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

Fisicaro, N; Londrigan, SL; Brady, JL; Salvaris, E; Nottle, MB; O&#39;Connell, PJ; Robson, SC; d&#39;Apice, AJF; Lew, AM; Cowan, PJ

Author(s): Fisicaro, N; Londrigan, SL; Brady, JL; Salvaris, E; Nottle, MB; O'Connell, PJ; Robson, SC; d'Apice, AJF; Lew, AM; Cowan, PJ;
Details: Publication Year 2011-03,Volume 18,Issue #2,Page 121-130
Journal Title: XENOTRANSPLANTATION
Publication Type: Journal Article
Abstract: Background: 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.
Publisher: WILEY-BLACKWELL
Keywords: THERAPEUTIC LEVELS; MULTIPLE PROTEINS; 2A PEPTIDE; CELL LINE; IN-VIVO; EXPRESSION; ANTIBODY; VECTOR; PIGS; DELIVERY
Publisher's Version: https://doi.org/10.1111/j.1399-3089.2011.00631.x
Terms of Use/Rights Notice: Refer to copyright notice on published article.

Creation Date: 2011-03-01 12:00:00