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Am J Physiol Cell Physiol 285: C489-C498, 2003. First published March 26, 2003; doi:10.1152/ajpcell.00045.2003
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METHODS IN CELL PHYSIOLOGY

Specific iNOS-targeted antisense knockdown in endothelial cells

Karsten Hemmrich, Christoph V. Suschek, Guido Lerzynski, Oliver Schnorr, and Victoria Kolb-Bachofen

Research Group Immunobiology, Heinrich-Heine-University of Düsseldorf, D-40001 Düsseldorf, Germany

Submitted 3 February 2003 ; accepted in final form 21 March 2003

ABSTRACT

The inhibition of inducible nitric oxide synthase (iNOS) expression via antisense oligonucleotides (AS-ODN) may represent a highly specific tool. Endothelial cells (EC) represent prime candidate cells for in vivo application, and we therefore aimed at optimizing this technique for effectiveness and specificity in primary nontransformed rat EC. EC or L929 fibroblasts were incubated with iNOS-specific ODN optimizing all experimental steps. We find that ODN uptake, as analyzed by fluorescence microscopy and labeled ODN, was absolutely dependent on vehicle presence, and among the vehicles tested, Lipofectin displayed negligible toxicity and good uptake. In addition, omission of serum was also essential, a factor that might limit its use in vivo. Moreover, intranuclear accumulation of AS-ODN appeared crucial for successive inhibition. The impact of ODN on iNOS mRNA, protein, and enzyme activity was specific and resulted in >95% inhibition of protein formation. In conclusion, in this article we provide a protocol for an optimized AS-mediated knockdown, representing a specific and efficient instrument for blocking of iNOS formation and allowing for studying the impact of iNOS expression on endothelial function. We also expose application problems of this technique when working in inflammatory conditions.

endothelium; inflammation; nitric oxide; inducible nitric oxide synthase


Address for reprint requests and other correspondence: V. Kolb-Bachofen, Research Group Immunobiology, Heinrich-Heine-Univ. of Düsseldorf, Gebäude 23.12, Postfach 10 10 07, D-40001 Düsseldorf, Germany (E-mail: bachofen{at}uni-duesseldorf.de).




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