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Evidence for expression of promoterless GFP cassette: Is GFP an ideal reporter gene in biotechnology science?

Green fluorescent protein (GFP) has played an important role in biochemistry and cell biology as a reporter gene. It has been used to assess the potency of promoters for recombinant protein production. This investigation reveals evidences suggesting that the gfp GFP gene (EGFP) could be expressed wi...

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Autores principales: Mohammadi, Zahra, Karamzadeh, Arezou, Tabatabaiefar, Mohammad Amin, Khanahmad, Hossein, Shariati, Laleh
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Wolters Kluwer - Medknow 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6714119/
https://www.ncbi.nlm.nih.gov/pubmed/31516512
http://dx.doi.org/10.4103/1735-5362.263559
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author Mohammadi, Zahra
Karamzadeh, Arezou
Tabatabaiefar, Mohammad Amin
Khanahmad, Hossein
Shariati, Laleh
author_facet Mohammadi, Zahra
Karamzadeh, Arezou
Tabatabaiefar, Mohammad Amin
Khanahmad, Hossein
Shariati, Laleh
author_sort Mohammadi, Zahra
collection PubMed
description Green fluorescent protein (GFP) has played an important role in biochemistry and cell biology as a reporter gene. It has been used to assess the potency of promoters for recombinant protein production. This investigation reveals evidences suggesting that the gfp GFP gene (EGFP) could be expressed without the promoter. In a study, a pLenti-F/GFP vector was constructed with the purpose to allow GFP expression in transduced cells but not in packaging cells; however, after transfecting the HEK293T cell line, GFP gene was expressed, compared to pLOX/CWgfp-transfected cells showed expression lag, lower levels and reduced percentage of GFP expression in the cells. This unexpected result could be due to auto transduction in packaging cell, possible retrotransposon activity in the cell line, possible contamination of pLenti-F/GFP with the pLOX/CWgfp and possible presence of a promoter within backbone of the vector. All the possibilities were ruled out. To exclude the possibility that a sequence within the region might act as a promoter, the fragment to be transfected was minimized to a region containing “from the start of the GFP gene to 5’LTR R”. The GFP gene was again expressed. Therefore, our findings suggest the EGFP does not need promoter for expression. This should appeal to the researchers designing GFP based assays to evaluate the potency of promoters, since possible aberrant expression may have a potential to influence on the results of a planned experiment.
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spelling pubmed-67141192019-09-12 Evidence for expression of promoterless GFP cassette: Is GFP an ideal reporter gene in biotechnology science? Mohammadi, Zahra Karamzadeh, Arezou Tabatabaiefar, Mohammad Amin Khanahmad, Hossein Shariati, Laleh Res Pharm Sci Original Article Green fluorescent protein (GFP) has played an important role in biochemistry and cell biology as a reporter gene. It has been used to assess the potency of promoters for recombinant protein production. This investigation reveals evidences suggesting that the gfp GFP gene (EGFP) could be expressed without the promoter. In a study, a pLenti-F/GFP vector was constructed with the purpose to allow GFP expression in transduced cells but not in packaging cells; however, after transfecting the HEK293T cell line, GFP gene was expressed, compared to pLOX/CWgfp-transfected cells showed expression lag, lower levels and reduced percentage of GFP expression in the cells. This unexpected result could be due to auto transduction in packaging cell, possible retrotransposon activity in the cell line, possible contamination of pLenti-F/GFP with the pLOX/CWgfp and possible presence of a promoter within backbone of the vector. All the possibilities were ruled out. To exclude the possibility that a sequence within the region might act as a promoter, the fragment to be transfected was minimized to a region containing “from the start of the GFP gene to 5’LTR R”. The GFP gene was again expressed. Therefore, our findings suggest the EGFP does not need promoter for expression. This should appeal to the researchers designing GFP based assays to evaluate the potency of promoters, since possible aberrant expression may have a potential to influence on the results of a planned experiment. Wolters Kluwer - Medknow 2019-08 /pmc/articles/PMC6714119/ /pubmed/31516512 http://dx.doi.org/10.4103/1735-5362.263559 Text en Copyright: © 2019 Research in Pharmaceutical Sciences http://creativecommons.org/licenses/by-nc-sa/4.0 This is an open access journal, and articles are distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as appropriate credit is given and the new creations are licensed under the identical terms.
spellingShingle Original Article
Mohammadi, Zahra
Karamzadeh, Arezou
Tabatabaiefar, Mohammad Amin
Khanahmad, Hossein
Shariati, Laleh
Evidence for expression of promoterless GFP cassette: Is GFP an ideal reporter gene in biotechnology science?
title Evidence for expression of promoterless GFP cassette: Is GFP an ideal reporter gene in biotechnology science?
title_full Evidence for expression of promoterless GFP cassette: Is GFP an ideal reporter gene in biotechnology science?
title_fullStr Evidence for expression of promoterless GFP cassette: Is GFP an ideal reporter gene in biotechnology science?
title_full_unstemmed Evidence for expression of promoterless GFP cassette: Is GFP an ideal reporter gene in biotechnology science?
title_short Evidence for expression of promoterless GFP cassette: Is GFP an ideal reporter gene in biotechnology science?
title_sort evidence for expression of promoterless gfp cassette: is gfp an ideal reporter gene in biotechnology science?
topic Original Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6714119/
https://www.ncbi.nlm.nih.gov/pubmed/31516512
http://dx.doi.org/10.4103/1735-5362.263559
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