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

作者：Ebrahimzadeh Vesal Reza; Shokrgozar Mohammad Ali; Nayernia Karim; Teimoori Toolabi Ladan; Miryounesi Mohammad; Nourashrafeddin Seyedmehdi; Ranji Najmeh; Modarressi Mohammad Hosein^*

来源：Iranian Journal of Basic Medical Sciences, 2014, 17(8): 566-570.

• Differentiation
• Germ-line cells
• Mouse embryonic stem cell
• Vector-based system

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. %26lt;br%26gt;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(-5)M. 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. %26lt;br%26gt;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. %26lt;br%26gt;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.

• 出版日期2014-8