T2a Peptide-Linked Adenoviral Vectors for Polycistronic Gene Expression

t2a peptide

The t2a peptide is an auto-cleaving peptide that is found in the viral genome of many Picornaviridae viruses, including rotavirus. This peptide has two key advantages over the traditional IRES sequence: it is short (60 bp) and provides stoichiometric expression of genes flanking it. However, the t2a peptide has not yet been widely used in polycistronic gene expression due to limited availability and complex cloning procedures.

In our previous studies, we showed that the t2a peptide can efficiently drive transgene expression in human cultured cells and zebrafish embryos. In this study, we tested whether the results from our zebrafish and human cell line experiments would also be reproduced in mouse liver tissue using a t2a peptide-linked adenoviral vector.

First, we established a set of Golden Gate cloning-compatible modular vectors for assembling 2A peptide-linked tricistronic expression cassettes. Each module contains an inward-facing two BsaI restriction enzyme sites and a customizable 4 bp-overhang to enable directional linear assembly in plants.

We then selected oligonucleotides encoding the t2a peptide P2A, T2A, E2A and F2A from Bioneer (Daejeon, Korea) and individually inserted them into the SphI/BglII sites of a pCS4+ plasmid. The resulting pCS4+-2A constructs were digested with BglII and SphI and the 2A peptide and CDS overlapping fragments were ligated using the Gibson method.

The recombinant adenovirus was electroporated into HEK293 A packaging cells and amplified through four freeze-thaw-vortex cycles. The resulting infectious adenovirus was then titrated and injected into mouse tail veins. Western blot and confocal microscopy analyses revealed that P2A has the highest cleavage efficiency in mouse liver, followed by T2A and then E2A.


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