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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 Ghaderi 1 , Farzaneh Sabahi 1 , * , Majid Sadeghi-Zadeh 2 , Zahra Khanlari 1 , Azam Jamaati 1 , Dawood Mousavi-Nasab 1 , Nasrin Majidi-Gharenaz 3 , Mehdi Ajorloo 1 , Maryam Fazeli 1

1 Dept. of Virology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran

2 Dept. of Genetics, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran

3 Dept. of Anatomy, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran

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.

ARTICLE INFORMATION

International Journal of Cancer Management: 7 (3); e80542
Published Online: September 30, 2014
Article Type: Research Article
Received: March 11, 2014
Accepted: June 11, 2014

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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.

Keywords

T7 RNA polymerase Cancer gene therapy Oncolytic RNA viruses

© 2014, Author(s). This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License (http://creativecommons.org/licenses/by-nc/4.0/) which permits copy and redistribute the material just in noncommercial usages, provided the original work is properly cited.

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