Self-cleaving 2A peptides allow for expression of multiple genes in Dictyostelium discoideum

Abstract

The social amoeba Dictyostelium discoideum is a model for a wide range of biological processes including chemotaxis, cell-cell communication, phagocytosis, and development. Interrogating these processes with modern genetic tools often requires the expression of multiple transgenes. While it is possible to transfect multiple transcriptional units, the use of separate promoters and terminators for each gene leads to large plasmid sizes and possible interference between units. In other eukaryotic systems this challenge has been addressed through polycistronic expression mediated by 2A viral peptides, permitting efficient, co-regulated gene expression. Here, we screen the most commonly used 2A peptides, porcine teschovirus-1 2A (P2A), Thosea asigna virus 2A (T2A), equine rhinitis A virus 2A (E2A), and foot-and-mouth disease virus 2A (F2A), for activity in D. discoideum. We find that these 2A sequences are effective but that, unexpectedly, combining the coding sequences of two proteins into a single transcript leads to notable decreases in expression level with strain-dependent magnitudes, suggesting unexplored constraints of gene expression in D. discoideum that merit further investigation. Our results show that P2A is the optimal sequence for polycistronic expression, opening up new possibilities for genetic engineering in this model system.

Single cell discovery of m6A RNA modifications in the hippocampus

“…S1A). We argued that this low signal is because Egfp is located at the C-terminal of one long RNA transcript(Zhu et al 2023). We therefore changed the order of the expression cassette on either side of P2A, moving the Egfp to the N-terminus, to make the Egfp - P2A - Apobec1 - Yth - HA ( E-Yth ) and Egfp - P2A - Apobec1 - Yth mut - HA ( E - Yth mut ) plasmids.…”

Protective efficacy of a recombinant adenovirus expressing novel dual F and HN proteins of bovine parainfluenza virus type 3

“…P2A is capable of e ciently cleaving adjacent proteins through ribosomal skipping, with a high e ciency rate of 99%. This technique was commonly employed in the construction of multi-gene expression vectors [22], which had been successfully utilized in the construction of genetic engineering vaccines [23,24].…”