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

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 Stra۸-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 Stra۸ gene promoter (-۱۴۰۰ to +۷) was inserted in ScaI/HindIII multiple cloning site of pEGFP-۱ 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 ۱۰-۵M. The expression of protamine ۱ (Prm۱) gene was checked as post meiotic marker in differentiated mESCs after ۵, ۱۰, ۱۵, ۲۱ and ۳۰ days after RA induction. Results: The PCR amplification by specific primers for Stra۸-EGFP fusion gene was detected in DNA sample from mESCs after electroporation and puromycin treatment.  GFP-positive mESC colonies were observed after ۷۲ hr RA induction. The protamine ۱ gene was expressed after ۲۱ 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 Stra۸-EGFP. The Stra۸ 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