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Versatile co-expression of graft-protective proteins using 2A-linked cassettes

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dc.contributor.authorFisicaro, N.
dc.contributor.authorLondrigan, S.
dc.contributor.authorBrady, J.
dc.contributor.authorSalvaris, E.
dc.contributor.authorNottle, M.
dc.contributor.authorO'Connell, P.
dc.contributor.authorRobson, S.
dc.contributor.authord'Apice, A.
dc.contributor.authorLew, A.
dc.contributor.authorCowan, P.
dc.date.issued2011
dc.description.abstractBackground: Expression of multiple graft-protective proteins targeted to different locations (i.e., intracellular, cell surface, and secreted) has become an increasingly important goal in xenotransplantation. The 2A “ribosome skip” signal is used as a linker to enable transgene co-expression, but some studies have shown that post-translational modification and trafficking of 2A-linked proteins may be adversely affected depending on their position relative to 2A. We tested whether several relevant proteins, subject to a range of processing and localization mechanisms, could be efficiently co-expressed using the 2A system. Methods: Six expression cassettes were constructed, each containing up to four 2A-linked open reading frames, encoding combinations of human CD55, thrombomodulin (TBM), CD39, CTLA4-Ig and hygromycin resistance. Each linker incorporated a furin cleavage site to remove the carboxy-terminal extension that remains on upstream proteins after 2A processing. The cassettes were used to produce vectors for transfection, adenoviral transduction and transgenesis. Expression was detected by flow cytometry and/or Western blotting. Results: All proteins were expressed in the appropriate location following transient transfection of COS-7 cells, irrespective of the number of linked genes. The percentage of stable transfectants expressing a linked gene was increased 10-fold (from 4–5% to 58–67%) by incorporating the hygromycin resistance gene into the cassette. Stable transfection of transgenic GalT KO pig fibroblasts with a hygromycin- TBM-CD39 construct resulted in surface expression of both TBM and CD39 by the majority of hygromycin-resistant cells. Expression was maintained after flow cytometric sorting and expansion. Adenoviral transduction of NIT-1 mouse insulinoma cells with a TBM-CD39 construct resulted in strong expression of both genes on the cell surface. Mice transgenic for 3-gene (CD55- TBM-CD39) or 4-gene (CD55- TBM-CTLA4Ig-CD39) constructs expressed all genes except CD55. Conclusions: These results confirm the versatility of the 2A system, and demonstrate that careful construct design can minimize potential problems with post-translational modification and trafficking. In addition, incorporation of a selection marker into the 2A-linked chain can dramatically increase the proportion of stable transfectants expressing proteins of interest. This provides a powerful method for the rapid modification of existing genetically modified pigs.
dc.description.statementofresponsibilityNella Fisicaro, Sarah L. Londrigan, Jamie L. Brady, Evelyn Salvaris, Mark B. Nottle, Philip J. O'Connell, Simon C. Robson, Anthony J. F. d'Apice, Andrew M. Lew and Peter J. Cowan
dc.identifier.citationXenotransplantation, 2011; 18(2):121-130
dc.identifier.doi10.1111/j.1399-3089.2011.00631.x
dc.identifier.issn0908-665X
dc.identifier.issn1399-3089
dc.identifier.orcidNottle, M. [0000-0001-7625-5542]
dc.identifier.urihttp://hdl.handle.net/2440/68515
dc.language.isoen
dc.publisherBlackwell Munksgaard
dc.rights© 2011 John Wiley & Sons A/S
dc.source.urihttps://doi.org/10.1111/j.1399-3089.2011.00631.x
dc.subject2A
dc.subjectgenetic modification
dc.subjecttransgene co-expression
dc.subjectxenotransplantation
dc.titleVersatile co-expression of graft-protective proteins using 2A-linked cassettes
dc.typeJournal article
pubs.publication-statusPublished

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