title: Rational Development of A Polycistronic Plasmid with A CpG-Free Bacterial Backbone as A Potential Tool for Direct Reprogramming.
abstract: OBJECTIVE
  Induced pluripotent stem cells are generated from somatic cells by direct reprogramming. These reprogrammed pluripotent cells have different applications in biomedical fields such as regenerative medicine. Although viral vectors are widely used for efficient reprogramming, they have limited applications in the clinic due to the risk for immunogenicity and insertional mutagenesis. Accordingly, we designed and developed a small, non-integrating plasmid named pLENSO/Zeo as a 2A-mediated polycistronic expression vector.
  
  
  METHODS
  In this experimental study, we developed a single plasmid which includes a single expression cassette containing open reading frames of humanLIN28, NANOG, SOX2andOCT4along with anEGFPreporter gene. Each reprogramming factor is separated by an intervening sequence that encodes a 2A self-processing peptide. The reprogramming cassette is located downstream of a CMV promoter. The vector is easily propagated in theE. coliGT115 strain through a CpG-depleted vector backbone. We evaluated the stability of the constructed vector bioinformatically, and its ability to stoichiometric expression of the reprogramming factors using quantitative molecular methods analysis after transient transfection into HEK293 cells.
  
  
  RESULTS
  In the present study, we developed a nonviral episomal vector named pLENSO/ Zeo. Our results demonstrated the general structural stability of the plasmid DNA. This relatively small vector showed concomitant, high-level expression of the four reprogramming factors with similar titers, which are considered as the critical parameters for efficient and consistent reprogramming.
  
  
  CONCLUSIONS
  According to our experimental results, this stable extrachromosomal plasmid expresses reliable amounts of four reprogramming factors simultaneously. Consequently, these promising results encouraged us to evaluate the capability of pLENSO/Zeo as a simple and feasible tool for generation of induced pluripotent stem cells from primary cells in the future.
authors.affiliation: Department of Pharmaceutical Biotechnology and Isfahan Pharmaceutical Sciences Research Center, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, Iran; Department of Molecular Biotechnology, Cell Science Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran.
authors.forename: Kianoush
authors.initials: K
authors.lastname: Dormiani
authors.affiliation: Department of Pharmaceutical Biotechnology and Isfahan Pharmaceutical Sciences Research Center, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, Iran.
authors.forename: Hamid
authors.initials: H
authors.lastname: Mir Mohammad Sadeghi
authors.affiliation: Department of Pharmaceutical Biotechnology and Isfahan Pharmaceutical Sciences Research Center, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, Iran.
authors.forename: Hojjat
authors.initials: H
authors.lastname: Sadeghi-Aliabadi
authors.affiliation: Department of Molecular Biotechnology, Cell Science Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran.
authors.forename: Mahboobeh
authors.initials: M
authors.lastname: Forouzanfar
authors.affiliation: Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran; Department of Developmental Biology, University of Science and Culture, ACECR, Tehran, Iran.
authors.forename: Hossein
authors.initials: H
authors.lastname: Baharvand
authors.affiliation: Department of Molecular Biotechnology, Cell Science Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran; Department of Biology, School of Sciences, University of Isfahan, Isfahan, Iran.
authors.forename: Kamran
authors.initials: K
authors.lastname: Ghaedi
authors.affiliation: Department of Molecular Biotechnology, Cell Science Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran.
authors.forename: Mohammad Hossein
authors.initials: MH
authors.lastname: Nasr-Esfahani
keywords: 2A Peptide; CpG Dinucleotide; Extrachromosomal Plasmid; Polycistronic; Reprogramming