Towards Optimising the Production of and Expression from Polycistronic Vectors in Embryonic Stem Cells Steven Y. Gao1, Michelle M. Jack1,2, Christopher O’Neill1* 1 Centre for Developmental and Regenerative Medicine, Kolling Institute for Medical Research, Sydney Medical School, University of Sydney, Sydney, NSW, Australia, 2 Department of Endocrinology, Royal North Shore Hospital, Sydney, NSW, Australia 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. Citation: Gao SY, Jack MM, O’Neill C (2012) Towards Optimising the Production of and Expression from Polycistronic Vectors in Embryonic Stem Cells. PLoS ONE 7(11): e48668. doi:10.1371/journal.pone.0048668 Editor: Andre Van Wijnen, University of Massachusetts Medical, United States of America Received June 3, 2012; Accepted September 28, 2012; Published November 6, 2012 Copyright: 2012 Gao et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: 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. Competing Interests: The authors have declared that no competing interests exist. * E-mail: chris.oneill@sydney.edu.au Introduction Various approaches have been employed to co-express multiple proteins in cells. These include the use of internal ribosomal entry site (IRES) elements [1], dual promoter systems [2] or transfection of multiple vectors [3]. Each of these is associated with a number of limitations such as uneven or unreliable protein expression levels, silencing of some promoters [4,5], or increased toxicity to cells (with multiple transfections) [6]. An alternative strategy is the use of the self-processing viral 2A peptides, which are reported to allow the efficient expression of multiple genes under the control of a single promoter [7,8]. The 2A peptides are members of the cis-acting-hydrolase elements (CHYSELs) family. These elements were first identified in picornaviruses, and were subsequently found in a number of other viral systems [9]. The most studied 2A peptide sequence is from foot-and-mouth disease virus (F2A) [10]. Other commonly used 2A elements are from the equine rhinitis A virus (E2A) and Thosea asigna virus (T2A) [11]. All commonly used 2A peptides share a highly conserved 18 amino acid region responsible for cleavage. This occurs at the C- terminus of the sequence, between the 2A glycine and 2B proline [11,12]. The cleavage is achieved through a putative ‘‘ribosomal skip’’ mechanism [13]. The cleavage efficiency of the 2A peptide- linked proteins are generally high, however, it has been demonstrated that the efficiency can be influenced by the identity of the protein at the N-terminus of the peptide [5]. The cleavage efficiency can be improved by placing a Gly-Ser-Gly linker between the N-terminal protein and the 2A peptide [11]. This ‘‘flexible’’ linker creates a space between the N-terminus protein and the peptide, favouring a conformation of the peptide which facilitates efficient cleavage [14]. Self-processing 2A peptides have been used to achieve co- expression of heterogeneous proteins and show cleavage efficiency in a range of differentiated cell lines and embryonic stem cells [4,7,8,11,15,16,17]. Expression of up to five genes linked by 2A sequences under the control of a single promoter has been demonstrated [18], but the efficiency of expression of individual proteins from a polycistronic construct may be lower than their expression from monocistronic constructs [8], although a definitive reason for this reduced efficiency has not been shown. The cleavage of 2A peptides may be affected by the nature of the protein attached to its N-terminus [19,20], the order of the genes used in a specific expression cassette [21], the variant of the 2A peptide used (even if it was derived from the same peptide) [22], and the expression environment [20]. The P2A peptide is reported to have the highest cleavage efficiency in an in vivo environment [23], however, there remains relatively limited PLOS ONE | www.plosone.org 1 November 2012 | Volume 7 | Issue 11 | e48668