A vector-based system for the differentiation of mouse embryonic stem cells toward germ-line cells
- سال انتشار: 1393
- محل انتشار: مجله علوم پایه پزشکی ایران، دوره: 17، شماره: 8
- کد COI اختصاصی: JR_IJBMS-17-8_006
- زبان مقاله: انگلیسی
- تعداد مشاهده: 361
نویسندگان
Department of Medical Genetics, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran
National Cell Bank of Iran, Pasteur Institute of Iran, Tehran, Iran
Institute of Human Genetics, North East England, Stem Cell Institute, International Center for Life, Newcastle University, Newcastle, UK
Molecular Medicine Department, Biotechnology Research Center, Pasteur Institute of Iran
Genomic Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran
Magee-Womens Research Institute & Foundation, University of Pittsburgh Medical Sciences, Pittsburgh, PA ۱۵۲۱۳, USA
Department of Genetics, Faculty of Sciences, Islamic Azad University, Rasht Branch, Rasht, Iran
Department of Medical Genetics, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran
چکیده
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.کلیدواژه ها
Differentiation, Germ-line cells, Mouse embryonic stem cell, Vector-based systemاطلاعات بیشتر در مورد COI
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