Molecular Biology Core Laboratory

分子生物学核心实验室

• 批准号: 7217725
• 负责人: DAVID W. RUSSELL
• 金额: $ 71.58万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-07-01 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7217725
• 关键词: Acrylamide; Acrylamides; Actins; Agar Gel Electrophoresis; Age; Aliquot; American Type Culture Collection; Amino Acid Sequence; Appendix; Archives; Autoradiography; Biotin; Blood; Blood specimen; Budgets; Buffers; Cataloging; Catalogs; Cells; Charge; Chromatography; Chromosome Mapping; Collection; Communities; Complementary DNA; Complementary RNA; Computer software; Contracts; Core Facility; Custom; Cyclophilins; DNA; DNA Binding; DNA Sequence; DNA Sequencing Facility; Data; Databases; Deposition; Digestion; Electronic Mail; Electrophoresis; Endopeptidase K; Facility Construction Funding Category; Future; Gel; Gene Expression; Gene Proteins; Gene Structure; Genes; Genetic; Genetic Engineering; Genomic Library; Genomics; Genotype; Glyceraldehyde 3-Phosphate; Glycerol; Hour; Human; Human Resources; Immunoblotting; Immunoprecipitation; Individual; Institution; Knock-out; Knockout Mice; Label; Laboratories; Length; Libraries; Los Angeles; Mails; Maintenance; Measures; Mediation; Messenger RNA; Methods; Modeling; Molecular Biology; Molecular Genetics; Mus; Numbers; Oligonucleotide Microarrays; Oligonucleotide Primers; Oligonucleotides; Operative Surgical Procedures; Oxidoreductase; Pathology; Pattern; Peptide Sequence Determination; Peptidylprolyl Isomerase; Pharmacologic Substance; Plasmids; Polyacrylamide Gel Electrophoresis; Polymerase Chain Reaction; Population; Power Sources; Preparation; Price; Primer Extension; Procedures; Process; Program Research Project Grants; Protein Analysis; Proteins; Protocols documentation; RNA; RNA Probes; Rate; Reaction; Recombinant Proteins; Recombinants; Research; Research Personnel; Research Project Grants; Reverse Transcription; Ribosomal Proteins; Robot; Robotics; Roentgen Rays; Running; Sampling; Scanning; Sequence Analysis; Services; Signal Transduction; Site; Sodium Dodecyl Sulfate-PAGE; Specimen; Standards of Weights and Measures; System; Tail; Techniques; Technology; Time; Tissue Extracts; Tissue-Specific Gene Expression; Tissues; Transgenes; Transgenic Mice; Transgenic Organisms; Upper arm; Variant; Work; base; cDNA Library; cost; day; design; experience; gel electrophoresis; genetic pedigree; laboratory facility; lipid metabolism; mutant; polyacrylamide gels; programs; protein purification; research study; restriction enzyme; satisfaction; sex; tissue/cell culture

The Molecular Biology Core Laboratory is directed by Dr. Russell. He will be assisted in day-to-day operations by Dr. Jonathan Cohen, who is an expert in high-throughput DMA sequencing. This Core laboratory provides support for acrylamide gel electrophoresis of proteins, DMA sequencing, analysis of gene expression by oligonucleotide microarray hybridization and real-time polymerase chain reactions (PCRs), oligonucleotide procurement, genomic DMA and RNA isolation, and the maintenance and storage of bacterial strains, plasmids, and purified proteins used within this Program Project. Four experienced technicians, Kevin Anderson (100% time), Emily Brown (100%), Jeffrey Cormier (100%), and Scott Clark (25%) will perform the duties associated with this Core. The laboratory facility is located within the Department of Molecular Genetics. For the analysis of proteins, 30 vertical electrophoresis units and 12 multi-outlet power supplies are available. SDS polyacrylamide gels are either prepared (25% of gels) or purchased pre-poured (75% of gels) and run by a single technician (Emily Brown). Following electrophoresis, individual Investigators working on the different Research Projects process the gels for autoradiography, immunoblotting, or protein sequencing. A darkroom that contains a Konica automatic X-ray developer is used to process all autoradiograms and chemilumigrams. The services of the facility are used extensively to assess protein purification, immunoprecipitation from cells, expression of recombinant proteins, and immunoblotting from cultured cells, tissues, and recombinant hosts. These techniques and methods are crucial to our studies on the expression and purification of essentially all genes and proteins with which we work. In addition, the determination