Rational Development of A Polycistronic Plasmid with A CpG-Free Bacterial Backbone as A Potential Tool for Direct Reprogramming
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 li...
| Main Authors: | , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Royan Institute (ACECR), Tehran
2016-10-01
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| Series: | Cell Journal |
| Subjects: | |
| Online Access: | http://celljournal.org/journal/article/6882/download |
| Summary: | 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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| ISSN: | 2228-5806 2228-5814 |