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First siRNA library screening in hard-to-transfect HUVEC cells.

Zumbansen M, Altrogge LM, Spottke NU, Spicker S, Offizier SM, Domzalski SB, St Amand AL, Toell A, Leake D, Mueller-Hartmann HA - J RNAi Gene Silencing (2009)

Bottom Line: Of the 37 primary hits, down-regulation of 33 led to reduced proliferation or increased cell death, while down-regulation of two allowed for better cell viability.Our results demonstrate that the Nucleofector(R) 96-well Shuttle(R) System allows the delivery of siRNA libraries in cell types previously considered to be difficult to transfect.Thus, identification and validation of gene targets can now be conducted in primary cells, as the selection of cell types is not limited to those accessible by lipid-mediated transfection.

View Article: PubMed Central - PubMed

Affiliation: Lonza Cologne AG, Nattermannallee 1, 50829 Cologne, Germany.

ABSTRACT
Meaningful RNAi-based data for target gene identification are strongly dependent on the use of a biologically relevant cell type and efficient delivery of highly functional siRNA reagents into the selected cell type. Here we report the use of the Amaxa(R) Nucleofector(R) 96-well Shuttle(R) System for siRNA screening in primary cells. Lonza's Clonetics(R) HUVEC-Human Umbilical Vein Endothelial Cells were transfected with Thermo Scientific Dharmacon siGENOME(R) siRNA Libraries targeting protein kinases and cell cycle related genes and screened for genes important for cell viability. Of the 37 primary hits, down-regulation of 33 led to reduced proliferation or increased cell death, while down-regulation of two allowed for better cell viability. The validated four genes out of the 16 strongest primary hits (COPB2, PYCS, CDK4 and MYC) influenced cell proliferation to varying degrees, reflecting differing importance for survival of HUVEC cells. Our results demonstrate that the Nucleofector(R) 96-well Shuttle(R) System allows the delivery of siRNA libraries in cell types previously considered to be difficult to transfect. Thus, identification and validation of gene targets can now be conducted in primary cells, as the selection of cell types is not limited to those accessible by lipid-mediated transfection.

No MeSH data available.


Determination of optimal assay conditions. In three independent experiments, HUVEC cells were transfected with 20 pmol SMARTpool® siRNA targeting PLK-1 (A, B) or CHEK-1 (B, C) and siGENOME® non-targeting control. Cell viability was analyzed at different time points post Nucleofection® (A/B: 24, 48, 72 and 96 hrs; C: 72 hrs). Values were normalized to the negative control samples (A, B) or to untreated cells (C). The rightmost dots in C represents the mean and SD of the 60 individual values.
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Figure 1: Determination of optimal assay conditions. In three independent experiments, HUVEC cells were transfected with 20 pmol SMARTpool® siRNA targeting PLK-1 (A, B) or CHEK-1 (B, C) and siGENOME® non-targeting control. Cell viability was analyzed at different time points post Nucleofection® (A/B: 24, 48, 72 and 96 hrs; C: 72 hrs). Values were normalized to the negative control samples (A, B) or to untreated cells (C). The rightmost dots in C represents the mean and SD of the 60 individual values.

Mentions: For the kinase and cell cycle screen in HUVEC Cells, siRNA reagents targeting polo-like kinase 1 (PLK-1) and Cell Cycle Check-point Kinase 1 (CHK-1 or CHEK-1) were selected as positive controls to set up the viability assay. PLK-1 is a key regulator of mitotic progression in mammalian cells and the knock-down of PLK-1 is known to induce apoptosis in cancer cells (Spänkuch-Schmitt et al, 2002; Reagan-Shaw and Ahmad, 2005). CHEK-1 is involved in the DNA damage response and is also required for cell proliferation and survival. CHEK-1 knockdown by siRNA has been reported to induce mitotic arrest (Tang et al, 2006). As such, down-regulation of PLK-1 and/or CHEK-1 is expected to decrease cell viability. Using PLK-1, post-transfection plating densities were adjusted to allow for significant discrimination of positive and negative control samples on the phenotypic level. This was achieved by plating HUVEC cells at a low cell density of 2 × 103 per well for 3-4 days after transfection (Figure 1A). As shown in Figure 1B, the phenotypic effect for PLK-1 silencing was weaker and built up slower than silencing of CHEK-1, thus representing potential differences expected for “strong” and “weak” library targets. It has been reported earlier that PLK-1 depletion by siRNA transfection exerts a strong effect on cancer cell lines, but not primary cells (Spänkuch-Schmitt et al, 2002; Reagan-Shaw and Ahmad, 2005). However, as the down-regulation of PLK-1 mRNA was not demonstrated in the previous reports, it could not be excluded that the underlying cause was more of an issue of inefficient transfection of the siRNA rather than the significance of PLK-1 roles in cell survival in primary cells. Nevertheless, despite a PLK-1 mRNA knockdown of more than 90% (data not shown), cell survival of HUVEC cells was diminished only to approximately 50% after 96 hrs, suggesting that persistent PLK-1 may indeed be a factor in survivability of cancer cells. An analysis time point of 72 hrs suited well for both targets. In pilot screens for further determination of assay robustness, controls were plated into the central 60 wells of a 96-well culture plate and analyzed for cell viability. Wells in the outer rows were filled with medium to avoid potential edge effects. Z' factors of both positive controls (CHEK-1: 0.55; PLK-1: 0.22; Figure 1C; data for PLK-1 not shown) reflected a suitable window for discrimination of potential hits with different phenotypic strength in the subsequent screen from background.


