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

Area of Science:

• Xenotransplantation research
• Genetic engineering and molecular biology
• Immunology and regenerative medicine

Background:

• Achieving expression of multiple graft-protective proteins in xenotransplantation is crucial.
• The 2A

Purpose of the Study:

• To assess the efficiency of the 2A system for co-expressing multiple graft-protective proteins.
• To investigate the impact of protein position on 2A-linked expression and trafficking.
• To evaluate the utility of incorporating a selection marker into 2A constructs.

Main Methods:

• Constructed six expression cassettes with up to four 2A-linked genes (CD55, thrombomodulin, CD39, CTLA4-Ig, hygromycin resistance).
• Incorporated furin cleavage sites into linkers for improved protein processing.
• Utilized transfection, adenoviral transduction, and transgenesis for vector delivery.
• Detected protein expression via flow cytometry and Western blotting.

Main Results:

• All proteins were expressed in correct locations after transient transfection, regardless of gene linkage.
• Including hygromycin resistance gene increased stable transfectant expression 10-fold (4-5% to 58-67%).
• Transgenic pig fibroblasts and adenovirally transduced mouse cells demonstrated successful surface expression of linked proteins.

Conclusions:

• The 2A system is versatile for co-expressing multiple proteins.
• Careful construct design mitigates issues with post-translational modification and trafficking.
• Selection markers integrated into 2A constructs significantly enhance stable transfectant generation for genetic modification.