Ice In Vitro Inbred Strain Individual Infection Injection of therapeutic agent Institution Knock-out Knockout Mice Lab-On-A-Chips Label Laboratories Laboratory Personnel Lentivirus Vector Letters Libraries Link Location Luciferases Luminescent Measurements Mammalian Cell Measurement Medical center Mentors Messenger RNA Methodology Methods Michigan Microarray Analysis Microfluidics Microinjections Micromanipulation Modeling Molecular Molecular Biology Molecular Biology Techniques Molecular Genetics Molecular Profiling Monitor Mouse Strains Mus Mutation Northern Blotting Oligonucleotides Operative Surgical Procedures Organism Other Genetics Pathway interactions Pattern Peptides Performance Phenotype Physiologic pulse Physiological Plaque Assay Plasmid Cloning Vector Plasmids Polymerase Chain Reaction Preparation Process Production Proteins Protocols documentation PubMed Quantitative Reverse Transcriptase PCR RNA RNA Interference RNA analysis Radioimmunoassay Rat Strains Rattus Reagent Recombinants Recording of previous events Recovery Renilla Luciferases Reporter Reporter Genes Research Research Design Research Infrastructure Research Personnel Resources Retroviral Vector Retroviridae Reverse Transcriptase Polymerase Chain Reaction Review Committee Rodent Role SNP genotyping Safety Sampling Scanning Scheme Scientist Sequence Analysis Services Shuttle Vectors Somatostatin Source Southern Blotting Specific Pathogen Frees Specimen Spottings Stem cells Structure System Targeted Research Techniques Technology Time Tissues Training Training Programs Training and Education Transcript Transfection Transgenes Transgenic Animals Transgenic Mice Transgenic Organisms United States National Institutes of Health Universities Validation Vertebral column Viral Viral Vector Virus Virus Diseases Work Xenopus laevis Zebrafish assisted reproduction base beta-Galactosidase blastocyst cDNA Arrays cell type chromatin immunoprecipitation cost data acquisition data mining design design and construction discount embryonic stem cell experience expression vector gastrointestinal genetic strain genomic tools germ free condition high throughput analysis in vivo in vivo Model indexing instrument interest investigator training laser capture microdissection medical schools meetings member mouse genome next generation sequencing pathogen plasmid DNA pluripotency programs promoter recombinase research study skills success theories tool transduction efficiency transgene expression vector web site zygote

项目摘要

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,