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Cell Biol Int Rep (2010). 2011;18(1):e00010. doi: 10.1042/CBR20110003. Epub 2011 Apr 26.

Selective silencing of DNA topoisomerase IIβ in human mesenchymal stem cells by siRNAs (small interfering RNAs).

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1
Department of Biology, Fatih University, Stem Cell Research Laboratory, 34500 Buyukcekmece, Istanbul, Turkey.

Abstract

hMSCs (human mesenchymal stem cells) express two isoforms of DNA topo II (topoisomerase II). Although both isoforms have the same catalytic activity, they are specialized for different functions in the cell: while topo IIα is essential for chromosome segregation in mitotic cells, topo IIβ is involved in more specific cellular functions. A number of inhibitors are available that inhibit the catalytic activity of both topo II isoforms. However, in order to investigate the isoform-specific inhibition of these two enzymes, it is necessary to use other techniques such as siRNA (small interfering RNA) interference to selectively silence either one of the isoforms individually. Depending on the lipid charge densities and protein varieties of the cell membrane, previous studies have demonstrated that transfection efficiencies of siRNAs to hMSCs are very low. In the study reported here, we demonstrate the use of Lipofectamine RNAiMAX as an efficient transfection reagent to introduce siRNAs into human mesenchymal stem cells with significantly great efficiency to silence topo IIβ selectively. A high level of transfection efficiency (80%) was achieved by using unlabelled topo IIβ-specific siRNA oligos. Specifically, it was confirmed repeatedly that green labelled siRNAs interfere with the transfection of siRNAs. The reagent induced minimal cytotoxicity (3.5-4.5%), and cell viability of the transfected hMSCs decreased 20-30% compared with untreated cells, depending on the concentration of the reagent.

KEYWORDS:

DMEM, Dulbecco's modified Eagle's medium; DNA topoisomerase IIβ; GFP, green fluorescent protein; HEK, human embryonic kidney; LDH, lactate dehydrogenase; MSC, mesenchymal stem cell; MSC-FBS, MSC-qualified fetal bovine serum; PE, phycoerythrin; RNAi, RNA interference; RNAiMAX; hMSC, human mesenchymal stem cell; human mesenchymal stem cell; siRNA transfection; siRNA, small interfering RNA; topo II, topoisomerase II

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