PMID: 9558486Apr 29, 1998Paper

Flow cytometric and microscopic characterization of the uptake and distribution of phosphorothioate oligonucleotides in human keratinocytes

Archives of Dermatological Research
M WingensP E van Erp

Abstract

Gene-specific inhibition by antisense oligonucleotides has been successful in a large number of systems. In an attempt to use this strategy for the modulation of skin disease-specific gene expression, we studied oligonucleotide uptake in cultured human keratinocytes. This study revealed a heterogeneous uptake of fluorescently labeled phosphorothioate oligonucleotides. Flow cytometric and microscopic analysis showed two fluorescent cell populations with differences in intensity: a 'bright' population of highly fluorescent small cells and a 'dim' population of less fluorescent but larger cells. The heterogeneity in uptake between these two populations was not a result of differences in cell cycle phases of the keratinocytes, as shown by flow cytometric sorting and measurements of relative DNA content. In both populations the oligonucleotides were transported intracellularly and were mainly located in the cytoplasm. A typically speckled localization pattern was demonstrated by confocal laser scanning microscopy. We used propidium iodide (PI) to assess viability, and showed that in nonviable (PI-permeable) keratinocytes the oligonucleotides accumulated in the nucleus. The use of a lipidfection reagent also changed the intracellular...Continue Reading

Citations

Jul 13, 2001·Pharmacology & Therapeutics·C J Wraight, P J White
Oct 18, 2000·Advanced Drug Delivery Reviews·R M Brand, P L Iversen
Jul 27, 2001·Journal of Photochemistry and Photobiology. B, Biology·M SasakiA Ito
Dec 19, 2003·Expert Opinion on Biological Therapy·P J WhiteC J Wraight
May 23, 2012·Journal of the American Chemical Society·Heera Krishna, Marvin H Caruthers

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