Volume 9, Issue 1 (2018)
JMBS 2018, 9(1): 1-8 |
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Rasekhi M, Bakhshande B, Sadeghizadeh M, Salimi A, Soleimani M. Production of Recombinant HEK293T with miR-1 Overexpression as a Biological Model for Cardiac Studies. JMBS 2018; 9 (1) :1-8
URL: http://biot.modares.ac.ir/article-22-13983-en.html
URL: http://biot.modares.ac.ir/article-22-13983-en.html
1- National Institute of Genetic Engineering & Biotechnology, Tehran, Iran
2- Biotechnology Department, Science Faculty, University of Tehran, Tehran, Iran, Biotechnology Department, No. 13, Shafiee Alley, Qods Street, Enqelab Sq., Tehran, Iran , b.bakhshandeh@ut.ac.ir
3- Genetic Department, Biological Sciences Faculty, Tarbiat Modares University, Tehran, Iran
4- Nanobiotechnology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran
5- Hematology Departmen, Medical Sciences Faculty, Tarbiat Modares University, Tehran, Iran
2- Biotechnology Department, Science Faculty, University of Tehran, Tehran, Iran, Biotechnology Department, No. 13, Shafiee Alley, Qods Street, Enqelab Sq., Tehran, Iran , b.bakhshandeh@ut.ac.ir
3- Genetic Department, Biological Sciences Faculty, Tarbiat Modares University, Tehran, Iran
4- Nanobiotechnology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran
5- Hematology Departmen, Medical Sciences Faculty, Tarbiat Modares University, Tehran, Iran
Abstract: (9783 Views)
Aims: The induction of artificial over-expression of miRNAs is an appropriate approach to more effective cell differentiation. The significant role of microRNA-1(miR-1) has been reported in the development and differentiation of cardiac cells. Lentivirus is an effective vector for stable cell line production. The aim of this study was the production of recombinant HEK293T with miR-1 overexpression as a biological model for cardiac studies.
Materials & Methods: In this experimental study, HEK 293T cells were cultured in DMEM medium with 10% Fetal Bovine Serum (FBS) and L-glutamine 2mM and Penicillin-Streptomycin 1X in incubator medium. After cloning of miR-1 gene, recombinant clones were selected and the recombination was confirmed by sequencing. The miR-1 carrying vector and auxiliary vectors were packaged in the HEK293T to produce the recombinant virus. The infection of HEK293T by recombinant virus was performed in order to achieve stable cell line. Then, GFP fluorescent marker evaluated the efficiency of transfection and effective virus dilution. Finally, the alteration in expression level of miR-1 was assessed by qPCR. Data analysis was performed by comparing the threshold cycle and Pfaffl method.
Findings: The most GFP expression was detected in transfected cells by 150 micromole dilution. GFP fluorescent marker facilitated optimization and purification of recombinant cells. qPCR investigation demonstrated the significant increase in expression of miR-1 in transfected cells in comparison to controls.
Conclusion: The stable recombinant HEK293T miR-1 over-expressing cell line in lentivirus can be utilized as a suitable biological model for investigation of cardiac evolution and development processes.
Materials & Methods: In this experimental study, HEK 293T cells were cultured in DMEM medium with 10% Fetal Bovine Serum (FBS) and L-glutamine 2mM and Penicillin-Streptomycin 1X in incubator medium. After cloning of miR-1 gene, recombinant clones were selected and the recombination was confirmed by sequencing. The miR-1 carrying vector and auxiliary vectors were packaged in the HEK293T to produce the recombinant virus. The infection of HEK293T by recombinant virus was performed in order to achieve stable cell line. Then, GFP fluorescent marker evaluated the efficiency of transfection and effective virus dilution. Finally, the alteration in expression level of miR-1 was assessed by qPCR. Data analysis was performed by comparing the threshold cycle and Pfaffl method.
Findings: The most GFP expression was detected in transfected cells by 150 micromole dilution. GFP fluorescent marker facilitated optimization and purification of recombinant cells. qPCR investigation demonstrated the significant increase in expression of miR-1 in transfected cells in comparison to controls.
Conclusion: The stable recombinant HEK293T miR-1 over-expressing cell line in lentivirus can be utilized as a suitable biological model for investigation of cardiac evolution and development processes.
Article Type: Research Paper |
Subject:
Agricultural Biotechnology
Received: 2016/03/12 | Accepted: 2018/01/27 | Published: 2018/05/22
Received: 2016/03/12 | Accepted: 2018/01/27 | Published: 2018/05/22
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