First siRNA library screening in hard-to-transfect HUVEC cells.

Zumbansen M, Altrogge LM, Spottke NU, Spicker S, Offizier SM, Domzalski SB, St Amand AL, Toell A, Leake D, Mueller-Hartmann HA - J RNAi Gene Silencing (2009)

Determination of optimal assay conditions. In three independent experiments, HUVEC cells were transfected with 20 pmol SMARTpool® siRNA targeting PLK-1 (A, B) or CHEK-1 (B, C) and siGENOME® non-targeting control. Cell viability was analyzed at different time points post Nucleofection® (A/B: 24, 48, 72 and 96 hrs; C: 72 hrs). Values were normalized to the negative control samples (A, B) or to untreated cells (C). The rightmost dots in C represents the mean and SD of the 60 individual values.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2902142&req=5

Figure 1: Determination of optimal assay conditions. In three independent experiments, HUVEC cells were transfected with 20 pmol SMARTpool® siRNA targeting PLK-1 (A, B) or CHEK-1 (B, C) and siGENOME® non-targeting control. Cell viability was analyzed at different time points post Nucleofection® (A/B: 24, 48, 72 and 96 hrs; C: 72 hrs). Values were normalized to the negative control samples (A, B) or to untreated cells (C). The rightmost dots in C represents the mean and SD of the 60 individual values.
Mentions: For the kinase and cell cycle screen in HUVEC Cells, siRNA reagents targeting polo-like kinase 1 (PLK-1) and Cell Cycle Check-point Kinase 1 (CHK-1 or CHEK-1) were selected as positive controls to set up the viability assay. PLK-1 is a key regulator of mitotic progression in mammalian cells and the knock-down of PLK-1 is known to induce apoptosis in cancer cells (Spänkuch-Schmitt et al, 2002; Reagan-Shaw and Ahmad, 2005). CHEK-1 is involved in the DNA damage response and is also required for cell proliferation and survival. CHEK-1 knockdown by siRNA has been reported to induce mitotic arrest (Tang et al, 2006). As such, down-regulation of PLK-1 and/or CHEK-1 is expected to decrease cell viability. Using PLK-1, post-transfection plating densities were adjusted to allow for significant discrimination of positive and negative control samples on the phenotypic level. This was achieved by plating HUVEC cells at a low cell density of 2 × 103 per well for 3-4 days after transfection (Figure 1A). As shown in Figure 1B, the phenotypic effect for PLK-1 silencing was weaker and built up slower than silencing of CHEK-1, thus representing potential differences expected for “strong” and “weak” library targets. It has been reported earlier that PLK-1 depletion by siRNA transfection exerts a strong effect on cancer cell lines, but not primary cells (Spänkuch-Schmitt et al, 2002; Reagan-Shaw and Ahmad, 2005). However, as the down-regulation of PLK-1 mRNA was not demonstrated in the previous reports, it could not be excluded that the underlying cause was more of an issue of inefficient transfection of the siRNA rather than the significance of PLK-1 roles in cell survival in primary cells. Nevertheless, despite a PLK-1 mRNA knockdown of more than 90% (data not shown), cell survival of HUVEC cells was diminished only to approximately 50% after 96 hrs, suggesting that persistent PLK-1 may indeed be a factor in survivability of cancer cells. An analysis time point of 72 hrs suited well for both targets. In pilot screens for further determination of assay robustness, controls were plated into the central 60 wells of a 96-well culture plate and analyzed for cell viability. Wells in the outer rows were filled with medium to avoid potential edge effects. Z' factors of both positive controls (CHEK-1: 0.55; PLK-1: 0.22; Figure 1C; data for PLK-1 not shown) reflected a suitable window for discrimination of potential hits with different phenotypic strength in the subsequent screen from background.

Bottom Line: Of the 37 primary hits, down-regulation of 33 led to reduced proliferation or increased cell death, while down-regulation of two allowed for better cell viability.Our results demonstrate that the Nucleofector(R) 96-well Shuttle(R) System allows the delivery of siRNA libraries in cell types previously considered to be difficult to transfect.Thus, identification and validation of gene targets can now be conducted in primary cells, as the selection of cell types is not limited to those accessible by lipid-mediated transfection.

View Article: PubMed Central - PubMed

Affiliation: Lonza Cologne AG, Nattermannallee 1, 50829 Cologne, Germany.

ABSTRACT
Meaningful RNAi-based data for target gene identification are strongly dependent on the use of a biologically relevant cell type and efficient delivery of highly functional siRNA reagents into the selected cell type. Here we report the use of the Amaxa(R) Nucleofector(R) 96-well Shuttle(R) System for siRNA screening in primary cells. Lonza's Clonetics(R) HUVEC-Human Umbilical Vein Endothelial Cells were transfected with Thermo Scientific Dharmacon siGENOME(R) siRNA Libraries targeting protein kinases and cell cycle related genes and screened for genes important for cell viability. Of the 37 primary hits, down-regulation of 33 led to reduced proliferation or increased cell death, while down-regulation of two allowed for better cell viability. The validated four genes out of the 16 strongest primary hits (COPB2, PYCS, CDK4 and MYC) influenced cell proliferation to varying degrees, reflecting differing importance for survival of HUVEC cells. Our results demonstrate that the Nucleofector(R) 96-well Shuttle(R) System allows the delivery of siRNA libraries in cell types previously considered to be difficult to transfect. Thus, identification and validation of gene targets can now be conducted in primary cells, as the selection of cell types is not limited to those accessible by lipid-mediated transfection.

No MeSH data available.