Stable suppression of MDR1 gene expression and function by RNAi in Caco-2 cells.
Vector-based RNAi was used to establish a stable Caco-2 cell line with a persistent knockdown of multidrug resistant gene 1 (MDR1) and P-glycoprotein (P-gp). Several positive clones were collected, many of which showed significantly reduced levels of MDR1 mRNA and P-gp compared to wt Caco-2 cells. Selected clones were sub-cultivated for six passages and real-time PCR showed that MDR1 expression remained significantly reduced (up to 96%) over this period of time. RNAi-MDR1 clones frozen long term also kept their low MDR1 expression levels when re-cultured. Permeability studies were performed across RNAi-MDR1 clone cell monolayers, and the efflux of cyclosporine A, digoxin, vinblastine, and vincristine showed 58%, 61%, 91%, and 78% decrease in active transport, respectively, compared to wt Caco-2 cells. This stably modified Caco-2 cell line provides a novel tool for studies on MDR1 and other ABC transporter protein gene cellular functions.
Biovitrum AB, In Vitro Sciences, Preclinical R&D, Lindhagensgatan 133, SE-112 76 Stockholm, Sweden. trine.celius@biovitrum.com <trine.celius@biovitrum.com>
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Celius
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Garberg
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Lundgren
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics* / Q000378:metabolism*; D000818:Animals; D018938:Caco-2 Cells; D002463:Cell Membrane Permeability; D005786:Gene Expression Regulation; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D006801:Humans; D009690:Nucleic Acid Conformation; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D034741:RNA, Small Interfering