Enhanced Transgene Expression by Optimization of Poly A in Transfected CHO Cells

Abstract

The generation of the stable, high-level recombinant protein-producing cell lines remains a significant challenge in the biopharmaceutical industry. 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. In this study, we investigated the effects of different poly A elements on transgene expression in Chinese hamster ovary (CHO) cells. 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. 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. However, qPCR results showed that the eGFP expression at protein level was not related to the gene copy number and mRNA level. Importantly, the SV40 and Synt poly A elements decreased the variation of eGFP transgene expression. Furthermore, it also showed that the SV40 and Synt poly A elements induced higher levels of adalimumab expression. 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.

Implementation of a Novel Optogenetic Tool in Mammalian Cells Based on a Split T7 RNA Polymerase

“…This property is especially interesting if applied to studying resource allocation and gene expression burden. Recently, several studies have shown how burden in mammalian cells is generated through the sharing of cellular resources both at the transcriptional and translational level. , The ability to isolate exogenous genes from the cellular resources can be used, for example, in bioproduction where the stable generation of a protein over time can have dramatic consequences on its yield and downstream applications . We showed that using mOptoT7 to orthogonally generate RNA species (in an inducible manner) not only helps in reducing burden, but can also help in gaining a better understanding of the effect of transcriptional resources on burden as a whole.…”

“… 8 , 9 The ability to isolate exogenous genes from the cellular resources can be used, for example, in bioproduction where the stable generation of a protein over time can have dramatic consequences on its yield and downstream applications. 59 We showed that using mOptoT7 to orthogonally generate RNA species (in an inducible manner) not only helps in reducing burden, but can also help in gaining a better understanding of the effect of transcriptional resources on burden as a whole. In this context, the contribution of IRES sequences that are included in the mOptoT7 reporter are not considered in this study and require further investigation.…”

Stabilizing and Anti-Repressor Elements Effectively Increases Transgene Expression in Transfected CHO Cells

“…At 48-h post transfection, the transfection efficiency of each expression vector was observed under a fluorescence microscope (ECLIPSE Ti; Nikon, Tokyo, Japan), and mean fluorescence intensity (MFI) was detected by flow cytometry as previously described ( Wang et al, 2019 ; Wang et al, 2022 ). Briefly, a total of 100,000 fluorescent events were obtained using a 530/15 bandpass filter for the green fluorescent signal, which was acquired with an emission wave length of 530 nm.…”

“…The establishment of transfected cell lines with high-level expression of the gene of interest (GOI) is often an arduous process due to the heterogeneity of transgene expression. To obtain stable and highly transfected GOI cell lines, cell line engineering, growth medium and production process modification, and expression vector design were performed ( Jia et al, 2018 ; Guo et al, 2020 ; Wang et al, 2022 ). Among these strategies, the expression vector design is an effective method to increase GOI expression level and stability.…”

“…These elements can be cloned into different sites of the expression vector, such as the flank of the expression box, upstream of the promoter, or downstream of poly A, which can improve the transcription efficiency and stability, to significantly reduce the time and cost required to produce many recombinant proteins. The MAR, UCOE, promoter, intron, insulator, selective marker, and poly A can be used to increase GOI expression levels in CHO cells ( Mariati Yeo et al, 2014 ; Chen et al, 2017 ; Zhao et al, 2017 ; Tian et al, 2018 ; Xu et al, 2018 ; Li et al, 2019 ; Wang et al, 2022 ). MAR can increase the expression level in CHO cells and reduce the variation of transgene expression among transformants ( Wang et al, 2008 ; Wang et al, 2010 ; Wang et al, 2012 ; Li et al, 2013 ; Zhao et al, 2017 ; Li et al, 2018 ; Tian et al, 2018 ; Li et al, 2019 ).…”