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


Technology Report

J RNAi Gene Silencing (2010), 6(1), 354-360

doi: jrgsxx

Published online: 29 October 2009

Full Text: (html | pdf ~465kb | refs)

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

Markus Zumbansen †, Ludger M Altrogge †, Nicole UE Spottke †, Sonja Spicker †, Sheila M Offizier †, Sandra BS Domzalski †, Allison L St Amand ‡, Andrea Toell †, Devin Leake ‡ and Herbert A Mueller-Hartmann †*

† Lonza Cologne AG, Nattermannallee 1, 50829 Cologne, Germany

‡ Thermo Fisher Scientific, Dharmacon Products, 2650 Crescent Drive, #100, Lafayette, CO 80026, USA

*Correspondence toHerbert Mueller-Hartmann, Email: Herbert.Mueller-Hartmann@lonza.com, Tel: +49 221 99199347, Fax: +49 221 99199199

Received: 26 March 2009, Revised: 16 September 2009, Accepted: 23 September 2009

© Copyright The Authors

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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 ® Nucleofector ® 96-well Shuttle ® System for siRNA screening in primary cells. Lonza’s Clonetics ® HUVEC-Human Umbilical Vein Endothelial Cells were transfected with Thermo Scientific Dharmacon siGENOME ® 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 ® 96-well Shuttle ® 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.

KEYWORDS: Nucleofection, RNAi, siRNA, primary cell, screening, transfection, HUVEC

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