Louisiana State University Health Sciences Center - Shreveport
 Office Of Research
Facilities
 
Animal Resource Facility
BRI Building
Computer Facility
Confocal Microscopy in the Research Core Facility
DNA Array (Chip) Analysis in the Research Core Facility
Flow Cytometry in the Research Core Facility
Fluorescence Microscopy in the Research Core Facility
Laser Capture Microdissection in the Research Core Facility
Mass Spectrometry in the Research Core Facility
Real-Time PCR in the Research Core Facility
Research Core Facility
Small Animal Imaging Facility



 

Animal Resource Facility

The mission of the Animal Resource Facility is to provide the highest level possible of animal husbandry care and veterinary services to assist the faculty of the LSU Health Sciences Center, Shreveport in their research endeavors involving animals; to ensure that all applicable laws, policies and standards of animal care and use are met by the institution; and to serve as a resource to the research program of the institution. The Animal Resource Facility is one component of the overall animal care and use program of the institution. The facility is composed of two areas. The main facility is located in the School of Medicine Building and in the adjoining Biomedical Research Institute Building. A satellite farm facility is located in Stonewall, Louisiana. The facility is staffed by the Director (a veterinarian with research and laboratory animal medicine training), an Assistant Director (B.S., certified as a Laboratory Animal Technologist by the American Association for Laboratory Animal Science (AALAS)), a clinical veterinarian (with research training and board certified by the American College of Laboratory Animal Medicine), a veterinary technician (A.S. degree, registered veterinary technician, and certified as an Assistant Laboratory Animal Technician by AALAS), a staff of 12.5 animal care technicians (approximately 50% are certified at some level by AALAS), and 2 secretaries. The facility is able to provide housing for rodents, rabbits and a limited number of other species. Special capabilities include a 3-room surgical suite for survival surgical procedures on larger animals, a Biosafety Level 3 Suite with accommodations for 2 simultaneous projects, a transgenic mouse breeding facility, and specialized animal procedure rooms. The entire animal care and use program is fully accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care, International (AAALAC). In addition, the program maintains a current assurance with the Office of Laboratory Animal Welfare (OLAW) of the National Institutes of Health and is registered as part of the LSU System with the U.S. Department of Agriculture. Faculty Member in Charge: V. Hugh Price, D.V.M., Director, Animal Resources

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BRI Building

BRI Building BRI Building BRI Building BRI Building BRI Building BRI Building BRI Building BRI Building BRI Building BRI Building

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Computer Facility

The computer facilities of LSUHSC-S are excellent and provide access to many advanced state and national resources. All faculty offices and laboratories have wired (and optionally, wireless) network access to a variety of services such as clinical information systems and repositories, statistical programs, the Wisconsin DNA software, E-mail, Medline, and other library databases. Internet and Internet 2 access is presently provided, but in April, 2005, the research community will have access to the National Lambda Rail (NLR) and LONI (Louisiana Optical Network Initiative), the most advanced state network backbone in the country and one of the most advanced in the world. LONI will also provide access to the state’s Grid Computing infrastructure. Faculty Member in Charge: Lee Bairnsfather, Ph.D.

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Confocal Microscopy in the Research Core Facility

The confocal microscopy component of the RCF is built around a Bio-Rad Radiance 2000/AGR-3 (Q) with a three-channel, three-laser, three-detector system mounted on a Nikon TE 300 inverted microscope. The instrument has three lasers with ATOF for balancing the laser lines: a 25-mw argon ion laser emitting at 457, 476, 488, 514nm; a 1-mw green HeNe laser emitting at 543nm; and a red diode laser emitting at 638nm. The software consists of Lasersharp I 32-bit acquisition, display, and 3D volume rendering. The TE300 inverted microscope has fluorescence and differential interference contrast (DIC) capacity with 10, 20 and 40X CF infinity plan fluor dry objectives, as well as CF1 plan fluor 40X oil (N.A. 1.30), DF1 plan apo 60X oil (N. A. 1.40) and CF1 plan apo 100X oil (N.A. 1.40) objectives. A separate computer running MetaMorph image analysis software is available for additional image analysis. Scientific Advisor: Bryan Bellaire, Ph.D.

