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Cloning of human SOX2 in lentiviral vector and transduction of HEK293T cell line
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E. Darbari   , Z.S. Soheili |
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| Keywords: Key words : SOX2 Transcription Factor, Cloning Vectors, Green Fluorescent Proteins |
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Full-Text [PDF 578 kb]
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| Abstract (HTML) (6932 Views)
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Type of Study: Research |
Subject:
Biotechnology
Published: 2015/07/5
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Full-Text: (2510 Views) |
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Sci J Iran Blood Transfus Organ 2015; 12(2): 101-110
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Cloning of human SOX2 in lentiviral vector and
transduction of HEK293T cell line
Darbari E.1, Soheili Z.S.2
1College of Science, University of Tehran, Tehran, Iran
2National Institute of Genetic Engineering and Biotechnology, Tehran, Iran
Abstract
Background and Objectives
Recently the regenerative stem cell-based therapy has held great promise in treating severe degenerative diseases. SOX2 gene is highly conserved among species and plays an important role in maintenance of self renewal capacity in stem cells. SOX2 concomitant with OCT4, cMYC, and KLF4 is recruited to reprogram somatic cells towards stem cells. Today SOX2 is frequently being used in induced pluripotent stem cells generation and desired cell based therapies. The aim of this study was to clone SOX2 gene in lentiviral vector and evaluate HEK293T transduction.
Materials and Methods
In the present experimental study, the coding sequence of human SOX2 gene was synthesized and received in pUC57 cloning vector. The synthesized cDNA was sub-cloned into pLEX- MCS Lentiviral vector. Lentiviral particles were produced in HEK293T cells and subsequently the HEK293T were transducted and infected cells were selected by their resistance to puromycin in the culture. Expression of SOX2 was evaluated in infected HEK293 cells by using Real-Time PCR.
Results
Constructs expressing SOX2 gene were produced and confirmed. HEK293T was successfully transducted by lentiviral particles and after 24 hours more than 90% of HEK293T represented the expression of GFP. Real-Time PCR confirmed SOX2 expression in infected HEK293 cells.
Conclusions
HEK293T wase transducted and expressed GFP and SOX2 successfully.
Key words: SOX2 Transcription Factor, Cloning Vectors, Green Fluorescent Proteins
Received: 1 Feb 2015
Accepted: 20 May 2015
Correspondence: Soheili ZS., PhD of Biochemistry. Assistant Professor of National Institute of Genetic Engineering and Biotechnology.
P.O.Box: 14956-161, Tehran, Iran. Tel: (+9821) 44787379; Fax: (+9821) 44787399
E-mail: soheili@nigeb.ac.ir
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