© 1995 American Heart Association, Inc.
Articles |
Reversible Permeabilization
A Novel Technique for the Intracellular Introduction of Antisense Oligodeoxynucleotides Into Intact Smooth Muscle
From the Departments of Anesthesiology (R.E.L.), Molecular Physiology and Biological Physics (R.E.L., A.P.S., G.K.O., A.V.S.), Medicine (A.P.S.), and Pathology (A.V.S.), University of Virginia Health Sciences Center, Charlottesville.
Correspondence to Ryan E. Lesh, MD, Department of Molecular Physiology and Biological Physics, University of Virginia Health Sciences Center, Box 449, Charlottesville, VA 22908.
Abstract Antisense oligodeoxynucleotides (ODNs) have been used to modify gene expression in vitro and are also promising therapeutic agents. Although there are numerous reports of antisense ODN–mediated changes in protein expression of cultured cells, use of these compounds to achieve antisense regulation of specific proteins in intact tissue has been limited. The aims of this study were (1) to define organ culture conditions for ileum smooth muscle that would permit long-term maintenance of force-generating capabilities and normal ultrastructure and (2) to develop a method for efficient introduction of antisense ODNs into intact tissue. Sheets of ODN-containing, reversibly permeabilized rat outer longitudinal ileum were maintained in a serum-free organ culture medium for 1 week without significant decreases in tension response to membrane depolarization or carbachol stimulation; the G protein–coupled calcium sensitization pathway was also intact after 7 days. Reversible permeabilization, a method previously used to load smooth and cardiac muscle with aequorin and heparin, was effective for loading >95% of ileum smooth muscle cells with a fluorescein-conjugated antisense ODN (5'-AAGGGCCATTTTGTT-FITC-3'). Confocal microscopy of reversibly permeabilized smooth muscle loaded with fluorescent antisense ODNs revealed intense nuclear fluorescence and less intense, homogeneous, cytoplasmic fluorescence. Internally radiolabeled ODNs (homologous to the above sequence) showed complete degradation between 4 and 16 hours after introduction into the cells. In summary, we have demonstrated methods for long-term organ culture and high-efficiency introduction of antisense ODNs into intact smooth muscle sheets. Such methods have broad potential utility for investigating many questions in smooth muscle biology. At present, however, a major limitation of this approach is the short half-life of phosphorothioated ODNs.
Key Words: antisense oligodeoxynucleotides • smooth muscle • reversible permeabilization • confocal microscopy • electron microscopy
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