of tissue-specific expression patterns of genes under study via immunoblotting in transgenic and knockout mice is heavily dependent on this aspect of the Core. As summarized in Table 1 of the Program Introduction, DMA sequencing was used to analyze the cDNA and gene structures of a large number of normal and mutant DNAs that were cloned for the first time by investigators working on this Program Project Grant (see pages 162-163). Through 2003, all of our DMA sequencing was performed on-site by personnel in the Molecular Biology Core; however, in that year, a large institution-wide DNA sequencing core facility was opened at UT Southwestern. Because of the large volume of sequencing done by this facility, their costs ($5 per sequencing reaction) were lower than ours, and we switched to using their services. At the present time, DNA samples to be sequenced (plasmids, cloned genomic DMAs, and PCR products) are prepared by individual Investigators using rigidly standardized purification protocols and given to Mr. Cormier, who then personally delivers the samples once per day to the sequencing facility. DNA sequence data are returned to Mr. Cormier electronically and thereafter dispensed to Investigators in the Program Project. The turnaround time required to sequence a given DNA sample by the facility is generally less than 24 hours, and the accuracy of the sequence data provided is very high. High-throughput sequencing of human genomic DNA for Research Project 4 is budgeted separately (see Research Project 4). A significant aspect of our research in transgenic and knockout mice involves determining how the presence of a transgene or the loss of an endogenous gene alters the expression of other genes. To this end, the Molecular Biology Core performs large numbers of microarray experiments using chips from Affymetrix that are estimated to contain 34,000 gene sequences. In a typical experiment, total RNA is prepared from one or more tissues of isogenic age- and sex-matched wild type and knockout mice by Scott Clark, and the quality of the preparation is assessed by reverse transcription and agarose gel electrophoresis. If the RNA is judged intact, Mr. Anderson prepares complementary RNA probes labeled with biotin from each sample and hybridizes them to individual chips. After washing, each chip is scanned and variations in the hybridization signal intensity are determined and compared between chips to derive a readout of differential gene expression. This data is returned to individual Investigators of the Program Project via electronic mail in a spreadsheet format, which can be manipulated to display gene products that are increased or decreased in level in the two starting RNA populations. We have a contract with Affymetrix that provides access to a large number of chips at a cost of $435 each. An on-campus core facility charges us $90 per hybridization reaction to measure signal intensity on hybridized chips. A second gene expression service provided by the Molecular Biology Core involves quantitation of single mRNAs by real-time PCR. For a given target mRNA, Mr. Jeffrey Cormier first designs four pairs of oligonucleotide primers using software (Primer Express¿) provided by Applied Biosystems. The efficiency with which each primer pair amplifies the target mRNA is determined and those pairs that match the efficiency of primers used to amplify internal standard mRNAs (cyclophilin, [5-actin, glyceraldehyde phosphate dehydrogenase, 36-B4 ribosomal protein) are chosen for future use. We currently have a collection of over 500 primer pairs corresponding to mRNAs that encode proteins involved in lipid metabolism. To measure gene expression, a Program Project Investigator indicates to Mr. Cormier which mRNAs he/she wishes to measure and provides him with total RNA samples extracted from the tissues of transgenic, knockout, or control mice. Mr. Cormier then converts the RNA into cDNA by reverse transcription and uses a Beckman Biomek 2000 robot to set up triplicate real-time PCR reactions in 384-well microtiter plates for each mRNA in the RNA samples. Multiple plates are then analyzed sequentially on an Applied Biosystems Model 7900HT real-time PCR machine. Each plate is subjected to a 1.7 hour thermocycler routine during which data are collected continuously from the amplification