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Nucleic Acids Research, 1994, Vol. 22, No. 14 2830-2836
© 1994

METHODS

In vivo generation of highly abundant sequence-specific oligonucleotides for antisense and triplex gene regulation

Sarah B. Noonberg1,2,*, Gary K. Scott3, Marvin R. Garovoy4, Christopher C. Benz5 and C.Anthony Hunt1,5

1Bioengineering Graduate Group, University of California, Berkeley/University of California San Francisco 2School of Medicine San Francisco, CA 94143, USA 3Cancer Research Institute San Francisco, CA 94143, USA 4Department of Surgery San Francisco, CA 94143, USA 5Departments of Pharmacy and Pharmaceutical Chemistry, University of California San Francisco, CA 94143, USA

*To whom correspondence should be addressed at: Box 0128, Cancer Research Institute, University of California, San Francisco, San Francisco, CA 94143, USA

Received February 25, 1994. Revised June 16, 1994. Accepted June 16, 1994.

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 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 106 copies per cell, reach steady-state levels of expression within 9 hours post-transfection, and are still readily detectable 7 days post-transfection. addition, these oligonucleotides are retained in the nucleus, obtain a 5' 7-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.


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