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,

A.定义分子生物学核心的目的是促进分子遗传学工具的应用胃肠道肽及其生理功能的研究。在各个方面，分子生物学提供基本服务的核心功能，并为所有中心研究人员提供教育资源。尽管基本的分子技术可通过商业来源广泛用于中心研究人员，但仍然有合并资源和受过良好训练的人员提供的特定服务，这些服务最能提供例如可以由中心核心设施提供的。在以前的资金期间，四个核心提供了程序：转基因啮齿动物计划，微阵列计划，病毒矢量计划和分子技术程序。在最后一次添加了微阵列基因芯片程序在当前资金期间提供了竞争性更新和分子技术计划循环以在调查人员拥有可以执行的技术中提供教育和培训实验室接受了一些理论和实用培训。后者可以自由地作为对中心成员的教育资源对染色质结构分析等技术感兴趣。研究人员需要提供基本分子技术的帮助，例如定量RT-PCR等复杂的技术，例如染色质免疫沉淀试验（CHIP）和CHLP-SEQ，可以接受核心董事（商人博士）和她的助手的培训（先生 Tessier）。图1显示了每个中心调查人员的数量该核心提供的程序。总而言之，这四个程序的特定目标分子生物学核心如下： 1。转基因啮齿动物计划的目的是促进小鼠和大鼠模型的生成研究中的调节肽生理环境。转基因和胚胎（ES）细胞技术将通过集中核心向所有中心成员提供密歇根大学医学中心的设施。转基因程序的具体目标如下：一个。作为转基因和ES细胞研究各个方面的教育资源 DNA构造对畜牧业的设计和表型的分析。 b。通过重新组合来制备专业的BAC构建体以产生转基因小鼠，例如组织 CRE重组酶或GFP的特异性表达。 c。从中心研究人员提供的克隆DNA的转基因小鼠和大鼠。 d。用靶向基因突变产生小鼠ES细胞克隆。 e。扩展ES克隆。 f。将ES细胞克隆对胚泡进行显微注射。 g。提供设备和试剂，向其实验室人员希望的调查人员中心自行进行胚泡注射或ES细胞的产生。 h。提供技术来协助啮齿动物菌落管理，包括小鼠应变冷冻保存和恢复，通过胚胎转移将小鼠转化为SPF状态以及体外受精（IVF）对于无法可靠产生后代的遗传线。我。监视向中心成员提供服务的公平分配。 2。病毒矢量程序的目的是促进使用病毒矢量在体外和体内基因转移到哺乳动物细胞中。最近的资金期间，最近重点的重点转变为雌雄病病毒载体的腺病毒载体。转变与基因转移到茎的兴趣增加有关细胞和原代细胞不经常分裂。在以前的资助期间，提供的服务是扩展到包括含有基因沉默技术的慢病毒载体。向量程序的特定目标如下：一个。作为使用病毒向量进行基因转移的教育资源。 b。作为一种教育资源，以使调查人员满足OSEH安全要求使用病毒载体所需。 c。培训中心调查人员如何在研究中构建和种植病毒库存。 d。为中心调查人员提供媒介和设施的访问权限，以构建各种病毒向量。 e。提供试剂量数量的病毒载体，以中心研究者。 3。微阵列基因芯片计划的目的是协助中心科学家使用高发射 RNA分析和基因分型方法用于描述肠道激素在健康和疾病。该程序包括现有Affymetrix和cDNA微阵列中的单独组件 Core，为密歇根大学中央和医学院隶属的所有调查员提供服务校园以及几个由NIH资助的中心。微阵列基因芯片程序的目标是以下内容：一个。积极协助中心研究人员进行基因表达的设计，性能和分析使用基于寡核苷酸和cDNA的微阵列技术进行分析实验。这些服务包括微阵列和Genechip的探针准备，标签，杂交，洗涤和数据采集实验。 b。在微阵列获得的数据的统计分析中提供专业知识和帮助处理大型数据集，以及用于数据挖掘大型数据集； c。提供使用定量聚合酶验证微阵列获得数据的手段链反应技术。 d。为基因组研究提供工具，包括SNP单倍型服务和Affymetrix HUSNP 转基因服务。 4。分子技术培训计划的目的是为中心研究人员提供理论背后并获得分子技术实用训练，例如蛋白质-DNA相互作用和染色质免疫沉淀试验（CHIP）和CHLP-SEQ。后者将与 DNA测序核心（Robert Lyons，董事）和生物信息学核心（詹姆斯·卡瓦科利（James Cavacoli），导演）。 [看博士的支持信。里昂和卡瓦科利]。该计划的具体目标如下：一个。作为用于应用基因表达和DNA蛋白的教育资源相互作用技术，例如电泳迁移率分析（EMSA，凝胶移位），DNA足迹，DNA 甲基化，转染，启动子构建，定量PCR，报告基因测定，芯片分析。 b。培训研究人员采用专业技术，例如DNA-蛋白结合和CHIP分析。 c。监视到微阵列程序的应用程序流。 d。提供和维护设备以分析记者基因测定。 e。向调查人员传授如何附加“底漆适配器”以生成下一步的库在提交DNA测序核心之前，生成测序（NGS）。 f。为了对胃蛋白和生长抑素进行放射免疫测定，