title: Development of a siRNA and shRNA screening system based on a kinase fusion protein.
abstract: RNA interference (RNAi) is one of the processes in the cell that regulates mRNA expression levels. RNAi can be exploited to experimentally knockdown the expression of one or more genes in cell lines or even in cells in vivo and also became an interesting tool to develop new therapeutic approaches. One of the major challenges of using RNAi is selecting effective shRNAs or siRNAs that sufficiently down-regulate the expression of the target gene. Here, we describe a system to select functional shRNAs or siRNAs that makes use of the leukemia cell line Ba/F3 that is dependent on the expression of a mutant form of the PDGFRα kinase for its proliferation and survival. The basis of this system is the generation of an expression construct, where part of the open reading frame of the gene of interest is linked to the mutant PDGFRα. Thus, shRNAs or siRNAs that effectively target the gene of interest also result in a reduction of the expression of the mutant PDGFRα protein, which can be detected by a reduction of the proliferation of the cells. We demonstrate that this validation system can be used for the selection of effective siRNAs as well as shRNAs. Unlike other systems, the system described here is not dependent on obtaining high-transduction efficiencies, and nonspecific effects of the siRNAs or shRNAs can be detected by comparing the effects in the presence or absence of the growth factor interleukin-3.
authors.affiliation: Center for the Biology of Disease, VIB, B-3000 Leuven, Belgium.
authors.forename: Sofie
authors.initials: S
authors.lastname: Pieraets
authors.forename: Luk
authors.initials: L
authors.lastname: Cox
authors.forename: Olga
authors.initials: O
authors.lastname: Gielen
authors.forename: Jan
authors.initials: J
authors.lastname: Cools
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D006801:Humans; D007377:Interleukin-3 / Q000378:metabolism; D051379:Mice; D016366:Open Reading Frames; D034741:RNA, Small Interfering / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D017479:Receptors, Platelet-Derived Growth Factor / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D007377:Interleukin-3; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; C099852:PDGF receptor tyrosine kinase; D017479:Receptors, Platelet-Derived Growth Factor