Enrichment strategies for siRNA-transfected cells in an untransfected background.
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 24h 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.
Narz
Frank
F
QIAGEN, QIAGEN Str. 1, 40724 Hilden, Germany.
Hübner
Silke
S
Magyar
Silvia
S
Bielke
Wolfgang
W
Weber
Martin
M
Dennig
Jörg
J
D015704:CD4 Antigens / Q000378:metabolism; D045744:Cell Line, Tumor; D004351:Drug Resistance / Q000235:genetics; D006801:Humans; D034741:RNA, Small Interfering / Q000235:genetics* / Q000378:metabolism*; D014162:Transfection / Q000379:methods*
D015704:CD4 Antigens; D034741:RNA, Small Interfering