First siRNA library screening in hard-to-transfect HUVEC cells.
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.
Zumbansen
Markus
M
Lonza Cologne AG, Nattermannallee 1, 50829 Cologne, Germany.
Altrogge
Ludger M
LM
Spottke
Nicole Ue
NU
Spicker
Sonja
S
Offizier
Sheila M
SM
Domzalski
Sandra Bs
SB
St Amand
Allison L
AL
Toell
Andrea
A
Leake
Devin
D
Mueller-Hartmann
Herbert A
HA