A vector-based system for the differentiation of mouse embryonic stem cells toward germ-line cells
نویسندگان
1 Department of Medical Genetics, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran
2 Institute of Human Genetics, North East England, Stem Cell Institute, International Center for Life, Newcastle University, Newcastle, UK
3 Genomic Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran
4 Department of Genetics, Faculty of Sciences, Islamic Azad University, Rasht Branch, Rasht, Iran
5 Department of Medical Genetics, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran
6 National Cell Bank of Iran, Pasteur Institute of Iran, Tehran, Iran
7 Molecular Medicine Department, Biotechnology Research Center, Pasteur Institute of Iran
8 Magee-Womens Research Institute & Foundation, University of Pittsburgh Medical Sciences, Pittsburgh, PA 15213, USA
doi
10.22038/ijbms.2014.3190چکیده
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.
