title: Versatile co-expression of graft-protective proteins using 2A-linked cassettes.
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.
authors.affiliation: Immunology Research Centre, St Vincent's Hospital, Melbourne, Victoria, Australia.
authors.forename: Nella
authors.initials: N
authors.lastname: Fisicaro
authors.forename: Sarah L
authors.initials: SL
authors.lastname: Londrigan
authors.forename: Jamie L
authors.initials: JL
authors.lastname: Brady
authors.forename: Evelyn
authors.initials: E
authors.lastname: Salvaris
authors.forename: Mark B
authors.initials: MB
authors.lastname: Nottle
authors.forename: Philip J
authors.initials: PJ
authors.lastname: O'Connell
authors.forename: Simon C
authors.initials: SC
authors.lastname: Robson
authors.forename: Anthony J F
authors.initials: AJ
authors.lastname: d'Apice
authors.forename: Andrew M
authors.initials: AM
authors.lastname: Lew
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Cowan
mesh_terms: D000069594:Abatacept; D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D030801:Animals, Genetically Modified; D015703:Antigens, CD / Q000235:genetics* / Q000378:metabolism; D001081:Apyrase / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D018958:CD55 Antigens / Q000235:genetics* / Q000378:metabolism; D019556:COS Cells; D045744:Cell Line, Tumor; D002522:Chlorocebus aethiops; D004251:DNA Transposable Elements / Q000235:genetics*; D005347:Fibroblasts / Q000378:metabolism / Q000473:pathology; D005700:Galactosyltransferases / Q000235:genetics; D005786:Gene Expression Regulation; D055786:Gene Knockout Techniques; D006085:Graft Survival / Q000235:genetics*; D006801:Humans; D018796:Immunoconjugates / Q000235:genetics* / Q000378:metabolism; D007340:Insulinoma / Q000378:metabolism / Q000473:pathology; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D010190:Pancreatic Neoplasms / Q000378:metabolism / Q000473:pathology; D011499:Protein Processing, Post-Translational; D013552:Swine; D018180:Thrombomodulin / Q000235:genetics* / Q000378:metabolism; D014162:Transfection; D014183:Transplantation, Heterologous / Q000379:methods*
chemical_list: D015703:Antigens, CD; D018958:CD55 Antigens; D004251:DNA Transposable Elements; D018796:Immunoconjugates; D018180:Thrombomodulin; D000069594:Abatacept; D005700:Galactosyltransferases; C530267:alpha-1,3-galactosyltransferase 1, porcine; D001081:Apyrase; C067869:CD39 antigen