| Autor: |
Roberto A Zúñiga, Matías Gutiérrez-González, Norberto Collazo, Pablo Hérnan Sotelo, Carolina H Ribeiro, Claudia Altamirano, Carmen Lorenzo, Juan Carlos Aguillón, María Carmen Molina |
| Jazyk: |
angličtina |
| Rok vydání: |
2019 |
| Předmět: |
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| Zdroj: |
Journal of Biological Engineering, Vol 13, Iss 1, Pp 1-12 (2019) |
| Druh dokumentu: |
article |
| ISSN: |
1754-1611 |
| DOI: |
10.1186/s13036-019-0187-y |
| Popis: |
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. |
| Databáze: |
Directory of Open Access Journals |
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