![]() Therefore, the proper selection of these elements can increase recombinant protein expression levels and stability ( Lee JS et al., 2018 Lee CP et al., 2018 Yeo et al., 2017). The expression of a heterologous gene in host cells depends on some components in the expression vector, such as an enhancer, promoter, intron, poly A, integration site, and regulatory sequences. Among these approaches, expression vector optimization is an effective strategy to increase transgene expression level and stability. To achieve stable, high recombinant protein production, cell line engineering, growth medium modification, and the incorporation of cis-acting elements were performed, and obtained the ideal purpose ( Jia et al., 2018 Guo et al., 2020). However, the main bottlenecks during recombinant protein production are low levels, variability of transgene expression, and unstable of expression level during long-time culture ( Santilli et al., 2011 Jazayeri et al., 2018). In conclusion, SV40 poly A and Synt poly A are stronger elements that increase stable transgene expression and decrease the variation of expression, and the choice of suitable poly A element is helpful to improve the expression of recombinant protein.Ĭhinese hamster ovary (CHO) cells are the most frequently used expression system for the production of recombinant therapeutic proteins ( Tripathi and Shrivastava 2019 Kuo et al., 2018). Furthermore, it also showed that the SV40 and Synt poly A elements induced higher levels of adalimumab expression. Importantly, the SV40 and Synt poly A elements decreased the variation of eGFP transgene expression. However, qPCR results showed that the eGFP expression at protein level was not related to the gene copy number and mRNA level. The results indicated the SV40 and Synt poly A sequences can significant improve eGFP transgene expression in stable transfected CHO cells and maintain long-term expression. Five poly A elements, including bovine growth hormone (BGH), mutant BGH, herpes simplex virus type 1 thymidine kinase (HSV-TK), SV40, and a synthetic (Synt) poly A, were cloned into the expression vector and transfected into CHO cells. In this study, we investigated the effects of different poly A elements on transgene expression in Chinese hamster ovary (CHO) cells. Expression vector optimization is an effective strategy to increase transgene expression levels and stability, and the choice of suitable poly A element is crucial for the expression of recombinant protein. We therefore conclude that an internal polyadenylation signal in lentiviral vectors has a highly beneficial effect on transgene expression, but reduces viral titer in a promoter-dependent manner.The generation of the stable, high-level recombinant protein-producing cell lines remains a significant challenge in the biopharmaceutical industry. When the CMV and EF1alpha promoters were used, functional viral titer decreased 8- to 9-fold in the presence of the polyadenylation signal, but titer was not affected when transgene expression was driven by the beta-actin promoter or tissue-specific PSA/Pb promoter. We show that mOrange expression levels in virus-infected HEK-293, LNCaP, and primary prostate epithelial cells were increased 3- to 6.5-fold when an internal polyadenylation signal was present. Therefore, we have assessed the effect of a strong internal polyadenylation signal on both transgene expression levels in virus-infected cells and functional viral titer, in a series of eight self-inactivating lentiviruses expressing the mOrange transgene under the control of the constitutive cytomegalovirus (CMV), elongation factor 1alpha (EF1alpha), and beta-actin promoters or the highly tissue-specific prostate-specific antigen/probasin hybrid (PSA/Pb) promoter with or without a simian virus 40 (SV40) early polyadenylation signal downstream of the mOrange-coding sequence. The lack of the polyadenylation signal, however, has the potential to reduce stability and translation efficiency of transgene mRNA. In lentiviral gene delivery systems, transgene expression cassettes are commonly cloned without a polyadenylation signal to prevent disruption of full-length lentiviral genomes on mRNA maturation in producer cells.
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