Overexpression of urokinase receptor in mammalian cells following administration of the in vitro transcribed encoding mRNA.
The ability to overexpress physiologically important proteins in cultured mammalian cells after delivering the encoding mRNAs could have important applications for analyzing their in vivo functions. To explore the potential of this approach, urokinase-type plasminogen activator receptor (uPAR), a membrane protein extensively modified post-translationally, was selected. The uPAR-encoding mRNAs, containing different 5' and 3' untranslated regions (UTR) were tested in cultured human osteosarcoma (HOS) cells following a cationic lipid-mediated delivery. The most effective structure was the capped and polyadenylated transcript containing Xenopus beta-globin 5' and 3' UTRs. Delivering this mRNA to HOS cells resulted in a significant increase of uPAR expression in 89% of the cells, measured by flow cytometry. Using a radioligand binding assay, the increase in functional uPAR levels was found to be up eight- to 11-fold between 8 and 48 h and up three-fold at 72 h after delivery. A similar increase in uPAR levels was achievable in a number of mammalian cell lines. Surprisingly, poly(A)-tailed mRNA leading to a uPAR production highest in magnitude and duration did not demonstrate increased intracellular stability compared with other tested mRNAs. Thus, the exceptional translational performance is not likely the result of an increased mRNA half-life. These results demonstrate that, after delivery of selected mRNAs into mammalian cells, immediate and significant overexpression of a post-translationally modified protein is achievable.
Karikó
K
K
Department of Neurosurgery, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA.
Kuo
A
A
Barnathan
E
E
D015152:Blotting, Northern; D005434:Flow Cytometry; D006801:Humans; D008081:Liposomes; D012333:RNA, Messenger / Q000235:genetics*; D011956:Receptors, Cell Surface / Q000378:metabolism*; D055293:Receptors, Urokinase Plasminogen Activator; D014158:Transcription, Genetic; D014162:Transfection
D008081:Liposomes; C524898:PLAUR protein, human; D012333:RNA, Messenger; D011956:Receptors, Cell Surface; D055293:Receptors, Urokinase Plasminogen Activator