Author/Authors :
Ebrahimzadeh-Vesal, Reza tehran university of medical sciences tums - Faculty of Medicine - Department of Medical Genetics, تهران, ايران , Shokrgozar, Mohammad Ali Pasteur Institute of Iran - National Cell Bank of Iran, ايران , Nayernia, Karim Newcastle University - Institute of Human Genetics, North East England ,Stem Cell Institute, International Center for Life, UK , Teimoori-Toolabi, Ladan Pasteur Institute of Iran - Biotechnology Research Center - Molecular Medicine Department, ايران , Miryounesi, Mohammad shahid beheshti university of medical sciences - Genomic Research Center, تهران, ايران , Nourashrafeddin, mehdi University of Pittsburgh Medical Sciences - Magee-Womens Research Institute Foundation, USA , Ranji, Najmeh islamic azad university - Faculty of Sciences - Department of Genetics, ايران , Modarressi, Mohammad Hosein tehran university of medical sciences tums - Faculty of Medicine - Department of Medical Genetics, تهران, ايران
Abstract :
Objective(s): To culture the in 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. The electroporation was 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.
Keywords :
Differentiation , Germ , line cells , Mouse embryonic stem cell , Vector , based system
