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      Drug Design, Development and Therapy (submit here)

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      Enhancing the Butyrylcholinesterase Activity in HEK-293 Cell Line by Dual-Promoter Vector Decorated on Lipofectamine


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          Human butyrylcholinesterase (BChE) serves as a bio scavenger to counteract organophosphate poisoning. It is also a potential drug candidate in several therapeutic fields. Therefore, in the present study, we constructed a new dual-promoter plasmid consisting of Cytomegalovirus (CMV) and human elongation factor 1α (EF-1α) promoters and transfected that into HEK-293 cells using Lipofectamine to enhance the BChE secretion.


          The new dual-promoter construction (pBudCE dual BChE) including two copies of the BChE gene was designed and transfected into cells by liposomal structures. The cloned plasmids were evaluated by enzyme digestion and gel electrophoresis analysis. Experimental groups were categorized into the cells transfected by pBudCE dual BChE (treatment), pCMV (positive control) vectors, and nontransfected cells (negative control). BChE gene expression was evaluated by qRT-PCR and the enzyme activity was assessed using modified Ellman’s method. The freeze-thaw process was carried out for analyzing the stability of the pBudCE dual BChE vector.


          Validation examination of the cloned plasmids confirmed the successful cloning process. The gene expression level and Ellman’s method value in pBudCE dual BChE was higher than the other groups. CMV promoter has also increased the enzyme activity, although the difference was not significant compared with the control group. Interestingly, freeze-thaw cycles followed by several passages did not affect the enzyme activity.


          The designed construction with CMV and EF-1α promoters could increase BChE gene expression and the activity of the BChE enzyme in HEK-293 cell line. Large-scale production of BChE enzyme can be achieved by using dual-promoter plasmid construction compared to a single-promoter vector to be used in clinical trials.

          Most cited references33

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          Management of acute organophosphorus pesticide poisoning

          Summary Organophosphorus pesticide self-poisoning is an important clinical problem in rural regions of the developing world, and kills an estimated 200 000 people every year. Unintentional poisoning kills far fewer people but is a problem in places where highly toxic organophosphorus pesticides are available. Medical management is difficult, with case fatality generally more than 15%. We describe the limited evidence that can guide therapy and the factors that should be considered when designing further clinical studies. 50 years after first use, we still do not know how the core treatments—atropine, oximes, and diazepam—should best be given. Important constraints in the collection of useful data have included the late recognition of great variability in activity and action of the individual pesticides, and the care needed cholinesterase assays for results to be comparable between studies. However, consensus suggests that early resuscitation with atropine, oxygen, respiratory support, and fluids is needed to improve oxygen delivery to tissues. The role of oximes is not completely clear; they might benefit only patients poisoned by specific pesticides or patients with moderate poisoning. Small studies suggest benefit from new treatments such as magnesium sulphate, but much larger trials are needed. Gastric lavage could have a role but should only be undertaken once the patient is stable. Randomised controlled trials are underway in rural Asia to assess the effectiveness of these therapies. However, some organophosphorus pesticides might prove very difficult to treat with current therapies, such that bans on particular pesticides could be the only method to substantially reduce the case fatality after poisoning. Improved medical management of organophosphorus poisoning should result in a reduction in worldwide deaths from suicide.
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            Systematic Comparison of Constitutive Promoters and the Doxycycline-Inducible Promoter

            Constitutive promoters are used routinely to drive ectopic gene expression. Here, we carried out a systematic comparison of eight commonly used constitutive promoters (SV40, CMV, UBC, EF1A, PGK and CAGG for mammalian systems, and COPIA and ACT5C for Drosophila systems). We also included in the comparison the TRE promoter, which can be activated by the rtTA transcriptional activator in a doxycycline-inducible manner. To make our findings representative, we conducted the comparison in a variety of cell types derived from several species. We found that these promoters vary considerably from one another in their strength. Most promoters have fairly consistent strengths across different cell types, but the CMV promoter can vary considerably from cell type to cell type. At maximal induction, the TRE promoter is comparable to a strong constitutive promoter. These results should facilitate more rational choices of promoters in ectopic gene expression studies.
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              HEK293 cell line: a vehicle for the expression of recombinant proteins.

              The HEK cell line has been extensively used as an expression tool for recombinant proteins since it was generated over 25 years ago. Although of epithelial origin, its biochemical machinery is capable of carrying out most of the post-translational folding and processing required to generate functional, mature protein from a wide spectrum of both mammalian and non-mammalian nucleic acids. Though popular as a transient expression system, this cell type has also seen wide use in stably transfected forms (i.e. transformed cells) to study a variety of cell-biological questions in neurobiology. The principal attributes which have made the HEK cell a popular choice among electrophysiologists to study isolated receptor channels include; its quick and easy reproduction and maintenance; amenability to transfection using a wide variety of methods; high efficiency of transfection and protein production; faithful translation and processing of proteins; and small cell size with minimal processes appropriate for voltage-clamp experimentation. These, and other attributes, also mean that complementary biochemical/cell biological evaluations of expressed proteins can be performed in concert with functional analyses to establish detailed pharmacological and biophysical profiles for the action of new drugs and their targets. The increased amount of sequence information available from the human genome has placed greater emphasis upon heterologous cell expression systems as targets for high throughput structure-function evaluation of novel drug targets and disease markers. Here we have highlighted some of the innate characteristics of the HEK cell in order that its suitability as a vehicle for the expression of a gene product can be assessed for particular needs. We have also detailed some of the standard methods used for transfection and obtaining functional data from electrophysiological recording techniques.

                Author and article information

                Drug Des Devel Ther
                Drug Des Devel Ther
                Drug Design, Development and Therapy
                04 September 2020
                : 14
                : 3589-3599
                [1 ]Department of Anatomy, Afzalipour School of Medicine, Kerman University of Medical Sciences , Kerman, Iran
                [2 ]Pharmaceutics Research Centre, Institute of Neuropharmacology, Kerman University of Medical Sciences , Kerman, Iran
                [3 ]Department of Clinical Sciences, Faculty of Veterinary Medicine, Shahid Bahonar University of Kerman , Kerman, Iran
                [4 ]Neuroscience Research Centre, Institute of Neuropharmacology, Kerman University of Medical Sciences , Kerman, Iran
                [5 ]Afzal Research Institute (NGO), Kerman University of Medical Sciences , Kerman, Iran
                Author notes
                Correspondence: Seyed Noureddin Nematollahi-Mahani Tel +983 433257343 Email nnematollahi@kmu.ac.ir
                Author information
                © 2020 Mirzaie et al.

                This work is published and licensed by Dove Medical Press Limited. The full terms of this license are available at https://www.dovepress.com/terms.php and incorporate the Creative Commons Attribution – Non Commercial (unported, v3.0) License ( http://creativecommons.org/licenses/by-nc/3.0/). By accessing the work you hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. For permission for commercial use of this work, please see paragraphs 4.2 and 5 of our Terms ( https://www.dovepress.com/terms.php).

                : 06 May 2020
                : 06 August 2020
                Page count
                Figures: 6, Tables: 3, References: 37, Pages: 11
                Funded by: the Kerman University of Medical Sciences (KMU);
                This work was a part of the PhD thesis that was supported by the Kerman University of Medical Sciences (KMU) and submitted under the ethic approval code: IR.KMU.REC.1398.294.
                Original Research

                Pharmacology & Pharmaceutical medicine
                dual-promoter,vector,recombinant protein,drug delivery,ellman’s method,hek-293


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