A vector-based system for the differentiation of mouse embryonic stem cells toward germ-line cells

Authors

  • Karim Nayernia Institute of Human Genetics, North East England, Stem Cell Institute, International Center for Life, Newcastle University, Newcastle, UK
  • Mohammad Ali hokrgozar National Cell Bank of Iran, Pasteur Institute of Iran, Tehran, Iran
  • Mohammad Hosein Modarressi Department of Medical Genetics, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran
  • Mohammad Miryounesi Genomic Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran
  • Najmeh Ranji Department of Genetics, Faculty of Sciences, Islamic Azad University, Rasht Branch, Rasht, Iran
  • Reza Ebrahimzadeh-Vesal Department of Medical Genetics, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran
  • Seyedmehdi Nourashrafeddin Magee-Womens Research Institute & Foundation, University of Pittsburgh Medical Sciences, Pittsburgh, PA 15213, USA
Abstract:

Objective(s):To culture thein vitro mouse embryonic stem cells (mESCs) and to direct their  differentiation to germ-line cells; in present study we used a vector backbone containing the fusion construct Stra8-EGFP to select differentiated ES cells that entered meiosis.  Retinoic acid was used to differentiate embryonic stem cells to germ cells. Materials and Methods: A fragment of Stra8 gene promoter (-1400 to +7) was inserted in ScaI/HindIII multiple cloning site of pEGFP-1 vector. Theelectroporationwas done on embryonic stem cells and positive colonies were selected as puromycin-resistant after three weeks of treatment with puromycin. All-trans retinoic acid (RA) was used for differentiation of mESCs at final concentration of 10-5M. The expression of protamine 1 (Prm1) gene was checked as post meiotic marker in differentiated mESCs after 5, 10, 15, 21 and 30 days after RA induction. Results: The PCR amplification by specific primers for Stra8-EGFP fusion gene was detected in DNA sample from mESCs after electroporation and puromycin treatment.  GFP-positive mESC colonies were observed after 72 hr RA induction. The protamine 1 gene was expressed after 21 days of RA induction. Conclusion: In this study, we demonstrated the in vitro generation of mouse embryonic stem cells to germ cells by using a backbone vector containing the fusion gene Stra8-EGFP. The Stra8 gene is a retinoic acid-responsive protein and is able to regulate meiotic initiation.

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Journal title

volume 17  issue 8

pages  566- 570

publication date 2014-08-01

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