reactions. The machine has a robotic arm that automatically inserts and removes microtiter plates at the beginning and end of the thermocycler routine, allowing us to perform real-time PCR on a 24-hour basis. The capacity of this system is large, and last year we performed -300,000 real-time PCR reactions, which makes this machine and service the most heavily requested in the Molecular Biology Core. In a relatively few instances, real-time PCR is also used to genotype transgenic mice. Custom oligonucleotides are purchased from Integrated DNA Technologies (Coralville, IA). Our current rate of oligonucleotide purchase is approximately 5000 per year, a number that allows us to negotiate an excellent price for this service (23 cents/base for unpurified oligonucleotides, 25 nanomole amounts). The average length of each primer is 25 bases, and approximately 80% of primers are used as obtained (without further purification) and -20% after purification by polyacrylamide gel electrophoresis by the company. An occasional oligonucleotide primer is purified in our laboratories by preparative acrylamide gel electrophoresis and Sep-Pak Ci8 chromatography prior to use. Large numbers of oligonucleotide primers are used for PCR, DNA sequencing, primer extension, genetic engineering, mutagenesis, genetic mapping, and DNA binding and hybridization experiments by all investigators on this Program Project. Preparation of genomic DNA from cells, tissues, mouse tails, and blood samples is carried out by Mr. Kevin Anderson using proteinase K and specialized buffers from Viagen Biotech (Los Angeles, CA). We have used these kits for three years and have found that the yields of DNA are about 2-fold higher, that the time of preparation is short, that the method is safer, and that the quality of genomic DNA prepared is better than that isolated by other extraction methods or kits. The isolation of genomic DNA has proven especially useful in recent years in our genetic studies in which large multigenerational pedigrees (both human and mouse) are analyzed. In general, a given sample, whether obtained from cells, tissue, or blood, is ready for restriction enzyme digestion or amplification within 16 hours of receipt. Additional DNA extraction kits are used in specialized cases, such as when small amounts of sample are available or when DNA must be isolated from fixed specimens obtained from pathology. Because of the large number of bacterial strains, plasmids, and purified proteins that are used in our experiments, it is necessary to have a central facility to catalogue and store these materials. Once a cloned DNA is isolated and characterized, a sample of the bacterial strain harboring the plasmid is stored in multiple aliquots at -70¿C in medium with glycerol. At the same time, the description of the plasmid is entered into a central data base. In this manner, each new isolate or different construction that is utilized in our studies is made available to others when needed. In addition to the individual clones that are stored, aliquots of cDNA and genomic libraries that have been prepared in the course of our experiments are also maintained. Purified proteins are treated similarly and stored in small aliquots in a large dedicated -70¿C freezer. Organized maintenance of bacterial strains and libraries is also necessary to meet the frequent requests that we receive for these materials from other investigators. We have mailed out a voluminous number of aliquots of different cDNAs, genes, and specialized plasmid constructs to laboratories throughout the world. In addition to these clones, we have provided samples of cDNA libraries to other investigators as detailed in the Appendix, Vol. ill. The volume of these requests and the general recipient satisfaction (virtually no repeat requests) testify to the need and accuracy of this vital service and archive facility. Many of the most commonly requested cDNA clones have been deposited in the ATCC collection for general distribution to the biomedical and pharmaceutical community (see Appendix, Vol. III). Specific Interactions between the Molecular Biology Core and Research Projects 1-6. All 6 Research Projects are dependent on this Core for providing support for recombinant DMA procedures, DNA sequencing, quantitative real-time PCR, and SDS polyacrylamide gel electrophoresis.

