Limits...
Ca2+ enrichment in culture medium potentiates effect of oligonucleotides.

Hori S, Yamamoto T, Waki R, Wada S, Wada F, Noda M, Obika S - Nucleic Acids Res. (2015)

Bottom Line: In addition, CEM reflects in vivo silencing activity more consistently than conventional transfection methods.The presence of these nanoparticles is critical for the efficient uptake of various oligonucleotides.In contrast, CEM is ineffective for plasmids, which are readily transfected via the conventional calcium phosphate method.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Pharmaceutical Sciences, Osaka University, 1-6, Yamadaoka, Suita, Osaka, 565-0871, Japan.

No MeSH data available.


Related in: MedlinePlus

CEM effect on cellular uptake of 2′, 4′-BNA ASO (A) Flow cytometric analysis of ApoB-ASO delivery into Huh-7 cells. Cells were treated with Cy3-labeled ApoB-ASO with or without CaCl2 for 2 h in phenol red-free media and washed twice with PBS. Cells (10 000 events) were analyzed using a FACSCalibur flow cytometer and the mean fluorescence intensity of Cy3 was calculated using CellQuest Pro software. Each data point represents the mean ± SD of three independent experiments. Statistical comparisons of results were performed by Student's t-tests, *P < 0.0005. (B) Addition of CaCl2 at different stages of cellular uptake or intracellular trafficking of ZsGreen1-ASO in ZsG-N1–2R/HLE cells. CaCl2 was added to the medium before addition of ZsGreen1-ASO (‘Pre’) and/or together with addition of ASO for 7 h (‘TF’), and/or after removal of ASO and washing of the cells (‘Post’). At 72 h after ASO addition, the knockdown activity of the ASO was analyzed. Relative fluorescence intensity was measured and is presented as the percentage relative to the UTC. Each data point represents the mean ± SD of three independent experiments.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4627064&req=5

Figure 4: CEM effect on cellular uptake of 2′, 4′-BNA ASO (A) Flow cytometric analysis of ApoB-ASO delivery into Huh-7 cells. Cells were treated with Cy3-labeled ApoB-ASO with or without CaCl2 for 2 h in phenol red-free media and washed twice with PBS. Cells (10 000 events) were analyzed using a FACSCalibur flow cytometer and the mean fluorescence intensity of Cy3 was calculated using CellQuest Pro software. Each data point represents the mean ± SD of three independent experiments. Statistical comparisons of results were performed by Student's t-tests, *P < 0.0005. (B) Addition of CaCl2 at different stages of cellular uptake or intracellular trafficking of ZsGreen1-ASO in ZsG-N1–2R/HLE cells. CaCl2 was added to the medium before addition of ZsGreen1-ASO (‘Pre’) and/or together with addition of ASO for 7 h (‘TF’), and/or after removal of ASO and washing of the cells (‘Post’). At 72 h after ASO addition, the knockdown activity of the ASO was analyzed. Relative fluorescence intensity was measured and is presented as the percentage relative to the UTC. Each data point represents the mean ± SD of three independent experiments.

Mentions: ZsG-N1–2R/HLE cells were seeded at 1.2 × 104 cells/well in 96-well black plates (Corning; Corning, NY, USA) containing 10% FBS/DMEM. After 24 h, each ZsGreen1-ASO was added in 10% FBS/DMEM containing or lacking CaCl2 or MgCl2. In separate experiments, ZsGN1–120-BNA (15) was used either at 300 nM for 4 days (Figure 1A), from 5 nM to 5 μM for 4 days (Figure 1B) or at 1 μM for 5 days (Figure 1C). To examine if CEM affects the uptake and/or intracellular trafficking of ASO, CaCl2 was added to the medium before addition of ZsGN1–120-BNA (15) (‘Pre’ interval) and/or together with addition of ASO for 7 h (‘TF’ interval; expected to be the time of ASO uptake), and/or after removal of ASO and washing of the cells (‘Post’ interval). At 72 h after ASO addition, the knockdown activity of ASO was analyzed (Figure 4B). The fluorescence of ZsGreen1 and DsRed was measured using a microplate spectrofluorometer (SPECTRAmax GEMINI; Molecular Devices, Sunnyvale, CA, USA). Knockdown efficiency of ASO was calculated by dividing the fluorescence of ZsGreen1 by that of DsRed; relative fluorescence intensity (RFI) is presented as the percentage relative to the untreated control (UTC).


