Towards optimising the production of and expression from polycistronic vectors in embryonic stem cells

PLoS One. 2012;7(11):e48668. doi: 10.1371/journal.pone.0048668. Epub 2012 Nov 6.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Base Sequence
  • Cell Line
  • Embryonic Stem Cells / metabolism*
  • Gene Dosage / genetics
  • Gene Expression*
  • Genes / genetics*
  • Genetic Techniques*
  • Genetic Vectors / genetics*
  • Green Fluorescent Proteins / metabolism
  • Homeobox Protein Nkx-2.2
  • Mice
  • Molecular Sequence Data
  • Peptides / metabolism
  • Promoter Regions, Genetic

Substances

  • Homeobox Protein Nkx-2.2
  • Nkx2-2 protein, mouse
  • Peptides
  • enhanced green fluorescent protein
  • Green Fluorescent Proteins

Grant support

This work was supported in part by grants from the NHMRC to CO and by funds provided by the Favaloro bequest to the Department of Endocrinology. SG was supported by a Rotary Postgraduate Scholarship and a Lola Douglas Paediatric Research Scholarship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.