Cytoplasmically retargeted HSV1-tk/GFP reporter gene mutants for optimization of noninvasive molecular-genetic imaging.

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Title Cytoplasmically retargeted HSV1-tk/GFP reporter gene mutants for optimization of noninvasive molecular-genetic imaging.
Author Vider, Jelena
Journal Name Neoplasia
Year Published 2003
Place of publication United States of America
Publisher Neoplasia Press
Abstract To optimize the sensitivity of imaging HSV1-tk/GFP reporter gene expression, a series of HSV1-tk/GFP mutants was developed with altered nuclear localization and better cellular enzymatic activity, compared to that of the native HSV1-tk/GFP fusion protein (HSV1-tk/GFP). Several modifications of HSV1-tk/GFP reporter gene were performed, including targeted inactivating mutations in the nuclear localization signal (NLS), the addition of a nuclear export signal (NES), a combination of both mutation types, and a truncation of the first 135 bp of the native hsv1-tk coding sequence containing a "cryptic" testicular promoter and the NLS. A recombinant HSV1-tk/GFP protein and a highly sensitive sandwich enzyme-linked immunosorbent assay for HSV1-tk/GFP were developed to quantitate the amount of reporter gene product in different assays to allow normalization of the data. These different mutations resulted in various degrees of nuclear clearance, predominant cytoplasmic distribution, and increased total cellular enzymatic activity of the HSV1-tk/GFP mutants, compared to native HSV1-tk/GFP when expressed at the same levels. This appears to be the result of improved metabolic bioavailability of cytoplasmically retargeted mutant HSV1-tk/GFP enzymes for reaction with the radiolabeled probe (e.g., FIAU). The analysis of enzymatic properties of different HSV1-tk/GFP mutants using FIAU as a substrate revealed no significant differences from that of the native HSV1-tk/GFP. Improved total cellular enzymatic activity of cytoplasmically retargeted HSV1-tk/GFP mutants observed in vitro was confirmed by noninvasive imaging of transduced subcutaneous tumor xenografts bearing these reporters using [(131)I]FIAU and a gamma-camera.
Peer Reviewed Yes
Published Yes
Publisher URI
Copyright Statement Copyright 2003 Neoplasia Press. The attached file is reproduced here in accordance with the copyright policy of the publisher. Please refer to the journal's website for access to the definitive, published version.
Volume 5
Issue Number 3
Page from 245
Page to 254
ISSN 1522-8002
Date Accessioned 2012-04-11; 2012-07-12T23:15:33Z
Research Centre Heart Foundation Research Centre; Molecular Basis of Disease
Faculty Griffith Health Faculty
Subject Molecular Targets
Publication Type Journal Articles (Refereed Article)
Publication Type Code c1x

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