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

Abstract

Purpose: 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. Methods: 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. Results: 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. Conclusion: 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. Largescale 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.

Anemoside B4 Exerts Hypoglycemic Effect by Regulating the Expression of GLUT4 in HFD/STZ Rats

“…Referring to reference [ 29 ], the GLUT4 promoter recombinant plasmid was constructed using pEGFP-N1 plasmid as the vector, the CMV promoter of pEGFP-N1 plasmid was replaced by the GLUT4 promoter using double enzyme digestion (see Figure 7 a). Rat GLUT4 (Slc2a4, NM_012751) sequences of its gene starting area (−2300–100bp) were extracted from the EPD website ( ) accessed on 28 September 2021.…”

Synthesis, characterization and biological evaluation of pyrazole-based benzene sulfonamides as inhibitors of human carbonic anhydrase II, IX and XII

“…Then, cells were incubated for 24 hours in CO 2 incubator with 5% CO 2 . 11 2.3.4 Selection of transfected cells. For selection of transfected cells DMEM media was prepared by adding 200 mg mL −1 of hygromycin B.…”

Egr1/HSP70 Promoter-Driven Activation of Gene Expression for Synergistic Anti-Hepatoma Using PEI-MZF Nanoparticles and Radiation

“…The most common approach for solving this problem is two single promoter in series to form a dual promoter system. [15][16][17][18][19] Finn et al studies showed that the dual promoter can effectively increase the gene expression level with non-viral vector. 20 Thus it is more important in clinical application.…”