Development of a new promoter to avoid the silencing of genes in the production of recombinant antibodies in chinese hamster ovary cells

Abstract

Background The production of recombinant proteins in mammalian cell lines is one of the most important areas in biopharmaceutical industry. Viral transcriptional promoters are widely used to express recombinant proteins in mammalian cell lines. However, these promoters are susceptible to silencing, thus limiting protein productivity. Some CpG islands can avoid the silencing of housekeeping genes; for that reason, they have been used to increase the production of recombinant genes in cells of animal origin. In this study, we evaluated the CpG island of the promoter region of the β-actin gene of Cricetulus griseous (Chinese hamster), associated to the Cytomegalovirus (CMV) promoter, to increase recombinant antibodies production in Chinese Hamster Ovary (CHO) cells. Results We focused on the non-coding region of CpG island, which we called RegCG. RegCG behaved as a promoter, whose transcriptional activity was mainly commanded by the CAAT and CArG boxes of the proximal promoter. However, the transcription started mainly at the intronic region before the proximal transcription start site. While the CMV promoter was initially more powerful than RegCG, the latter promoter was more resistant to silencing than the CMV promoter in stable cell lines, and its activity was improved when combined with the CMV promoter. Thereby, the chimeric promoter was able to maintain the expression of recombinant antibodies in stable clones for 40 days at an average level 4 times higher than the CMV promoter. Finally, the chimeric promoter showed compatibility with a genetic amplification system by induction with methotrexate in cells deficient in the dihydrofolate reductase gene. Conclusions We have generated an efficient synthetic hybrid transcription promoter through the combination of RegCG with CMV, which, in stable cell lines, shows greater activity than when both promoters are used separately. Our chimeric promoter is compatible with a genetic amplification system in CHO DG44 cells and makes possible the generation of stable cell lines with high production of recombinant antibodies. We propose that this promoter can be a good alternative for the generation of clones expressing high amount of recombinant proteins, essential for industrial applications. Electronic supplementary material The online version of this article (10.1186/s13036-019-0187-y) contains supplementary material, which is available to authorized users.

Slight Variations in the Sequence Downstream of the Polyadenylation Signal Significantly Increase Transgene Expression in HEK293T and CHO Cells

“…We believe that the results of the present work may be interesting for use in the production of therapeutic proteins and viral vectors [ 104 , 105 , 106 ], since HEK293T and CHO are the most popular cell lines for these purposes [ 107 , 108 , 109 , 110 , 111 , 112 ]. Since one of the important tasks in this field is to increase production efficiency, the modification of sequences involved in the transcription termination of transgenes based on the results of the appropriate MPRA screenings may substantially contribute to solving this problem.…”

New function of a well‐known promoter: Enhancer activity of minimal CMV promoter enables efficient dual‐cassette transgene expression

“…This observation could be related to promoter suppression reported in double-promoter monocistronic expression vector. 47 Colella et al 47 with different variants of chicken β-actin promoter in CAG or CBh, 21,22,48 and the fusion of a 380-bp CMV enhancer fragment (À541 bp to À160 bp) with PDGFb promoter formed the neuronspecific CPDGFb. 23 The 120-bp cC element (À50 bp to +70 bp) studied here has only been used as a minimal promoter with very low baseline expression in combination with responsive elements to drive inducible transgene expression.…”

“…X03922.1). 20 Multiple fragments with different sizes from the enhancer region have been utilized to boost both ubiquitous and cell-specific promoters, including CBh and CAG, 21,22 as well as cardiomyocyte-, alveolar epithelia-or neuron-specific promoters. 23,24 Moreover, a 120-bp derivate of the core CMV promoter-extended region (cC element), containing nucleotides of -50 bp to +70 bp, has been employed as a minimal promoter for inducible transgene expression in mammalian cells in vitro.…”

Bioengineering tools for the production of pharmaceuticals: current perspective and future outlook

“…Inveterate enactment of C-fibres may compound infection pathology in rheumatoid joint pain through the antidromic discharge of pro-inflammatory neuropeptides [126]. An effective bioengineered crossbreed translation promoter through the combination of RegCG with CMV, which, in steady cell lines, appears more noteworthy action than when both promoters are utilized independently at that point other promoters [127]. Hereditary circuits bioengineering devices have allowed a more focus on results and biomimetic reactions to neurotic conditions.…”