MOLECULAR BIOLOGY CORE

分子生物学核心

基本信息

批准号：
8588312
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
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/8588312
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 Genome Genomic DNA Genomics 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 Testing 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 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研究胃肠肽及其生理功能。从各个方面来说，分子生物学核心职能是提供基本服务并作为所有中心调查员的教育资源。尽管中心研究人员可以通过商业来源广泛获得基本的分子技术，仍然存在最好由集中资源和训练有素的人员提供的具体服务例如中心核心设施可以提供的那些。在上一个资助期间，四个核心提供了以下计划：转基因啮齿动物计划、微阵列计划、病毒载体计划和分子技术计划。微阵列基因芯片项目是在去年添加的在当前资助期间提供了竞争性更新和分子技术计划周期提供有关这些技术的教育和培训，这些技术可以由研究者自己进行实验室经过一定的理论和实践培训后。后者可作为为对染色质结构分析等技术感兴趣的中心成员提供的教育资源。需要基本分子技术（例如定量 RT-PCR 等）帮助的研究人员复杂的技术，例如染色质免疫沉淀测定 (ChIP) 和 ChlP-Seq，可以接收由核心总监（Dr. Merchant）和她的助理（Mr. 泰西尔）。图 1 显示了每个项目的中心调查员人数该核心提供的程序。总而言之，四个计划的具体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生成小鼠和大鼠模型来研究调节肽生理背景。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开放密歇根大学医学中心的设施。转基因计划的具体目标如下：一个。作为转基因和胚胎干细胞研究各个方面的教育资源，畜牧业 DNA 构建体设计和表型分析。 b.通过重组工程制备专门的 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.利用中心研究人员提供的克隆 DNA 制备转基因小鼠和大鼠。 d.产生具有目标基因突变的小鼠 ES 细胞克隆。 e.扩展ES克隆。 f.将 ES 细胞克隆显微注射到囊胚中。 g。向实验室人员希望的中心研究人员提供设备和试剂自行进行囊胚注射或 ES 细胞生产。 h.提供协助啮齿动物群体管理的技术，包括小鼠品系冷冻保存和恢复、通过胚胎移植将小鼠转变为 SPF 状态以及体外受精 (IVF) 用于不能可靠产生后代的遗传系。我。监督向中心成员公平分配服务。 2. 病毒载体计划的目的是促进病毒载体在体外和体内基因的使用转移到哺乳动物细胞中。最近的资助期间，重点从腺病毒载体到慢病毒载体。这种转变与人们对基因转移到干细胞的兴趣增加有关不经常分裂的细胞和原代细胞。在上一个资助期间，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。 Vector 程序的具体目标如下：一个。作为使用病毒载体进行基因转移的教育资源。 b.作为中心调查人员满足 OSEH 安全要求的教育资源使用病毒载体所需的。 c.培训中心研究人员如何构建和培养用于研究的病毒储备。 d.为中心研究人员提供载体和设施来构建各种病毒向量。 e.向中心研究人员提供试剂大小的病毒载体。 3. 微阵列基因芯片计划的目的是协助中心科学家利用高通量技术 RNA 分析和基因分型方法用于描述肠道激素在健康和健康方面的作用疾病。该计划包含现有 Affymetrix 和 cDNA 微阵列中的一个单独组件 Core，为密歇根大学中央医学院附属的所有研究人员提供服务校园以及多个 NIH 资助的中心。微阵列基因芯片计划的目标是如下：一个。积极协助中心研究人员进行基因表达的设计、执行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。这些服务包括用于微阵列和基因芯片的探针制备、标记、杂交、清洗和数据采集实验。 b.为微阵列采集数据的统计分析提供专业知识和帮助，处理大型数据集，以及对大型数据集进行数据挖掘； c.提供使用定量聚合酶验证微阵列采集数据的方法链式反应技术。 d.提供基因组研究工具，包括 SNP 单倍型分析服务和 Affymetrix HuSNP 基因芯片服务。 4. 分子技术培训计划的目的是为中心研究人员提供分子技术背后的理论和实践培训，例如蛋白质-DNA 相互作用和染色质免疫沉淀分析 (ChIP) 和 ChlP-Seq。后者将与 DNA 测序核心（Robert Lyons，总监）和生物信息学核心（James Cavacoli，总监）。 [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。作为基因表达和 DNA 蛋白应用的教育资源相互作用技术，例如电泳迁移率变动分析（EMSA、凝胶变动）、DNA 足迹、DNA 甲基化、转染、启动子构建、定量 PCR、报告基因检测、ChIP 分析。 b.培训研究人员掌握专业技术，例如 DNA-蛋白质结合和 ChIP 测定。 c.监控微阵列计划的申请流程。 d.提供和维护分析报告基因检测的设备。 e.教导研究人员如何附加“引物适配器”来生成下一步的文库- 在提交给 DNA 测序核心之前进行一代测序 (NGS)。 f.为了进行胃泌素和生长抑素的放射免疫测定，