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



doi: 10.3389/fbioe.2022.840600.


eCollection 2022.

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Qin Li et al.


Front Bioeng Biotechnol.


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Abstract

Chinese hamster ovary (CHO) cells are currently the most widely used host cells for recombinant therapeutic protein (RTP) production. Currently, the RTP yields need to increase further to meet the market needs and reduce costs. In this study, three stabilizing and anti-repressor (SAR) elements from the human genome were selected, including human SAR7, SAR40, and SAR44 elements. SAR elements were cloned upstream of the promoter in the eukaryotic vector, followed by transfection into CHO cells, and were screened under G418 pressure. Flow cytometry was used to detect enhanced green fluorescent protein (eGFP) expression levels. The gene copy numbers and mRNA expression levels were determined through quantitative real-time PCR. Furthermore, the effect of the stronger SAR elements on adalimumab was investigated. The results showed that transgene expression levels in the SAR-containing vectors were higher than that of the control vector, and SAR7 and SAR40 significantly increased and maintained the long-term expression of the transgene in CHO cells. In addition, the transgene expression level increase was related with gene copy numbers and mRNA expression levels. Collectively, SAR elements can enhance the transgene expression and maintain the long-term expression of a transgene in transfected CHO cells, which may be used to increase recombinant protein production in CHO cells.


Keywords:

Chinese hamster ovary cell; epigenetic regulatory elements; green fluorescent protein; stabilizing and anti-repressor elements; transgene expression.

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures


FIGURE 1



FIGURE 1

Representation of the vectors containing different SARs. Schematic illustration of the expression vector containing different SARs which were inserted upstream of CMV. CMV, human cytomegalovirus IE gene promoter; eGFP, enhanced green fluorescence protein; IRES, internal ribosome entry site; poly A, polyadenylation signal. Neo, neomycin; Bla, blasticidin.


FIGURE 2



FIGURE 2

Transient expression of recombinant protein in transfected pools of different SARs. Three vectors were transfected into CHO cells, 48 h after transfection, and the transfection efficiencies were estimated. (A) The representative photos were observed by fluorescence microscopy. (B) Analysis of the transfection efficiency. (C) Analysis of the eGFP transient expression. The results are the mean values obtained for three independent experiments, and the standard deviation is indicated (*p < 0.05).


FIGURE 3



FIGURE 3

Analysis of MFI in stably transfected CHO cell line. (A) The MFI of eGFP of the different construct was measured by flow cytometry on day 20 under G418 selection. (B) Fold statistical analysis results of eGFP expression levels in the different constructs. The results are the mean values obtained for three independent experiments, and the standard deviation is indicated (*p < 0.05; **p < 0.05).


FIGURE 4



FIGURE 4

Analysis of the relative eGFP mRNA levels and eGFP copy number in stably transfected CHO cells. After screening under G418, the stable transfected CHO cell pools were acquired. (A) Total RNA was isolated and cDNA synthesis was performed, then the mRNA levels were detected using qRT-PCR. (B) Relative eGFP gene copy numbers were detected by qRT-PCR. (C) Correlation analysis between the relative eGFP copy (blue square), mRNA expression (red) and relative eGFP expression in long-term stable transfected CHO cells. Results were obtained from three independent experiments. The results are the mean values obtained for three independent experiments, and standard deviation is indicated (*p < 0.05; **p < 0.05).


FIGURE 5



FIGURE 5

Analysis of long-term stability of the transgene expression in transfected CHO cells. Cells were collected, and FACSCalibur estimated the eGFP fluorescence profile at generation 30, 60,90, and 120 after transfection. (A) The eGFP expression (MFI) on different days after transfection. (B) The retention of eGFP expression of different constructs. The results are the mean values obtained for three independent experiments, and the standard deviation is indicated.


FIGURE 6



FIGURE 6

Analysis of adalimumab antibody expression of different constructs in CHO cells. The vectors containing adalimumab antibody gene were transformed into CHO cells, respectively. Cells supernatant was collected for analysis. (A) Adalimumab antibody expression was detected by Western blot. Lane 1, pIRES-mAb-SAR40; Lane 2, pIRES-mAb-SAR7; Lane 3, pIRES-mAb. (B) Expression of the adalimumab antibody was detected by ELISA. (C) Relative expression of adalimumab antibody. The results are the mean values obtained for three independent experiments, and the standard deviation is indicated (*p < 0.05).

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