BioMMED Core Facilities

The LSU COBRE in Infectious Diseases is served by two MAIN Core Laboratories. The LSU COBRE Molecular Biology & Immunology Core Laboratory (MBIC) and the TNPRC Non-Human Primate and Laboratory Core Laboratory (NHPLCL). The LSU-MBIC:The LSU-MBIC is operationally housed within the existing laboratories of the Division of Biotechnology and Molecular Medicine (BioMMED). In addition, the LSU-MBIC collaborates with the FACS Facility and Microscopy Center of the LSU School of Veterinary Medicine. The LSU-MBIC concentrates its support functions on molecular biology/recombinant DNA services, bioinformatics and molecular evaluation of immunological parameters of mice. Molecular biological services and reagents includes standard services such as DNA sequencing, recombinant plasmid construction, and cDNA library construction. Advanced services will include processing and analysis of microarray experiments using advanced bioinformatics capabilities offered in conjuction with the Bionformatics Unit of the LSU Pennington Biomedical Research Center (PBRC-BU). In addition, LSU-MBIC will offer assistance with real-time PCR analysis of mouse cytokines, Fluorescent Activated Sorting (FACS) analysis, and confocal microscopy through the LSU School of Veterinary Medicine's Centralized Facilities dedicated to FACS and Microscopy. BioMMED is a Division within the School of Veterinary Medicine, Louisiana State University, Baton Rouge LA. It is financially supported by the School of Veterinary Medicine and the LSU Agricultural Center. BioMMED administers the LSU-Tulane Center for Experimental Infectious Disease Research supported by a five year program grant from the National Institutes of Health, National Center for Research Resources under the Center for Biomedical Research Excellence (COBRE) funding mechanism. In addition, BioMMED coordinates the Molecular/Cell Biology Core of the Idea Network for Biomedical Research Excellence (INBRE) (Tom Klei, PI).

The goals of BioMMED are:

1. To provide advanced molecular biological and bioinformatics capabilities to SVM, LSU  and  Louisiana-based researchers.
2. To develop in collaboration with LSU and Louisiana-based researchers molecular biological methodologies to ameliorate human and animal diseases.
3. To train SVM and LSU researchers in the effective use of molecular and analytical technologies through seminars, workshops and courses as well as "hands on training".

BioMMED is organized into five different Laboratories:

1) GeneLab ; 2)  Viral Vectors Laboratory ; 3) Protein and Antibody Production and Purification Laboratory ; 4) Cellular and Small Animal In vivo Imaging Laboratory; 5) Bioinformatics Laboratory. All BioMMED service functions are provided to LSU-based investigators on a charge back basis. Typically, LSU researchers are charged only the costs of supplies, while most of the personnel costs are provided via support of participating LSU units.

GeneLab :GeneLab provides molecular biology services include cloning and automated sequencing of genes, real-time quantitative PCR, cDNA library construction, and microarray production. GeneLab operates a new bioinformatics module, which is equipped with one iMac computer and three DELL PC computers. Available software include: Primer Express (PE Biosystems) for the design of TaqMan probes for real-time PCR, MacVector (Genetics Computer Group, Inc.) for analysis of DNA and protein sequences, FluorChem HD2 (Alpha Innotech) for the visualization and analysis of images such as those produced by ethidium bromide agarose electrophoresis of DNA fragments, SDS-PAGE analysis of proteins, immunoblots, etc. GeneLab has also additional software for assembly of large DNA sequences (Sequencher), analysis of blots (Alpha Innotech) and microarrays (AlphaReader 7000). GeneLab is an approved provider of all Applied Biosystems, Inc. molecular biological reagents through a freezer program and standing purchase orders offering substantial savings to Louisiana-based researchers. GeneLab undertakes specific research and training projects, which require expertise in gene cloning, PCR, DNA sequencing, cDNA library construction, gene expression and other molecular methods.

GeneLab performs custom research projects that require other biological experimentation in collaboration with faculty and using facilities of the School of Veterinary Medicine and LSU. Computer analysis of DNA and protein sequences as well as consultation on molecular biological research is provided free of charge. GeneLab provides support letters to LSU affiliated researchers for federal and state funding agencies on request.

RNA-based detection systems for cytokine gene expression. RNA detection approaches enable the detection of cytokine-specific mRNA in species in which antibody reagents are not available for ELISA or other immunoassay-based techniques, and have been used to address specific questions regarding gene regulation.The primary emphasis of this work in GeneLab has been on the development of quantitative reverse-transcription polymerase chain reaction (RT-PCR) assays. These assays are now performed using the "real-time" PCR capabilities of ABI 7500 and 7900 sequence detection systems located in the BioMMED Core Laboratory GeneLab. Also, plans are made to assist researchers in the utilization of microarray technologies to screen for the expression of multiple cytokine genes using commercially available arrays. The advantage of this approach, like that of the aforementioned cytokine bead array, is the ability to detect multiple cytokine mRNAs in a single sample. Once particular cytokine gene activity is identified, further analysis of gene expression can be pursued using other methods, as described above.

