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- Title
Versatile co-expression of graft-protective proteins using 2A-linked cassettes.
- Authors
Fisicaro, Nella; Londrigan, Sarah L.; Brady, Jamie L.; Salvaris, Evelyn; Nottle, Mark B.; O'Connell, Philip J.; Robson, Simon C.; d'Apice, Anthony J. F.; Lew, Andrew M.; Cowan, Peter J.
- Abstract
Fisicaro N, Londrigan SL, Brady JL, Salvaris E, Nottle MB, O'Connell PJ, Robson SC, d'Apice AJF, Lew AM, Cowan PJ. Versatile co-expression of graft-protective proteins using 2A-linked cassettes. Xenotransplantation 2011; 18: 121-130. © 2011 John Wiley & Sons A/S. 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. 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. 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. 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.
- Subjects
XENOTRANSPLANTATION; THROMBOMODULIN; GENE transfection; CYTOLOGICAL research; WESTERN immunoblotting; LABORATORY mice
- Publication
Xenotransplantation, 2011, Vol 18, Issue 2, p121
- ISSN
0908-665X
- Publication type
Article
- DOI
10.1111/j.1399-3089.2011.00631.x