分子生物学核心实验室由Russell博士领导，他将协助处理日常工作。 由高通量 DMA 测序专家 Jonathan Cohen 博士负责操作。 实验室为蛋白质丙烯酰胺凝胶电泳、DMA测序、基因分析提供支持 通过寡核苷酸微阵列杂交和实时聚合酶链反应（PCR）表达， 寡核苷酸采购、基因组 DMA 和 RNA 分离以及细菌的维护和储存 该计划项目中使用的菌株、质粒和纯化蛋白质，四名经验丰富的技术人员， 凯文·安德森 (100% 时间)、艾米莉·布朗 (100%)、杰弗里·科米尔 (100%) 和斯科特·克拉克 (25%) 将 履行与该核心相关的职责 实验室设施位于部门内。 分子遗传学。 用于蛋白质分析，配备 30 个垂直电泳单元和 12 个多插座电源 SDS 聚丙烯酰胺凝胶可以是制备好的（25% 的凝胶）或购买的预浇注的（75% 的凝胶）。 并由一名技术人员（Emily Brown）运行，电泳后，个别研究人员进行工作。 不同的研究项目处理凝胶以进行放射自显影、免疫印迹或蛋白质测序。 配备柯尼卡自动 X 射线显影机的暗室用于处理所有放射自显影图和 该设施的服务广泛用于评估蛋白质纯化， 细胞的免疫沉淀、重组蛋白的表达以及培养细胞的免疫印迹， 这些技术和方法对于我们的表达研究至关重要。 以及纯化我们所涉及的几乎所有基因和蛋白质。 通过转基因和基因敲除小鼠中的免疫印迹研究基因的组织特异性表达模式 严重依赖于核心的这一方面。 正如程序简介表1所总结的，DMA排序用于分析 首次克隆大量正常和突变DNA的cDNA和基因结构 由从事该计划项目资助的研究人员提供（参见第 162-163 页） 到 2003 年，我们所有的 DMA 项目均已完成。 测序是由分子生物学核心人员现场进行的，但在那一年，进行了大规模测序； 由于数据量大，在 UT 西南大学开设了全机构范围的 DNA 测序核心设施。 在该设施完成的测序中，他们的成本（每个测序反应 5 美元）比我们低，而且我们 目前，DNA 样本已被测序（质粒、克隆）。 基因组 DMA 和 PCR 产物）由个别研究人员使用严格标准化的方法制备 纯化方案并交给 Cormier 先生，然后他亲自将样品每天一次送到 DNA 序列数据以电子方式返回给 Cormier 先生，然后分发。 给计划项目的研究人员 对给定 DNA 样本进行测序所需的周转时间。 该设施一般小于24小时，提供的序列数据的准确性非常高。 研究项目 4 的人类基因组 DNA 高通量测序单独编列预算（参见 研究项目4）。 我们对转基因和基因敲除小鼠的研究的一个重要方面涉及确定 转基因的存在或内源基因的丢失会改变其他基因的表达。 最后，分子生物学核心使用来自以下公司的芯片进行大量微阵列实验： Affymetrix 估计包含 34,000 个基因序列 在典型实验中，总 RNA 为 由斯科特从同基因年龄和性别匹配的野生型和基因敲除小鼠的一种或多种组织制备 Clark，通过逆转录和琼脂糖凝胶评估制剂的质量 如果判断 RNA 完好，Anderson 先生会准备互补的 RNA 探针，并用 标记。 清洗后，对每个芯片进行扫描和杂交。 确定杂交信号强度的变化并在芯片之间进行比较，以获得 差异基因表达的读数将返回给该计划的各个研究人员。 通过电子邮件以电子表格格式进行项目，可以操纵该电子表格来显示基因产物 两个起始 RNA 群体中的水平增加或减少 我们与 Affymetrix 签订了合同。 提供对大量芯片的访问，每个芯片的费用为 435 美元。 每次杂交反应 90 美元，用于测量杂交芯片上的信号强度。 分子生物学核心提供的第二个基因表达服务涉及定量 对于给定的目标 mRNA，Jeffrey Cormier 先生首先设计了四对 mRNA。 使用Applied Biosystems 提供的软件(Primer Express¿) 进行寡核苷酸引物的效率。 确定每个引物对扩增目标 mRNA 的引物对，并确定与 用于扩增内标 mRNA 的引物效率（亲环蛋白、[5-肌动蛋白、磷酸甘油醛 脱氢酶、36-B4 核糖体蛋白）已被选择供将来使用。 