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

Kamaci N, Emnacar T, Karakas N, Arikan G, Tsutsui K, Isik S - Cell Biol Int Rep (2010) (2011)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: 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.

No MeSH data available.


Related in: MedlinePlus

GFP plasmid and siRNA transfection of HEK-293 and hMSCsMorphology of HEK-293 and hMSCs at third passage (A, B). GFP plasmid transfection of HEK-293 and hMSCs with Lipofectamine 2000 and FuGENE transfection reagents. While approximately 100% transfection efficiency was observed for HEK-293 cells, it was about 25% for Lipofectamine 2000 and 15% for FuGENE (C, D, E, F). Transfection with RNAiMAX transfection reagent by using Alexa Fluor 488-labelled siRNA oligos in HEK-293 and hMSCs with approximately 100% and 15% transfection efficiencies, respectively. All pictures were taken following 24 h of transfection (G, H).
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Figure 2: GFP plasmid and siRNA transfection of HEK-293 and hMSCsMorphology of HEK-293 and hMSCs at third passage (A, B). GFP plasmid transfection of HEK-293 and hMSCs with Lipofectamine 2000 and FuGENE transfection reagents. While approximately 100% transfection efficiency was observed for HEK-293 cells, it was about 25% for Lipofectamine 2000 and 15% for FuGENE (C, D, E, F). Transfection with RNAiMAX transfection reagent by using Alexa Fluor 488-labelled siRNA oligos in HEK-293 and hMSCs with approximately 100% and 15% transfection efficiencies, respectively. All pictures were taken following 24 h of transfection (G, H).

Mentions: In this study, hMSCs derived from bone marrow were chosen due to their high abundance in the bone marrow and also easiness to isolate and expand in the culture. After flow cytometry analysis, cells at third passage were subjected to the GFP–plasmid and siRNA transfection experiments in hMSCs. HEK 293 cell line was used as a positive control. Approximately 100% efficiency was observed for GFP plasmid transfection of HEK 293 with Lipofectamine 2000 and FuGENE HD transfection reagents. Increasing concentrations of reagent and lack of the serum resulted in higher cytotoxicity for HEK 293 cells (data not shown). However, for MSCs, efficiency did not exceed 25%. Alexa Fluor 488-labelled control siRNAs were delivered with Lipofectamine RNAiMAX and Lipofectamine 2000 to HEK 293 cells with about 95–100% efficiency. Unfortunately, this method was also insufficient for hMSCs. The possibility that the label at the end of oligo might be preventing efficient delivery of the siRNA into the cell led us to use unlabelled siRNAs (Figure 2).


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

Kamaci N, Emnacar T, Karakas N, Arikan G, Tsutsui K, Isik S - Cell Biol Int Rep (2010) (2011)

GFP plasmid and siRNA transfection of HEK-293 and hMSCsMorphology of HEK-293 and hMSCs at third passage (A, B). GFP plasmid transfection of HEK-293 and hMSCs with Lipofectamine 2000 and FuGENE transfection reagents. While approximately 100% transfection efficiency was observed for HEK-293 cells, it was about 25% for Lipofectamine 2000 and 15% for FuGENE (C, D, E, F). Transfection with RNAiMAX transfection reagent by using Alexa Fluor 488-labelled siRNA oligos in HEK-293 and hMSCs with approximately 100% and 15% transfection efficiencies, respectively. All pictures were taken following 24 h of transfection (G, H).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3475440&req=5

Figure 2: GFP plasmid and siRNA transfection of HEK-293 and hMSCsMorphology of HEK-293 and hMSCs at third passage (A, B). GFP plasmid transfection of HEK-293 and hMSCs with Lipofectamine 2000 and FuGENE transfection reagents. While approximately 100% transfection efficiency was observed for HEK-293 cells, it was about 25% for Lipofectamine 2000 and 15% for FuGENE (C, D, E, F). Transfection with RNAiMAX transfection reagent by using Alexa Fluor 488-labelled siRNA oligos in HEK-293 and hMSCs with approximately 100% and 15% transfection efficiencies, respectively. All pictures were taken following 24 h of transfection (G, H).
Mentions: In this study, hMSCs derived from bone marrow were chosen due to their high abundance in the bone marrow and also easiness to isolate and expand in the culture. After flow cytometry analysis, cells at third passage were subjected to the GFP–plasmid and siRNA transfection experiments in hMSCs. HEK 293 cell line was used as a positive control. Approximately 100% efficiency was observed for GFP plasmid transfection of HEK 293 with Lipofectamine 2000 and FuGENE HD transfection reagents. Increasing concentrations of reagent and lack of the serum resulted in higher cytotoxicity for HEK 293 cells (data not shown). However, for MSCs, efficiency did not exceed 25%. Alexa Fluor 488-labelled control siRNAs were delivered with Lipofectamine RNAiMAX and Lipofectamine 2000 to HEK 293 cells with about 95–100% efficiency. Unfortunately, this method was also insufficient for hMSCs. The possibility that the label at the end of oligo might be preventing efficient delivery of the siRNA into the cell led us to use unlabelled siRNAs (Figure 2).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: 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.

No MeSH data available.


Related in: MedlinePlus