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Molecular Biotechnology

, Volume 56, Issue 12, pp 1100–1109 | Cite as

Construction and Quantitative Evaluation of a Dual Specific Promoter System for Monitoring the Expression Status of Stra8 and c-kit Genes

  • Mahtab Dastpak
  • Maryam M. Matin
  • Moein Farshchian
  • Yvan Arsenijevic
  • Madjid Momeni-Moghaddam
  • Sajjad Sisakhtnezhad
  • Sohrab Boozarpour
  • Hamid Reza Bidkhori
  • Mahdi Mirahmadi
  • Ahmad Reza Bahrami
Research

Abstract

Applications of genetic constructs with multiple promoters, which are fused with reporter genes and simultaneous monitoring of various events in cells, have gained special attention in recent years. Lentiviral vectors, with their distinctive characteristics, have been considered to monitor the developmental changes of cells in vitro. In this study, we constructed a novel lentiviral vector (FUM-M), containing two germ cell-specific promoters (Stra8 and c-kit), fused with ZsGreen and DsRed2 reporter genes, and evaluated its efficiency in different cells following treatments with retinoic acid and DMSO. Several cell lines (P19, GC-1 spg and HEK293T) were transduced with this vector, and functional capabilities of the promoters were verified by flow cytometry and quantitative RT-PCR. Our results indicate that FUM-M shows dynamic behavior in the presence and absence of extrinsic factors. A correlation was also observed between the function of promoters, present in the lentiviral construct and the endogenous level of the Stra8 and c-kit mRNAs in the cells. In conclusion, we recommend this strategy, which needs further optimization of the constructs, as a beneficial and practical way to screen chemical inducers involved in cellular differentiation toward germ-like cells.

Keywords

Dual promoter Lentiviral vector Dynamic behavior Stra8 c-kit 

Notes

Acknowledgments

We are very grateful to Dr. Corrine Kostic (University of Lausanne, Switzerland) for their valuable discussions and to Ali Fallah and Muhammad Irfan-maqsood for their technical assistance and critical reading of this manuscript. This study was supported by the Grant no. 27672 from Ferdowsi University of Mashhad.

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Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Mahtab Dastpak
    • 1
    • 2
  • Maryam M. Matin
    • 1
    • 2
  • Moein Farshchian
    • 1
    • 2
  • Yvan Arsenijevic
    • 3
  • Madjid Momeni-Moghaddam
    • 2
    • 5
  • Sajjad Sisakhtnezhad
    • 1
    • 2
  • Sohrab Boozarpour
    • 1
    • 2
  • Hamid Reza Bidkhori
    • 4
  • Mahdi Mirahmadi
    • 4
  • Ahmad Reza Bahrami
    • 1
    • 2
  1. 1.Department of Biology, Faculty of ScienceFerdowsi University of Mashhad MashhadIran
  2. 2.Cell and Molecular Biotechnology Research Group, Institute of BiotechnologyFerdowsi University of MashhadMashhadIran
  3. 3.Unit of Gene Therapy and Stem Cell Biology, Service of Ophthalmology, Jules-Gonin Eye HospitalUniversity of LausanneLausanneSwitzerland
  4. 4.Department of Stem Cells and Regenerative MedicineACECRMashhadIran
  5. 5.Department of Biology, Faculty of SciencesHakim Sabzevari UniversitySabzevarIran

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