Molecular Biology Core Laboratory

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

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.