A convenient plasmid system for construction of helper-dependent adenoviral vectors and its application for analysis of the breast-cancer-specific mammaglobin promoter
| Autor: | Mary Hitt, Chang-Xin Shi, Frank L. Graham |
|---|---|
| Rok vydání: | 2006 |
| Předmět: |
Transgene
Genetic Vectors Molecular Sequence Data Breast Neoplasms Sensitivity and Specificity Adenoviridae Cell Line Mice Plasmid Mammaglobin Transduction Genetic Cell Line Tumor Gene Duplication Drug Discovery Genetics Animals Humans Uteroglobin Vector (molecular biology) Promoter Regions Genetic Enhancer Molecular Biology Genetics (clinical) Base Sequence biology Mammaglobin A Promoter Molecular biology Neoplasm Proteins Cell biology Restriction enzyme Organ Specificity biology.protein Molecular Medicine Expression cassette Helper Viruses Plasmids |
| Zdroj: | The Journal of Gene Medicine. 8:442-451 |
| ISSN: | 1521-2254 1099-498X |
| DOI: | 10.1002/jgm.867 |
| Popis: | Background Helper-dependent (HD) adenovirus (Ad) vectors, deleted of all viral coding sequences, have a higher cloning capacity, improved performance of tissue-specific promoters, and reduced toxicity in animals relative to first-generation Ad vectors, making these vectors promising tools for gene transfer in vitro and in vivo. However, the large size of HDAd precursor plasmids renders them relatively difficult to manipulate due to the paucity of unique restriction enzyme sites suitable for transgene insertion and to the size constraints imposed by the viral packaging machinery. Methods We have constructed a series of HDAd precursor plasmids that allows cassette insertion at a unique site in the vector backbone. We have tested whether these vector backbones will support the tissue-specificity of inserted expression cassettes in a study of the activity of the potentially breast-cancer-specific mammaglobin promoter and enhancer. Results We report here the generation of a series of HDAd precursor plasmids, both with and without an additional reporter expression cassette, that were designed to accommodate a wide range in size of inserted DNA. The system was validated for transcriptional targeting studies by demonstrating the tissue-specificity and activity of the mammaglobin promoter rescued using this precursor system. In addition, we have extended our previous studies on the mammaglobin promoter by demonstrating that two copies of the mammaglobin enhancer fused to the minimal promoter surpassed the activity of the single enhancer/promoter by at least 10-fold in breast cancer cells while maintaining only minimal expression in normal cells both in vitro and in a mouse tumor model. Conclusions This versatile plasmid system simplifies the construction of HDAd vectors and was valuable in demonstrating the targeting potential of the mammaglobin promoter for breast cancer gene therapy. Copyright © 2006 John Wiley & Sons, Ltd. |
| Databáze: | OpenAIRE |
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