Self-cleaving peptides 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 many 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 and find that all the screened 2A sequences are effective. However, combining the coding sequences of two proteins into a single transcript leads to notable strain-dependent decreases in expression level, suggesting additional factors regulate gene expression in D. discoideum that merit further investigation. Our results show that P2A is the optimal sequence for polycistronic expression in D. discoideum, opening up new possibilities for genetic engineering in this model system.

Atlas of Fshr Expression from Novel Reporter Mice

“…The 19 amino acids P2A sequences functions to cause ribosomal " skipping " during translation, resulting in a missing peptide bond and effectively separates the two proteins, e.g. Fshr and ZsGreen 23,24,25 . The injected zygotes were transferred into oviducts of Kunming pseudopregnant females to generate F0 mice.…”

Proliferation history and transcription factor levels drive direct conversion

“…Genes at upstream positions are expected to be translated at greater frequencies than at downstream positions (Mizuguchi et al, 2000). However, relative levels of expression from polycistronic cassettes are context- and encoding-dependent (Carey et al, 2011; Kim et al, 2011; Liu et al, 2017; Rosa et al, 2018; Wang Li et al, 2015; Zhu et al, 2023). Thus, while we had identified putatively optimal profiles of ectopic expression for the three transcription factors, we hypothesized that the ordering on a polycistronic cassette may not fully prescribe expression levels.…”

A versatile 2A peptide-based strategy for ectopic expression and endogenous gene tagging in Trypanosoma cruzi

“…First, 2A remnants at the C-terminal end may have potential deleterious effects on the target protein or affect their subcellular localization [ 49 ]. On the other hand, some specific 2A peptide-selectable marker configurations have been shown not to confer appropriate antibiotic resistance such as the neomycin resistance gene positioned after the sequence for the porcine teschovirus-1 2A (P2A) in Dictyostelium discoideum cells [ 50 ]. For these reasons and given that 2A peptides differ in length and sequence, a careful design of 2A fusion constructs as well as the optional insertion of linker sequences [ 51 ], may be key aspects to improve protein expression, cleavage efficiency and consequently, the selection process.…”