Enrichment strategies for siRNA-transfected cells in an untransfected background

• Published in: Journal of biotechnology Vol. 130; no. 3; pp. 209 - 212
• Main Authors: Narz, Frank, Hübner, Silke, Magyar, Silvia, Bielke, Wolfgang, Weber, Martin, Dennig, Jörg
• Format: Journal Article
• Language: English
• Published: Lausanne Elsevier B.V 30.06.2007; Amsterdam Elsevier; New York, NY
• Subjects: Biological and medical sciences; Biotechnology; CD4 Antigens - metabolism; Cell Line, Tumor; Cell separation; Drug Resistance - genetics; Fundamental and applied biological sciences. Psychology; Humans; RNA, Small Interfering - genetics; RNA, Small Interfering - metabolism; siRNA; Transfection; Transfection - methods; MAPK1; siRNA; Transfection; Cell separation; GAPDH; Small Interference RNA; Separation; RNA interference
• ISSN: 0168-1656; 1873-4863
• DOI: 10.1016/j.jbiotec.2007.04.018

Gene silencing experiments in difficult-to-transfect cells are often hampered by the presence of a background of untransfected cells. We present proof-of-concept data from two different strategies for enrichment of siRNA-transfected cells. In the first approach, a heterologous surface antigen is expressed from a plasmid that is co-transfected with an siRNA targeting an endogenous mRNA. The surface antigen is then used for enrichment of successfully transfected cells using antibody-coated magnetic particles. In the second strategy, a eukaryotic antibiotic resistance gene is expressed from a co-transfected plasmid. Addition of the corresponding antibiotic 24 h after transfection results in killing of untransfected cells, which can be washed away. Elimination of untransfected cells will allow more accurate interpretation of the effects of gene silencing.