In vivo generation of highly abundant sequence-specific oligonucleotides for antisense and triplex gene regulation
| Autor: | Sarah B. Noonberg, G K Scott, C C Benz, Marvin R. Garovoy, C. A. Hunt |
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| Jazyk: | angličtina |
| Rok vydání: | 1994 |
| Předmět: |
Molecular Sequence Data
Breast Neoplasms Kidney Transfection Cell Line RNA Small Nuclear Gene expression Genetics Tumor Cells Cultured Humans Cloning Molecular Gene Regulation of gene expression Expression vector biology Base Sequence Oligonucleotide Chimera Ribozyme RNA Oligonucleotides Antisense Molecular biology Cell biology Antisense RNA Kinetics Gene Expression Regulation Oligodeoxyribonucleotides biology.protein Dactinomycin Nucleic Acid Conformation Female |
| Popis: | Antisense and triplex oligonucleotides continue to demonstrate potential as mediators of gene-specific repression of protein synthesis. However, inefficient and heterogeneous cellular uptake, intracellular sequestration, and rapid intracellular and extracellular degradation represent obstacles to their eventual clinical utility. Efficient cellular delivery of targeted ribozymes can present similar problems. In this report we describe a system for circumventing these obstacles and producing large quantities of short, sequence-specific RNA oligonucleotides for use in these gene regulation strategies. The oligonucleotides are generated from a vector containing promoter, capping, and termination sequences from the human small nuclear U6 gene, surrounding a synthetic sequence incorporating the oligonucleotide of interest. In vivo, these oligonucleotides are produced constitutively and without cell type specificity in levels up to 5 x 10(6) copies per cell, reach steady-state levels of expression within 9 hours post-transfection, and are still readily detectable 7 days post-transfection. In addition, these oligonucleotides are retained in the nucleus, obtain a 5' gamma-monomethyl phosphate cap, and have an intracellular half-life of approximately one hour. This expression vector provides a novel and efficient method of intracellular delivery of antisense or triplex RNA oligonucleotides (and/or ribozymes) for gene regulation, as well as a cost-effective means of comparing the biological activity arising from a variety of different potential oligonucleotide sequences. |
| Databáze: | OpenAIRE |
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