500 个引物对，对应于编码参与脂质代谢的蛋白质的 mRNA。 基因表达，项目研究员向 Cormier 先生指出他/她希望表达哪些 mRNA 测量并向他提供从转基因、基因敲除或转基因组织中提取的总RNA样本。 然后 Cormier 先生通过逆转录将 RNA 转化为 cDNA，并使用 Beckman。 Biomek 2000 机器人在 384 孔微量滴定板中为每个 mRNA 建立一式三份实时 PCR 反应 然后在 Applied Biosystems 7900HT 型上依次分析多个板。 每个板都经过 1.7 小时的热循环程序，在此期间数据被保存。 该机器有一个机械臂，可以自动从扩增反应中连续收集。 在热循环仪程序的开始和结束时插入和移除微量滴定板，使我们能够 24小时进行实时PCR 这个系统的容量很大，去年我们就进行过。 -300,000 次实时 PCR 反应，这使得该机器和服务成为业内需求量最大的 分子生物学核心 在相对少数的情况下，实时 PCR 也用于转基因基因分型。 老鼠。 定制寡核苷酸购自 Integrated DNA Technologies（爱荷华州 Coralville）。 目前寡核苷酸的采购速度约为每年 5000 个，这个数字使我们能够进行谈判 这项服务的价格非常优惠（未纯化的寡核苷酸为 23 美分/碱基，25 纳摩尔量）。 每个引物的平均长度为25个碱基，大约80%的引物按原样使用（没有 进一步纯化）和公司聚丙烯酰胺凝胶电泳纯化后的-20%。 偶尔的寡核苷酸引物在我们的实验室通过制备型丙烯酰胺凝胶电泳进行纯化 和 Sep-Pak Ci8 色谱法在使用前使用大量寡核苷酸引物进行 PCR， DNA 测序、引物延伸、基因工程、诱变、遗传作图和 DNA 结合 以及该计划项目所有研究人员的杂交实验。 从细胞、组织、小鼠尾巴和血液样本中制备基因组 DNA 是由 Mr. Kevin Anderson 使用 Viagen Biotech（加利福尼亚州洛杉矶）的蛋白酶 K 和专用缓冲液。 使用这些试剂盒三年了，发现 DNA 的产量比之前的试剂盒高出约 2 倍。 制备时间短，方法更安全，制备的基因组DNA质量更好 与其他提取方法或试剂盒分离的基因组 DNA 相比，已被证明特别有效。 近年来在我们的遗传学研究中很有用，其中大型多代谱系（人类和 一般来说，给定的样品，无论是从细胞、组织还是血液中获得，都可以进行分析。 收到后 16 小时内进行限制性内切酶消化或扩增。另外还有 DNA 提取试剂盒。 在特殊情况下使用，例如当可用少量样本或必须提取 DNA 时 从病理学获得的固定标本中分离出来。 由于我们的实验中使用了大量的细菌菌株、质粒和纯化的蛋白质 实验后，有必要有一个中央设施来对这些材料进行克隆并进行分类和存储。 DNA 被分离和表征，含有质粒的细菌菌株样本被储存在多个容器中。 -70° 等分C 在含有甘油的培养基中同时，将质粒的描述输入到a中。 通过这种方式，我们的研究中使用的每个新的分离物或不同的构建都是如此。 除了储存的单个克隆外，还可以在需要时提供 cDNA 等分试样。 还保留了我们在实验过程中准备的基因组文库。 纯化的蛋白质经过类似处理并以小等份形式储存在大型专用-70¿ C冰箱。 对细菌菌株和文库的有组织的维护也是必要的，以满足频繁的需求 我们已寄出大量从其他调查人员处收到的有关这些材料的请求。 向实验室提供不同 cDNA、基因和专用质粒构建体的等分试样数量 除了这些克隆之外，我们还向其他研究人员提供了 cDNA 文库样本。 详细信息见附录，第 1 卷。 （几乎没有重复请求）证明了这一重要服务和档案设施的必要性和准确性。 最常见的 cDNA 克隆已存放在 ATCC 收藏中，用于一般用途 分发给生物医学和制药界（参见附录，第三卷）。 分子生物学核心与研究项目之间的具体相互作用 1-6 所有 6。 研究项目依赖于该核心为重组 DMA 程序、DNA 提供支持 测序、实时定量 PCR 和 SDS 聚丙烯酰胺凝胶电泳。