Up

Viral Vectors Laboratory :This laboratory provides custom baculovirus, adenovirus, vaccinia virus, herpes, and other recombinant virus construction for heterologous gene expression, and vaccine and gene therapy studies. For the production of proteins in eukaryotic cells the laboratory uses recombinant baculoviruses-based expression in insect cell cultures. For preparation of baculovirus we have used several different transfection kits based on the vector for the protein being used. Recently, we have concentrated on the use of the Bac-TO-Bac System (Invitrogen). In this system recombinant baculoviruses are made in E. Coli. The recombinant transfer vector transposes with a bacmid, in the presence of transposase expressed by another helper plasmid 4 also in the DH10Bac strain of E. coll. Recombinant bacmids can be selected by plating DH10Bac on kanamycin + gentamicin + tetracycline plates and choosing white colonies. A small culture of a positive colony is then grown, and the recombinant bacmid is harvested by miniprep. This purified bacmid DNA is used to transfect Sf9 cells, the transfected cells are assayed for recombinant protein production by indirect immunofluorescence and western blot assay and recombinant protein is purified according to the needs of the individual laboratory. Viral stocks are expanded in Sf9 cells (usually two rounds) and used to inoculate 60-80% confluent High 5 cells (Invitrogen, Carlsbad, CA) in serum-free medium (Express Five SFM, Life Technologies, Inc. Gaithersburg, MD) for protein production. Generally, High 5 insect cells are used for protein expression due to increased yields of protein compared to Sf insect cells. If the expressed protein is secreted, the media is collected for protein isolation, or if the expressed protein remains intracellular or membrane bound then the cell pellet is collected and lysed or appropriate membrane fraction solubilized, respectively. For proteins containing a HIS tag, an FPLC system is used to isolate the expressed protein.

Up

Protein and Antibody Production and Purification Laboratory :This laboratory concentrates on the production and purification of proteins and antibodies. The laboratory produces monospecific antibodies in rabbits and mice using conventional immunization methodologies using purified protein immunogens as well as genetic immunization methods. Antibodies are concentrated and purified using standard methodologies. The laboratory also provides on a limited basis the production and characterization of monoclonal antibodies. This laboratory concentrates most of its efforts on the purification of proteins after their expression using bacterial and eukaryotic expression systems. Current efforts are concentrated on the use of chromatographic methods for the purification of histidine-tagged proteins produced in bacteria and insect cells. Purification by FPLC is done using immobilized metal ion adsorption chromatography (IMAC). This technique employs a metal ion attached to the stationary phase, which is chelated by a 6-HIS tagged protein, and retained by the column. Several different metal ions can be used though nickel and cobalt is the most common. The sample is loaded at a slow flow rate to enhance binding. Attention is paid to the amount of protein being loaded to assure that the column is slightly overloaded as this lessens nonspecific binding of non-HIS tagged proteins. After loading, the column is washed to remove unbound proteins. The column is then eluted with a stepwise gradient of increasing imidazole (20 mM NaPO4, 0.5 M NaCI, 0-1 M Imidazole, 10% glycerol pH 7.9) concentration. The appropriate fractions are pooled and concentrated if desired. CCL has the capability to utilize other chromatographic purification strategies in addition to IMAC. This strategy can be employed, if desired, to provide a higher purity product. The Core has both 1 and 5 ml IMAC columns. Additional chromatographic methodologies that can be applied include Ion Exchange Chromatography (IEC) and reverse-phase chromatography, three HPLC systems (see below) that can be used in further protein purification. Recently, we purchased the fully automated chromatographic system AKTAExplorer FPLC system (Amersham/Pharmacia). The entire system is refrigerated and computer controlled using Unicorn software v3.10. HPLC equipment are available for further protein purification in collaboration with the Analytical Chemistry Laboratory of the LSU School of Veterinary Medicine.

Up

Cellular and Small Animal In vivo Imaging Laboratory:This laboratory focuses on imaging of fluorescent or chemiluminescent probe concentrations within live small rodents (mice, rats). Current equipment include special equipment for the visualization of fluorescent probe concentrations within mice or rats. Specific interests are centered on the visualization of various tumors tagged with fluorescent labels as well as tracking viruses expressing the green fluorescent protein (GFP). Equipment to be purchased include imaging and computing equipment for the visualization of chemiluminscent probes. BioMMED major equipment: BioMMED has 3 automated sequences (ABI377, 310, 310), three real-time PCR equipment (PE 7900, 7400), LightCycler (Roche), 2 MagnaPure automated nucleic acid extractors (Roche), microarray OmniGrid spotter (Gene Machines), microarray reader (AlphaReader 6000, Alpha Innotech), Imager station (Alpha Innotech Fluorochem 8000), 5 PCR (thermocyclers, a New Brunswick Fermentor, AktaExplorer Chromatography System, Cyclone phosphorimager, 2 four column DNA synthesizers (ABI), one Synergy peptide synthesizer (ABI), and other equipment. Additional equipment are available in the departments of Pathobiological Sciences and Comparative Biomedical Sciences that house centralized facilities such as the Organ and Tissue Culture Laboratory, the Glassware and Media Preparation Laboratory, the Electron Microscopy Laboratory, and the Analytical Chemistry Laboratory.

Up

Bioinformatics, Computational and Visualization LaboratoryThis Laboratory provides PC server based access to various software used for the analysis of nucleic acid sequences, microarray output data, etc. In the near future this laboratory may provide direct access to the Wisconsin package of software for the analysis of nucleic acids and proteins in collaboration with the LBRN program of the LSU College of Basic Sciences.

Up

Last Updated Wednesday, August 06 2008 @ 12:45 PM CDT|50,281 Hits