The studies in this program project all utilize modified herpes simplex virus (HSV)-based vectors to develop and/or test gene transfer as a method to deliver analgesic proteins. Three important issues will need to be addressed in order to fully evaluate the success of these studies. First we must be able to demonstrate the successful delivery of the HSV vectors to the intended tissues. Second, we must be able to assess the expression of the HSV transgenes at both the mRNA and protein levels. And third, we must be able to examine the biological effects of transgene expression.
The aim of this core is to provide standardized biochemical, molecular biological, and histological techniques to the three research projects so that they may adequately address these concerns. Towards this aim this core will perform: 1) Realtime quantitative PCR and reverse transcription PCR to measure vector load and transgene expression;2) In situ hybridization (ISH) and immunocytochemistry (ICC) to localize HSV vectors, assess transgene expression, and examine any changes in endogenous gene and protein expression;3) Enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), high performance liquid chromatography (HPLC), and protein biochemistry (including Western blot) to quantify changes in transgene or endogenous tissue proteins;and 4) Ca2+ influx assays to examine a cDNA library for genes that interact with the nociceptive-specific vanilloid receptor (VR1). By performing these various assays under the direction of a single core, we can increase quality control, decrease variation, and reduce costs across the entire program project. The core will be utilized by all projects.
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