In pharmaceutical biotechnology and recombinant protein production, due to the low efficacy of inserting the target gene into the host gene genome and its integration into the heterochromatin regions, which leads to the suppression of transcription as well as the instab
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In pharmaceutical biotechnology and recombinant protein production, due to the low efficacy of inserting the target gene into the host gene genome and its integration into the heterochromatin regions, which leads to the suppression of transcription as well as the instability of the expression of the desired sequence, achieving cells with highexpression is a challenge. To overcome the limitations transposons, which are mobile genetic elements and have the ability to cut and insert target fragments in certain regions of the genome with the “Cut and Paste” mechanism, are effective. This research aims to evaluate the effect of the PiggyBac transposon vector on the expression level of recombinant Erythropoietin (rEPO) protein and to find a cell line with high expression. First the optimized rEPO sequence based on CHO codon performance was cloned into the pOptiVECTM plasmid. To create the second expression vector, the EPO-IRES-DHFR fragment was inserted into the PB513B-1 plasmid, and then the homogenization steps in both vectors were confirmed using Colony PCR reaction, enzyme digestion, and Sanger sequencing. In order to create a stable cell line, both vectors were separately transfected into CHO DG44 cells and then screened. After confirming the insertion of the vectors into the genome of the target cell, the level of erythropoietin gene expression at the transcript and protein level was checked using qRT-PCR and western blotting tests, respectively, in two cell lines. The Real-Time PCR data indicate a 188-fold increase in erythropoietin gene transcript in the PB513B-1-EPO cell line compared to pOptiVEC-EPO. Additionally, the western blotting test's result confirmed the correct synthesis and secretion of this protein. Analysis of findings in this research revealed that the transposon element significantly increased the expression of the desired gene at the transcriptional level and had could create a cell line with high expression of the target protein.
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