What is it about?
A three-dimensional reconstruction analysis of localization of phosphodiester and phosphorothioate oligonucleotide antisense to type-1 plasminogen activator inhibitor (PAI-1) mRNA within endothelial cells is described.When EA.hy 926 cells were incubated with fluorescently labelled phosphodiester (PO-16) or phosphorothioate (PS-16) oligonucleotides at low, not cytotoxical concentrations, the relative brightness composition of the images of the particular samples was much higher for PS-16 than PO-16 and dependent upon the extracellular concentration and the incubation time.
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Why is it important?
The 3-D reconstructions based on the series of optical sections of the samples, spaced every 1.5 microns, showed the punctuate accumulation of the oligonucleotides and a striking difference in a spatial distribution between PO-16 and PS-16 within the cytoplasm. Even after 24 h incubation of endothelial cells with 2.5 microM of PO-16 and PS-16 oligonucleotides, there was a predominant oligonucleotide localization within the cytoplasm and only traces of oligonucleotides could be seen in the cell nucleus and/or perinuclear organelles.
Perspectives
We attempted to characterize endothelial cells using a laser confocal microscopy. We prepared fluorescein conjugates of both phosphodiester and phosphorothioate forms of the most inhibitory oligonucleotide antisense to PAI- mRNA and studied their uptake and distribution in endothelial cells applying a 3-D reconstruction. As far as we know such kind of spatial reconstruction has not beenyet used to study cellular uptake of antisense oligonucleotides.
Professor Elzbieta Wyroba
Nencki Institute of Experimental Biology
Read the Original
This page is a summary of: Internalization of oligodeoxynucleotide antisense to type-1 plasminogen activator inhibitor mRNA in endothelial cells: A three-dimensional reconstruction by confocal microscopy, Biology of the Cell, January 1996, Wiley,
DOI: 10.1111/j.1768-322x.1996.tb00964.x.
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