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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Program Projects (P01)
Project #
2P01DK044935-11A1
Application #
7083036
Study Section
Special Emphasis Panel (ZDK1-GRB-6 (J2))
Project Start
2006-08-01
Project End
2011-07-31
Budget Start
2006-08-01
Budget End
2007-07-31
Support Year
11
Fiscal Year
2006
Total Cost
$182,288
Indirect Cost
Name
University of Pittsburgh
Department
Type
DUNS #
004514360
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
Han, Fang; Miyagawa, Yoshitaka; Verlengia, Gianluca et al. (2018) Cellular Antisilencing Elements Support Transgene Expression from Herpes Simplex Virus Vectors in the Absence of Immediate Early Gene Expression. J Virol 92:
Verlengia, Gianluca; Miyagawa, Yoshitaka; Ingusci, Selene et al. (2017) Engineered HSV vector achieves safe long-term transgene expression in the central nervous system. Sci Rep 7:1507
Miyagawa, Yoshitaka; Verlengia, Gianluca; Reinhart, Bonnie et al. (2017) Deletion of the Virion Host Shut-off Gene Enhances Neuronal-Selective Transgene Expression from an HSV Vector Lacking Functional IE Genes. Mol Ther Methods Clin Dev 6:79-90
Laemmle, Lillian L; Cohen, Justus B; Glorioso, Joseph C (2016) Constitutive Expression of GATA4 Dramatically Increases the Cardiogenic Potential of D3 Mouse Embryonic Stem Cells. Open Biotechnol J 10:248-257
Goins, William F; Hall, Bonnie; Cohen, Justus B et al. (2016) Retargeting of herpes simplex virus (HSV) vectors. Curr Opin Virol 21:93-101
Reinhart, Bonnie; Goins, William F; Harel, Asaff et al. (2016) An HSV-based library screen identifies PP1? as a negative TRPV1 regulator with analgesic activity in models of pain. Mol Ther Methods Clin Dev 3:16040
Miyagawa, Yoshitaka; Marino, Pietro; Verlengia, Gianluca et al. (2015) Herpes simplex viral-vector design for efficient transduction of nonneuronal cells without cytotoxicity. Proc Natl Acad Sci U S A 112:E1632-41
Majima, Tsuyoshi; Funahashi, Yasuhito; Takai, Shun et al. (2015) Herpes Simplex Virus Vector-Mediated Gene Delivery of Poreless TRPV1 Channels Reduces Bladder Overactivity and Nociception in Rats. Hum Gene Ther 26:734-42
Sha, Huizi; Zou, Zhengyun; Xin, Kai et al. (2015) Tumor-penetrating peptide fused EGFR single-domain antibody enhances cancer drug penetration into 3D multicellular spheroids and facilitates effective gastric cancer therapy. J Control Release 200:188-200
Goins, William F; Huang, Shaohua; Cohen, Justus B et al. (2014) Engineering HSV-1 vectors for gene therapy. Methods Mol Biol 1144:63-79

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