Dormiani Kianoush, Mir Mohammad Sadeghi Hamid, Sadeghi-Aliabadi Hojjat, Forouzanfar Mahboobeh, Baharvand Hossein, Ghaedi Kamran, Nasr-Esfahani Mohammad Hossein
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.
Department of Pharmaceutical Biotechnology and Isfahan Pharmaceutical Sciences Research Center, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, Iran.
Cell J. 2017 Winter;18(4):565-581. doi: 10.22074/cellj.2016.4723. Epub 2016 Sep 26.
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.
In this experimental study, we developed a single plasmid which includes a single expression cassette containing open reading frames of human and along with an 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 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.
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.
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.
诱导多能干细胞是通过直接重编程从体细胞产生的。这些重编程的多能细胞在再生医学等生物医学领域有不同的应用。尽管病毒载体被广泛用于高效重编程，但由于存在免疫原性和插入诱变的风险，它们在临床上的应用有限。因此，我们设计并开发了一种名为pLENSO/Zeo的小型非整合质粒作为一种2A介导的多顺反子表达载体。
在本实验研究中，我们开发了一种单一质粒，其包含一个单一表达盒，该表达盒包含人类 和 的开放阅读框以及一个 报告基因。每个重编程因子由编码2A自加工肽的间隔序列隔开。重编程盒位于CMV启动子的下游。该载体通过不含CpG的载体骨架在GT115菌株中易于扩增。我们通过生物信息学评估了构建载体的稳定性，并在瞬时转染到HEK293细胞后，使用定量分子方法分析其重编程因子化学计量表达的能力。
在本研究中，我们开发了一种名为pLENSO/Zeo的非病毒附加体质粒。我们的结果证明了质粒DNA的总体结构稳定性。这种相对较小的载体显示出四种重编程因子以相似的滴度同时高水平表达，这被认为是高效且一致重编程的关键参数。
根据我们的实验结果，这种稳定的染色体外质粒可同时表达可靠量的四种重编程因子。因此，这些有前景的结果促使我们未来评估pLENSO/Zeo作为从原代细胞生成诱导多能干细胞的简单可行工具的能力。
