6121, 8113, 8141 MRB III
Faculty Coordinator: Aurea Pimenta, Ph.D.
8114C MRBIII
Tel: 615-936-3864
email:
This core facility provides equipment, methodology consultation, and reagents that assist in the introduction of transgenes for transient transfection, and in the localization of transgene and endogenous gene products. The coordinator meets with KC investigators to design DNA constructs for expression of reporter and specific transgenes, vectors for cloning by individual laboratories, methods for introducing genes into cells and embryos, and methods for the localization of transcripts in brain tissue. Equipment is available for use by all trained KC investigators and their laboratory personnel.
The Tissue Histology Laboratory provides equipment and laboratory space for KC investigators to prepare brain tissues for analysis using a variety of different methods. A library of mouse brain tissue sections on slides, ready for in situ hybridization is available. Mouse strain: C57Bl6/J. Ages: E18, P0, P7, P60, Adult.
The BioRad Gene Gun, maintained in the tissue histology Room 8113 MRB III, is used to transfect tissue slices and cultured cells. The BioRad Gene Pulser II and Amaxa Nucleofector systems are used to transfect DNA constructs into primary neuronal and glial cultures and cell lines.
The 830 Electroporation system for embryonic mice is housed in the mouse metabolism laboratory of the Vanderbilt Diabetes Center in dedicated space (843 Light Hall) to perform sterile surgeries on pregnant mice. This electroporator is outfitted with paddle and needle electrodes and is designed to introduce DNA constructs into live mouse embryos. The core service has been successful in introducing genes into forebrain and hindbrain regions in embryos from embryonic day 10.5-13.5. Training of laboratory personnel to master this complex technology is available. The transgene core service has established a newly outfitted mouse stereotaxic surgical suite in the new vivarium located in MCN. This suite contains a Kopf small animal stereotaxis, fine surgical instruments, Halothane anesthesia machine, and a controlled environmental platform for surgical recovery.
The Leica LMD laser dissection microscope is ideal for precise harvesting of small brain areas or groups of cells. It is equipped with a motorized stage, phase contrast and epifluorescence and a CCD camera and operated by a high-speed, dedicated computer. Material can be harvested from tissue cultures or brain sections, and the harvested materials can be used for downstream qPCR and DNA microarray analyses. Using epifluorescent microscopy it is possible to dissect FITC or Rhodamine-lableled immunostained material, allowing precise phenotypic characterization of targeted neuronal sub-phenotypes.
The coordinator provides training to any KC investigator, trainee, or laboratory staff to use specialized equipment that includes motorized and standard cryostats, temperature-controlled vibratome, freezing stage sliding microtome, slide histology work station, and in situ hybridization oven. Consultation is available to assist in experimental design for immunolocalization studies of proteins, and in situ hybridization for studies of mRNA localization. Complex strategies such as double and triple labeling of proteins and transcripts, using different fluorochromes, are done routinely in this core facility. The core also provides expertise in the use of the Biorad Gene Gun to label cells with lipophilic dyes for analysis of dendritic and axonal arbors.
Equipment Available: