title: A vector-based system for the differentiation of mouse embryonic stem cells toward germ-line cells.
abstract: OBJECTIVE
  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.
  
  
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Medical Genetics, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran.
authors.forename: Reza
authors.initials: R
authors.lastname: Ebrahimzadeh-Vesal
authors.affiliation: National Cell Bank of Iran, Pasteur Institute of Iran, Tehran, Iran.
authors.forename: Mohammad Ali
authors.initials: MA
authors.lastname: Shokrgozar
authors.affiliation: Institute of Human Genetics, North East England, Stem Cell Institute, International Center for Life, Newcastle University, Newcastle, UK.
authors.forename: Karim
authors.initials: K
authors.lastname: Nayernia
authors.affiliation: Molecular Medicine Department, Biotechnology Research Center, Pasteur Institute of Iran.
authors.forename: Ladan
authors.initials: L
authors.lastname: Teimoori-Toolabi
authors.affiliation: Genomic Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
authors.forename: Mohammad
authors.initials: M
authors.lastname: Miryounesi
authors.affiliation: Magee-Womens Research Institute & Foundation, University of Pittsburgh Medical Sciences, Pittsburgh, PA 15213, USA.
authors.forename: Seyedmehdi
authors.initials: S
authors.lastname: Nourashrafeddin
authors.affiliation: Department of Genetics, Faculty of Sciences, Islamic Azad University, Rasht Branch, Rasht, Iran.
authors.forename: Najmeh
authors.initials: N
authors.lastname: Ranji
authors.affiliation: Department of Medical Genetics, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran.
authors.forename: Mohammad Hosein
authors.initials: MH
authors.lastname: Modarressi
keywords: Differentiation; Germ-line cells; Mouse embryonic stem cell; Vector-based system