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Construction of a new minicircle DNA carrying an enhanced green florescent protein reporter gene for efficient expression into mammalian cell lines

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Abstract

The presence of a bacterial backbone in conventional eukaryotic expression plasmids may cause undesirable effects by triggering the immune responses in mammals and repression of episomal transgene expression. To avoid these problems, researchers have proposed the use of minicircle DNAs which are episomal vectors that have lost their bacterial backbone using a site-specific recombinase mediated recombination. In the present study, we have constructed a new minicircle DNA vector that carries an enhanced green florescent protein (EGFP) reporter gene using phage ΦC31 integrase-mediated recombination and homing endonuclease ISceI-mediated purification in E. coli. ΦC31 integrase expression was under the control of the araBAD promoter, whereas ISceI endonuclease was controlled by the tac promoter. This vector was transfected into CHO-K1 cells, which showed transient expression of EGFP up to 14 generations. Similar results were obtained upon transient transfection into HEK cells. In addition, PCR results on genomic DNA, demonstrated the EGFP-minicircle was episomal and did not integrate into the host genome. Our constructed parental plasmid expresses EGFP and could be used for the generation of episomal minicircle DNA with intent to carry out transient transfection of interested DNA fragments into the eukaryotic cells for various purposes.

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Acknowledgments

This study was funded by a Grant-in-aid of research from Royan Institute for Biotechnology in support of Nafiseh Sanei Ata-Abadi for obtaining her M.Sc. degree from the University of Isfahan.

Conflict of interest

None of the authors has any conflicts of interest to disclose and all authors support submission to this journal.

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Authors and Affiliations

  1. Department of Biology, School of Sciences, University of Isfahan, Isfahān, Iran

    Nafiseh Sanei Ata-Abadi & Kamran Ghaedi

  2. Department of Molecular Biotechnology at Cell Science Research Center, Royan Institute for Biotechnology, ACECR, Royan Street, Salman Street, Khorasgan, 81651-31378, Isfahān, Iran

    Nafiseh Sanei Ata-Abadi, Kianoush Dormiani, Yahya Khazaie, Kamran Ghaedi, Mahboobeh Forouzanfar, Liana Lachinani, Naeimeh Rezaei, Abbas Kiani-Esfahani & Mohammad Hossein Nasr-Esfahani

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  1. Nafiseh Sanei Ata-Abadi
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  2. Kianoush Dormiani
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  5. Mahboobeh Forouzanfar
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  8. Abbas Kiani-Esfahani
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  9. Mohammad Hossein Nasr-Esfahani
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Corresponding authors

Correspondence to Kamran Ghaedi or Mohammad Hossein Nasr-Esfahani.

Electronic supplementary material

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11033_2015_3864_MOESM1_ESM.doc

Supplementary Fig. 3 Dot blot of flow cytometry to measure the EGFP intensity.Flow cytometry was carried out to compare the rate of green fluorescence in transfected mycoplasma free HEK293 cells with semi-purified minicircle product (a) and in vitro completely purified minicircle (b).Supplementary material 1 (DOC 29 kb)

11033_2015_3864_MOESM2_ESM.tif

Supplementary Fig. 1 Dot blot of flow cytometry to measure the EGFP intensity.Flow cytometry was carried out to determine the rate of transfected CHO cells expressing EGFP. Please see materials and methods section for further elucidationSupplementary material 2 (TIFF 52 kb)

11033_2015_3864_MOESM3_ESM.tif

Supplementary Fig. 2 Dot blot of flow cytometry to measure the EGFP intensity.Flow cytometry was carried out to determine the rate of green fluorescence in transfected HEK cells different days post transfection as described in Materials and MethodsSupplementary material 3 (TIFF 588 kb)

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Sanei Ata-Abadi, N., Dormiani, K., Khazaie, Y. et al. Construction of a new minicircle DNA carrying an enhanced green florescent protein reporter gene for efficient expression into mammalian cell lines. Mol Biol Rep 42, 1175–1185 (2015). https://doi.org/10.1007/s11033-015-3864-z

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  • DOI: https://doi.org/10.1007/s11033-015-3864-z

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