Molecular Biology Core Laboratory
分子生物学核心实验室
基本信息
- 批准号:7217725
- 负责人:
- 金额:$ 71.58万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 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:
- 关键词:AcrylamideAcrylamidesActinsAgar Gel ElectrophoresisAgeAliquotAmerican Type Culture CollectionAmino Acid SequenceAppendixArchivesAutoradiographyBiotinBloodBlood specimenBudgetsBuffersCatalogingCatalogsCellsChargeChromatographyChromosome MappingCollectionCommunitiesComplementary DNAComplementary RNAComputer softwareContractsCore FacilityCustomCyclophilinsDNADNA BindingDNA SequenceDNA Sequencing FacilityDataDatabasesDepositionDigestionElectronic MailElectrophoresisEndopeptidase KFacility Construction Funding CategoryFutureGelGene ExpressionGene ProteinsGene StructureGenesGeneticGenetic EngineeringGenomic LibraryGenomicsGenotypeGlyceraldehyde 3-PhosphateGlycerolHourHumanHuman ResourcesImmunoblottingImmunoprecipitationIndividualInstitutionKnock-outKnockout MiceLabelLaboratoriesLengthLibrariesLos AngelesMailsMaintenanceMeasuresMediationMessenger RNAMethodsModelingMolecular BiologyMolecular GeneticsMusNumbersOligonucleotide MicroarraysOligonucleotide PrimersOligonucleotidesOperative Surgical ProceduresOxidoreductasePathologyPatternPeptide Sequence DeterminationPeptidylprolyl IsomerasePharmacologic SubstancePlasmidsPolyacrylamide Gel ElectrophoresisPolymerase Chain ReactionPopulationPower SourcesPreparationPricePrimer ExtensionProceduresProcessProgram Research Project GrantsProtein AnalysisProteinsProtocols documentationRNARNA ProbesRateReactionRecombinant ProteinsRecombinantsResearchResearch PersonnelResearch Project GrantsReverse TranscriptionRibosomal ProteinsRobotRoboticsRoentgen RaysRunningSamplingScanningSequence AnalysisServicesSignal TransductionSiteSodium Dodecyl Sulfate-PAGESpecimenStandards of Weights and MeasuresSystemTailTechniquesTechnologyTimeTissue ExtractsTissue-Specific Gene ExpressionTissuesTransgenesTransgenic MiceTransgenic OrganismsUpper armVariantWorkbasecDNA Librarycostdaydesignexperiencegel electrophoresisgenetic pedigreelaboratory facilitylipid metabolismmutantpolyacrylamide gelsprogramsprotein purificationresearch studyrestriction enzymesatisfactionsextissue/cell culture
项目摘要
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罗素博士领导。他的日常生活将得到协助
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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DAVID W. RUSSELL其他文献
Strand segregation or recombination
链分离或重组
- DOI:
10.1038/329678b0 - 发表时间:
1987-10-22 - 期刊:
- 影响因子:48.500
- 作者:
DAVID W. RUSSELL - 通讯作者:
DAVID W. RUSSELL
DAVID W. RUSSELL的其他文献
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{{ truncateString('DAVID W. RUSSELL', 18)}}的其他基金
2009 Lipids, Molecular & Cellular Biology of Gordon Research Conference
2009 脂质,分子
- 批准号:
7743873 - 财政年份:2009
- 资助金额:
$ 71.58万 - 项目类别:
Cholesterol and Oxysterol Metabolism in Brain and Liver
脑和肝脏中的胆固醇和氧甾醇代谢
- 批准号:
7217721 - 财政年份:2007
- 资助金额:
$ 71.58万 - 项目类别:
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- 批准号:
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