Rational Development of A Polycistronic Plasmid with A CpG-Free Bacterial Backbone as A Potential Tool for Direct Reprogramming

(Pages: 565-581)

Kianoush Dormiani, Pharm.D, ^1,2Hamid Mir Mohammad Sadeghi, Ph.D, ¹Hojjat Sadeghi-Aliabadi, Ph.D, ¹Mahboobeh Forouzanfar, M.Sc, ²Hossein Baharvand, Ph.D, ^3,4Kamran Ghaedi, Ph.D, ^2,5Mohammad Hossein Nasr-Esfahani, Ph.D, ^2,*

1-Department of Pharmaceutical Biotechnology and Isfahan Pharmaceutical Sciences Research Center, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, Iran

2-Department of Molecular Biotechnology, Cell Science Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran

3-Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran

4-Department of Developmental Biology, University of Science and Culture, ACECR, Tehran, Iran

5-Department of Biology, School of Sciences, University of Isfahan, Isfahan, Iran

*Corresponding Address: P.O.Box: 816513-1378 Department of Molecular Biotechnology Cell Science Research Center Royan Institute for Biotechnology ACECR Isfahan Iran Email:mh_nasr@royaninstitute.org

Any use, distribution, reproduction or abstract of this publication in any medium, with the exception of commercial purposes, is permitted provided the original work is properly cited 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 work is properly cited.

Dormiani Kianoush, Mir Mohammad Sadeghi Hamid, Sadeghi-Aliabadi Hojjat, Forouzanfar Mahboobeh, Baharvand Hossein, Ghaedi Kamran, Nasr-Esfahani Mohammad Hossein. Rational Development of A Polycistronic Plasmid with A CpG-Free Bacterial Backbone as A Potential Tool for Direct Reprogramming. Cell J. 2017; 18(4): 565-581.

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.

Materials and Methods

In this experimental study, we developed a single plasmid which includes a single expression cassette containing open reading frames of human LIN28, NANOG, SOX2 and OCT4 along with an EGFP reporter 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 the E. coli GT115 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.

Conclusion

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.

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