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DNA Array (Chip) Analysis in the Research Core Facility

The LSUHSC-S core facility includes an Affymetrix GeneChip Instrument and Bioinformatics Software System. The instrument system includes a recently upgraded GeneArray Scanner 3000 with workstation (Pentium III 500 MHz processor, 384 MB RAM, 25 GB hard drive, and 21” high resolution monitor), a new model 450 GeneChip Fluidics Station, and a GeneChip Hybridization Oven 640. The Bioinformatics System 1 consists of an Intell Xeon 4 x 450 MHz processor with 2 GB RAM and 110 GB hard drive array LIMS server, a second workstation, the GeneChip Analysis Suite, the GeneChip Data Mining Tool, GeneSpring and Spotfire analysis packages and a LIMS Access License. This system is suitable for gene expression studies using the Affymetrix GeneChip Probe Arrays available for analysis of full-length genes and EST clusters from human, mouse, rat, yeast, E. coli, and, C. elegans. These probe sets are three prime biased, but 20 fold redundant per gene for improved specificity. Arrays are also available for HuSNP Mapping Assays, HIV-1 protease and reverse transcriptase partial resequencing assays, p53 resequencing assays, and allelotyping of the 2D6 and 2C19 CYP450 genes. Scientific Advisor: Rona Scott, Ph.D.

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Flow Cytometry in the Research Core Facility

There are three instruments that make up the flow cytometry component of the RCF. FACSVantage SE. The FACSVantage flow cytometer/cell sorter is capable of 8-parameter (two laser light scatter and up to six fluorescent colors) analysis and cell sorting. It is equipped with three lasers for fluorochrome excitation: a) an Enterprise argon ion laser capable of simultaneous 488-nm and UV excitation; b) a helium/neon laser for 633-nm excitation; and c) a Spectrum laser, which is tunable to any one of 8 wavelengths (UV, 457 nm, 488 nm, 514 nm, 520 nm, 530 nm, 568 nm and 647 nm). This sorter is equipped with the TurboSort option, permitting high speed sorting up to 35,000 events per second. In addition the sorter is also equipped for direct sorting into tissue culture plates of any configuration, permitting direct cell cloning and low frequency response analyses. FACSCalibur. The FACSCalibur is an ultra sensitive flow cytometer, capable of 6-parameter (two laser light scatter and up to four fluorescent colors) analysis. It uses two lasers for fluorchrome excitation: a) an argon ion laser for 488-nm excitation; and b) a Red Diode laser for 635-nm excitation. BD LSR. This flow cytometer is capable of 8-parameter (two laser light scatter and up to six fluorescent colors) analysis. It has three lasers for excitation of flourochromes: a) an argon ion laser for 488-nm excitation; b) a helium/neon laser for 633-nm excitation; and c) a helium/cadmium laser for UV excitation. Workstations and Software. There are two Macintosh computers for off-line analysis of data. All three cytometers use CellQuest for data acquisition, and each workstation uses CellQuest for data analysis. In addition, both Attractors and FlowJo are available on the workstations for specific data analysis needs. For the analysis of cell cycle data, ModFit LT is available on both workstations. Scientific Advisor: Robert Chervenak, Ph.D.

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Fluorescence Microscopy in the Research Core Facility

The fluorescent microscope in the core facility is a Nikon TE300 inverted microscope, which has the capacity for FITC and rhodamine excitation-emission wavelengths, differential interference contrast (DIC) optics. Additionally, a flexible configuration allows imaging of a wide variety of specimens, including routine slides, tissue culture flasks, Petri dishes, and cell culture microslides. The objectives include 10, 20 and 40X CF infinity plan fluor dry objectives. High-resolution immersion objectives include a CF1 plan fluor 40X oil (N.A. 1.30), a DF1 plan apo 60X oil (N. A. 1.40) and a Cf1 plan apo 100X oil (N.A. 1.40). Images are captured with a Photometrics Synsys digital camera (Roper Photometrics, Tuscon, AZ) and analyzed with IPLab Spectrum image analysis software (Universal Imaging Corp, Trenton NJ). An additional digital imaging software package, Metamorph V4, is available for image analysis. Scientific Advisor: Bryan Bellaire, Ph.D.

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Laser Capture Microdissection in the Research Core Facility

