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International Journal of Cancer Management
The Official Journal of Cancer Research Center (CRC), Shahid Beheshti University of Medical Sciences
e-ISSN 2538-497X
p-ISSN 2538-4422
Construction of an eGFP Expression Plasmid under Control of T7 Promoter and IRES Sequence for Assay of T7 RNA Polymerase Activity in Mammalian Cell Lines
Authors
Mostafa Ghaderi1, Farzaneh Sabahi1,*, Majid Sadeghi-Zadeh2, Zahra Khanlari1, Azam Jamaati1, Dawood Mousavi-Nasab1, Nasrin Majidi-Gharenaz3, Mehdi Ajorloo1, Maryam Fazeli1
1Dept. of Virology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran
2Dept. of Genetics, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran
3Dept. of Anatomy, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran
*Corresponding Author: Corresponding author: Farzaneh Sabahi, Dept. of Virology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran, Tel: +98-2144829122, E-mail: Email: sabahi_f@modares.ac.ir
Published online:Sep 30, 2014
Article type:Research Article
Received:Mar 11, 2014
Accepted:Jun 11, 2014
How to Cite:Ghaderi M, Sabahi F, Sadeghi-Zadeh M, Khanlari Z, Jamaati A, et al. Construction of an eGFP Expression Plasmid under Control of T7 Promoter and IRES Sequence for Assay of T7 RNA Polymerase Activity in Mammalian Cell Lines. Int J Cancer Manag. 2014;7(3):e80542. doi:
Abstract
Background: Recently, the use of T7 RNA polymerase instead of other viral and cellular promoters is increasing due to high efficacy of transcription in the cell cytoplasm by this polymerase. In order to translate the transcripts produced by T7 RNA polymerase in mammalian cell lines, it is necessary to include Internal Ribosome Entry Site (IRES) sequences. In addition, if sequence of poly A signal would be included after interested gene, the rate of expression could be increased in the cells.
Methods: For expression of eGFP in HEK-293 and T7-BHK cells by T7 RNA polymerase, the sequence of eGFP as well as IRES sequences upstream of eGFP gene and poly A signal were inserted into a pUC57 plasmid. On the other hand, gene of T7 RNA polymerase was cloned into modified pIRES2- EGFP plasmid. Then, the constructed plasmids were transfected into HEK-293 cells. T7-BHK cell was used for control of T7 RNA polymerase activity.
Results: Our results showed that using T7 RNA polymerase for expression of foreign genes in mammalian cell lines is highly efficient.
Conclusion: Highly efficient eGFP expression in HEK-293 cells showed that T7 RNA polymerase could be used for cytoplasmic RNA transcription such as production of anti-cancer proteins and oncolytic viral genomic RNA by reverse genetics.
Highlights
Copyright
© 2014, Author(s). This open-access article is available under the Creative Commons Attribution 4.0 (CC BY 4.0) International License (https://creativecommons.org/licenses/by/4.0/), which allows for unrestricted use, distribution, and reproduction in any medium, provided that the original work is properly cited.
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