Design, Construction and Functional Evaluation of a Polycistronic Episomal Vector with The Aim of Human iPS Cell Induction

Background: Stem cell biology and personalized regenerative medicine are enormously influenced by induced pluripotent stem cells (iPSCs). Several approaches are accomplished for iPSCs induction. Amongst, nonviral episomal vectors provide a relatively easy, low-cost and low-risk way of introducing repro-gramming factors into the somatic cells. However, compared to integrated approaches, the efficiency of this method is very low. In the current study, we have constructed a polycistronic extrachromosomal plasmid containing Woodchuck Hepatitis Virus Posttranscriptional Regulatory (WPRE) element, which improves the efficiency of iPSCs generation.Materials and Methods: We developed a polycistronic plas-mid containing a single expression cassette with four human pluripotency transcription factors (LIN28, NANOG, SOX2, and OCT4) along with the EGFP reporter gene through using a 2A peptide sequence. WPRE was cloned in downstream of described polycistronic fragment, in a separate construct. These two plasmids were transfected into target cells and EGFP ex-pression was assessed at defined time points by fluorescent mi-croscopy and flow cytometry. The mRNA and Proteins Expres-sion Levels were determined by RT-qPCR and Western blotting. Results: Analysis of transfected cells showed that the expression level of proteins significantly increased in the presence of WPRE, which in turn gave rise to improvement in the ef-ficiency of iPSCs induction. Also, a similar amount of repro-gramming factor’s expression was evaluated by Western blot assay demonstrated an equivalent stoichiometric expression of 2a-mediated factors.Conclusion: We developed a stabilized extrachromosomal polycistronic plasmid as a simple and feasible tool for efficient iPSCs induction. This relatively small vector showed concomi-tant, high-level expression of the four reprogramming factors with similar titers, which are considered as the critical param-eters for efficient and consistent reprogramming. Moreover, Utilized WPRE element in the expression cassette increased the mRNA stability and consequently the yield of protein expres-sion in the target cell.

کلیدواژه ها:

iPSCs ، Extrachromosomal Plasmid ، Polycistronic ، 2A Peptide ، WPRE Element