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

Lookup NU author(s): Professor Karim Nayernia

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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(-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.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.

Publication metadata

Author(s): Ebrahimzadeh-Vesal R, Shokrgozar MA, Nayernia K, Teimoori-Toolabi L, Miryounesi M, Nourashrafeddin S, Ranji N, Modarressi MH

Publication type: Article

Publication status: Published

Journal: Iranian Journal of Basic Medical Sciences

Year: 2014

Volume: 17

Issue: 8

Pages: 566-570

Print publication date: 01/08/2014

ISSN (print): 2008-3866

ISSN (electronic): 2008-3874

Publisher: Mashhad University of Medical Sciences

URL: http://ijbms.mums.ac.ir/article_3190_391.html

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