Towards Optimising the Production of and Expression from Polycistronic Vectors in Embryonic Stem Cells

Abstract

Polycistronic vectors linked by self-processing 2A peptides have been successfully used in cellular reprogramming. The expression of these vectors has yet to be well documented in embryonic stem cells. In the present study, we generated expression cassettes containing combinatorial arrangements of 3 pancreatic transcriptions factors (Pdx1, Nkx2.2 and Ngn3) together with an eGFP reporter, all linked by self-processing 2A peptides. The study tested the utility of constructing complex expression cassettes by ligating multiple components, each flanked by unique restriction sites. This approach allowed flexible and efficient design and construction of a combinatorial array of polycistronic constructs, which were expressed after transient transfection into embryonic stem cells. The inclusion of EGFP provided for a convenient proxy measure of expression and showed that expression was similar regardless of EGFP’s position within a 2A polycistronic construct. Expression of terminal EGFP was 51% and 24% more efficient when linked by T2A compared to F2A or E2A peptides, respectively. The highest level of expression was achieved when all genes in a construct were linked exclusively by T2A peptides. This effect of T2A was independent of the type of promoter used, as a similar increase in terminal EGFP expression was observed when the polycistronic constructs were under the control of a CAG promoter compared to the CMV promoter, even though the GAG promoter was more efficient in this model than the CMV promoter. The study provides guidance on design strategies and methods for the efficient generation and expression of 2A polycistronic constructs in embryonic stem cells.

Golden Gate Cloning-Compatible DNA Replicon/2A-Mediated Polycistronic Vectors for Plants

“…In theory, the 2A-mediated polycistronic system allows for the production of multiple proteins at equimolar levels, as translation is carried out from a single polycistron, and peptides linked by 2A peptide are separated via a ribosome skipping mechanism. However, it should be noted that expression levels from 2A polycistronic constructs are dependent on the combined effects of various factors, including the number of transgenes, their relative positions in the construct, the order and types of 2A peptide, 2A coding sequences, as well as regulatory elements in the expression cassette (Donnelly et al, 2001a;de Felipe et al, 2010;Rothwell et al, 2010;Gao et al, 2012;Liu et al, 2017). With regards to the effects of transgene position, the results of this study are similar to those of a previous report which showed that no significant differences in transgene expression levels were observed when varying the positions within 2A polycistronic constructs in mouse embryonic stem cells (Gao et al, 2012).…”

Polycistronic gene expression in the model micro-organismUstilago maydis

“…This allowed fast pre-screening of homologous recombination events (see Materials and methods). Since it had previously been mentioned that linker sequences influence the expression of open reading frames connected by 2A peptides (Holst et al, 2006;Gao et al, 2012;Souza-Moreira et al, 2018), we tested different linker versions. We compared assemblies without linker (no linker; NL) with those comprising a short GSG linker sequence (L1) and a longer version of 18 amino acids (L2).…”

The Quest for Targets Executing MYC-Dependent Cell Transformation

“…To simultaneously overexpress multiple genes or interfering RNAs in single cells, several established techniques exist. They are based on different principles such as co-transfection of multiple plasmids, usage of bicistronic vectors containing an internal ribosomal-binding site, infection with retroviruses containing different envelope subtypes, or self-processing peptides ( 47 , 102 ).…”