Ca2+ enrichment in culture medium potentiates effect of oligonucleotides.

Hori S, Yamamoto T, Waki R, Wada S, Wada F, Noda M, Obika S - Nucleic Acids Res. (2015)

CEM effect on cellular uptake of 2′, 4′-BNA ASO (A) Flow cytometric analysis of ApoB-ASO delivery into Huh-7 cells. Cells were treated with Cy3-labeled ApoB-ASO with or without CaCl2 for 2 h in phenol red-free media and washed twice with PBS. Cells (10 000 events) were analyzed using a FACSCalibur flow cytometer and the mean fluorescence intensity of Cy3 was calculated using CellQuest Pro software. Each data point represents the mean ± SD of three independent experiments. Statistical comparisons of results were performed by Student's t-tests, *P < 0.0005. (B) Addition of CaCl2 at different stages of cellular uptake or intracellular trafficking of ZsGreen1-ASO in ZsG-N1–2R/HLE cells. CaCl2 was added to the medium before addition of ZsGreen1-ASO (‘Pre’) and/or together with addition of ASO for 7 h (‘TF’), and/or after removal of ASO and washing of the cells (‘Post’). At 72 h after ASO addition, the knockdown activity of the ASO was analyzed. Relative fluorescence intensity was measured and is presented as the percentage relative to the UTC. Each data point represents the mean ± SD of three independent experiments.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4627064&req=5

Figure 4: CEM effect on cellular uptake of 2′, 4′-BNA ASO (A) Flow cytometric analysis of ApoB-ASO delivery into Huh-7 cells. Cells were treated with Cy3-labeled ApoB-ASO with or without CaCl2 for 2 h in phenol red-free media and washed twice with PBS. Cells (10 000 events) were analyzed using a FACSCalibur flow cytometer and the mean fluorescence intensity of Cy3 was calculated using CellQuest Pro software. Each data point represents the mean ± SD of three independent experiments. Statistical comparisons of results were performed by Student's t-tests, *P < 0.0005. (B) Addition of CaCl2 at different stages of cellular uptake or intracellular trafficking of ZsGreen1-ASO in ZsG-N1–2R/HLE cells. CaCl2 was added to the medium before addition of ZsGreen1-ASO (‘Pre’) and/or together with addition of ASO for 7 h (‘TF’), and/or after removal of ASO and washing of the cells (‘Post’). At 72 h after ASO addition, the knockdown activity of the ASO was analyzed. Relative fluorescence intensity was measured and is presented as the percentage relative to the UTC. Each data point represents the mean ± SD of three independent experiments.
Mentions: ZsG-N1–2R/HLE cells were seeded at 1.2 × 104 cells/well in 96-well black plates (Corning; Corning, NY, USA) containing 10% FBS/DMEM. After 24 h, each ZsGreen1-ASO was added in 10% FBS/DMEM containing or lacking CaCl2 or MgCl2. In separate experiments, ZsGN1–120-BNA (15) was used either at 300 nM for 4 days (Figure 1A), from 5 nM to 5 μM for 4 days (Figure 1B) or at 1 μM for 5 days (Figure 1C). To examine if CEM affects the uptake and/or intracellular trafficking of ASO, CaCl2 was added to the medium before addition of ZsGN1–120-BNA (15) (‘Pre’ interval) and/or together with addition of ASO for 7 h (‘TF’ interval; expected to be the time of ASO uptake), and/or after removal of ASO and washing of the cells (‘Post’ interval). At 72 h after ASO addition, the knockdown activity of ASO was analyzed (Figure 4B). The fluorescence of ZsGreen1 and DsRed was measured using a microplate spectrofluorometer (SPECTRAmax GEMINI; Molecular Devices, Sunnyvale, CA, USA). Knockdown efficiency of ASO was calculated by dividing the fluorescence of ZsGreen1 by that of DsRed; relative fluorescence intensity (RFI) is presented as the percentage relative to the untreated control (UTC).

Bottom Line: In addition, CEM reflects in vivo silencing activity more consistently than conventional transfection methods.The presence of these nanoparticles is critical for the efficient uptake of various oligonucleotides.In contrast, CEM is ineffective for plasmids, which are readily transfected via the conventional calcium phosphate method.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Pharmaceutical Sciences, Osaka University, 1-6, Yamadaoka, Suita, Osaka, 565-0871, Japan.

No MeSH data available.


Related in: MedlinePlus