Major scientific and medical advances are transforming the field of translational laboratory research. Developments in gene sequencing and amplification techniques, among others, now allow investigators to extract DNA or RNA from tissue biopsies and cytological smears for pinpoint molecular analysis. The efficacy of these sophisticated genetic testing methods, however, depends on the purity and precision of the cell populations being analyzed. Simply homogenizing the biopsy sample results in an impure combination of healthy and diseased tissue. Using mechanical tools to manually separate cells of interest from the histologic section is time-consuming and extremely labor-intensive. None of these methods offers the ease, precision and efficiency necessary for modern molecular diagnosis. A new method, Laser Capture Microdissection (LCM), provides research and pathology laboratories with the ideal microdissection technology. LCM was conceived and first developed as a prototype research tool at the National Institute of Child Health and Human Development (NICHD) and the National Cancer Institute (NCI) of the NIH. LCM is being used in the Cancer Genome Anatomy Program (CGAP) to catalog the development of cells from a normal to a diseased state. It can be applied to any disease process which is accessible through tissue sampling, such as premalignant cancer lesions, multiple sclerosis, arteriosclerosis, and Alzheimer's disease. Research applications include: genomics (differential gene profiling, loss of heterozygosity, microsatellite instability, and gene quantification) and proteomics (two dimensional protein gels, western blotting, and immuno-quantification of proteins). The PixCell II® instrument performs Laser Capture Microdissection from heterogeneous tissue samples simply, quickly and precisely. In minutes the investigator can locate a single cell or large groups of cells and, using a simple aim-and-shoot method, extract them for subsequent molecular analysis. LCM preserves the exact morphologies of both the captured cells as well as the surrounding tissue. The PixCell II transfers cells from paraffin-embedded and frozen tissue samples, stained and immunolabelled slides. The entire process can be monitored and documented, and the images can be stored in an archiving workstation. Microdissection of fluorescently-stained cells is also possible with a fluorescence package that has been purchased. Scientific Advisor: Michael Mathis, Ph.D.

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Mass Spectrometry in the Research Core Facility

The mass spectrometry component of the RCF consists of a high resolution (10 ppm) Voyager DE PRO mass spectrometer. The particular type of mass spectrometry performed is Matrix-Assisted Laser Desorption Ionization – Time-Of-Flight (MALDI-TOF). Coupled to this instrument is a 2-GHz LeCroy computer for on-line database-searching. The third component is a robotic device (Symbiot) for spotting MALDI plates. Proteins excised from denaturing polyacrylamide gels are digested with trypsin or other proteolytic enzymes and identified by matching peptide masses to the theoretical peptides derived from all proteins in databases such as SwissProt and NCBI. This automated system is capable of identifying up to 70 proteins per hour. The entire system is marketed by Applied Biosystems as a package termed Proteomics Solution 1. The Research Core Facility has recently acquired a Finnigan LCQ Deca XP MAX ultra-high sensitivity quadruple 3-D ion trap mass spectrometer. The system includes an API source with electrospray ionization capabilities. Electrospray ionization allows for the measurement of a wide range of species, from small molecules, such as drugs and drug metabolites, to larger molecules, such as peptides and small proteins. Analyses that are difficult to measure on other mass spectrometers due to their inability to ionize are generally analyzed with greater ease with electrospray ionization. The current system has a mass range of m/z=15-4000, however, it can be upgraded to accommodate as large as m/z=20,000. The ion trap mass analyzer allows for MSn experiments (for n=1 to 10), thus, the instrument provides a wealth of data from one single experiment. In addition, electrospray ionization allows for the formation of multiply charged species, and formation of these species provides a substantial amount of data. As an example, when doubly charged peptides are formed, fragmentation can be achieved in either direction along the peptide backbone. The added data provided by the coupling of the ion trap mass analyzer with electrospray ionization provides the user with ample information for obtaining peptide/protein sequences. Further, the information obtained can be exploited to gain information about specific protein/peptide modifications. Finally, the new instrumentation is accompanied by several software packages that can be used for database mining (i.e., for protein/peptide identification), for identification of small molecules, and for mapping of protein/peptide modifications. Scientific Advisors: Robert Rhoads, Ph.D. and Tammy Dugas, Ph.D.

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Real-Time PCR in the Research Core Facility

Two real time PCR instruments are available in the RCF for PCR quantification of nucleic acids: an ABI Prism 7700 Sequence Detection System and a BioRad iCycler. Both instruments are capable of performing simultaneous measurements of several targets in a single tube by using different fluorescent dyes on the probes. A Macintosh with the ABI Prism and a PC with the iCycler are connected for acquisition. Two additional Macintosh computers and a PC computer are networked to a color laser printer for off-line analysis. This allows continual use of the cyclers for acquisition. These analysis computers contain the requisite software to perform the analysis and have PE-Express a program to design primers and probes for Taqman PCR. In the 5’-nuclease assay (also termed “Taqman”), the probe is a non-extendable oligonucleotide labeled on the 5’-end with a fluorescent donor molecule (such as FAM or VIC) and labeled on the 3’-end with a fluorescent quencher (such as TAMRA). If the probe is intact, the donor and quencher are in close proximity to one another and no light is emitted with stimulation by the light source. If the probe binds to PCR product between the forward and reverse primers, then the 5’-3’ exonuclease activity of Taq polymerase digests the probe. Now the donor fluorescent molecule is spatially separated from the quencher and light will be emitted upon stimulation. Thus, the fluorescent signal increases two-fold every PCR cycle until the enzymatic reaction becomes saturated. The user selects a threshold level in the linear range of PCR and the sequence detector program from PE Biosystems calculates the CT for each sample. Because measurement with Taqman always occurs in the linear range of PCR, there are no problems associated with endpoint measurement. The analysis has great sensitivity (>1,000 copies), a wide linear range (> 107-fold), superb reproducibility, and an excellent correlation coefficient (R>0.99) between input and output. Faculty member in charge: Mike Mathis, Ph.D.

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Research Core Facility

The LSUHSC Research Core Facility (RCF) provides ready access to the latest state-of-the-art biotechnologies for use in scientific research. The RCF allows the student to gain experience and training in methodologies that were developed only in the last few years and are in a process of evolution that will even expand their applications to biomedical research. For example, the use of DNA microarray methodology allows the investigator in a matter of days to discern the pattern of expression of thousands of genes at the level of messenger RNA production. Proteomics enables the scientist to understand exactly the profile of proteins being synthesized at a given time. Thus, one can determine how virus infection, or cancer, or a specific drug impacts overall gene expression and thereby identify specific genes or blocks of genes that are affected. Laser Capture Microdissection (LCM) permits the investigator to “capture” a single cell or group of cells, such as a cancer cell or a cell expressing a specific protein, from a section of tissue so that the gene expression profile or specific properties of that cell or group of cells may be determined by powerful assays such as RT-PCR using Real-Time PCR. Flow cytometry is a multi-faceted technology that allows the investigator to collect a subset of cells (“panning”) from a large number of cells based on the size or surface proteins being expressed by the cells of interest. In a matter of hours, millions of cells can be analyzed, and the numbers of each cell type in the total population can be determined. For example, the number of CD4 and CD8 T cells in a population of lymphocytes can be measured in a rapid and single assay, and each cell type could be collected. The above are but a few examples of the applications of some of the powerful technologies offered in the RCF. The RCF is located on the sixth floor of the Biomedical Research Institute and currently contains instrumentation for eight separate technologies. Each instrument is operated by a Research Associate and overseen by an LSUHSC-S scientific advisor. These advisors constitute the Scientific Advisory Board, the current membership of which is Robert Chervenak (Chair), Rona Scott, Bryan Bellaire, Mike Mathis, Robert Rhoads, Tammy Dugas, Nick Goeders and Shayne Barlow. Deborah Chervenak is the manager of the RCF laboratories.

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Small Animal Imaging Facility

The Small Animal Imaging Facility at Louisiana State University Health Sciences Center - Shreveport is a state-of-the-art center for molecular imaging. This resource is located within the barrier facility of the Animal Resources Program. Over 3000 square feet have been devoted to this facility. A group of administrators, scientists, radiation safety experts, and clinicians has been organized to aid in the direction of this facility. The center offers micro-positron emission tomography (microPET), micro-computed assisted tomography (microCT), and optical imaging, including in vivo chemiluminescence and fluorescence imaging capabilities. MicroPET provides high performance, functional imaging for studies involving animal models of disease, genetically engineered animals, pharmaceutical development, and radiotracer development. The facility has purchased a microPET R4 device from Concorde Microsystems Incorporated. This unit allows non-invasive serial and longitudinal studies to be performed in the same animal. Isotopes are provided to investigators in cooperation with the Biomedical Research Foundation (BRF) of Northwest Louisiana PET imaging center cyclotron. MicroCT generates anatomic reference for the MicroPET data sets, as well as provides the capability of bone morphology and density measurements, tumor identification and classification, fat pad distribution and volume measurements, as well as many other types of studies. The center has a MicroCT unit custom built by Imtek, Inc. This unit uses the same gantry as the microPET machine, which allows superimposition of images for anatomic correlation of functional PET studies. Resolution is up to 45 microns and real-time image reconstruction is possible with this unit. The Small Animal Imaging Facility offers two optical imaging options, photon detection from chemiluminescent systems such as luciferase, and detection of fluorescence from models containing systems such as green fluorescent protein. Both of these modalities are imaged from the Xenogen IVIS imaging system. This technology combines specially designed imaging chambers and software with a charged-coupled device (CCD) camera. Investigators will be able to monitor and record cellular and genetic activity within a living organism in real time. Faculty member in charge: Shayne Barlow, Ph.D., D.V.M

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