Approaches to Controlled Co-Amplification of Genes for Production of Biopharmaceuticals: Study of the Insertion and Amplification Dynamics of Genetic Cassettes in the Genome of Chinese Hamster Ovary Cells during Co-Expression of Compatible Pair of Plasmids
| Autor: | A I Vorobiev, Nadezhda A. Orlova, M. P. Kondrashova, Konstantin G. Skryabin, Alexander G. Gabibov, Ivan I. Vorobiev, Yu. А. Khodak, S. V. Kovnir |
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| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
Concatemer Genetic Vectors Green Fluorescent Proteins CHO Cells Biology Genome General Biochemistry Genetics and Molecular Biology Chinese hamster 03 medical and health sciences chemistry.chemical_compound Plasmid Cricetulus Cricetinae Animals Gene Chinese hamster ovary cell Gene Amplification General Medicine biology.organism_classification Molecular biology Housekeeping gene 030104 developmental biology Methotrexate chemistry mCherry Plasmids |
| Zdroj: | Bulletin of experimental biology and medicine. 163(2) |
| ISSN: | 1573-8221 |
| Popis: | Plasmid vector family p1.1 based on non-coding regions of Chinese hamster housekeeping gene EEF1A and concatemer of Epstein-Barr virus terminal repeat increases the frequency of genome integration and provides rapid amplification of the target genes in the genome. For a pair of fluorescent proteins eGFP and mCherry it was shown that p1.1 vectors bearing dihydrofolate reductase and glutamine synthetase selection markers upon co-transfection into CHO DG44 cell line allow obtaining a polyclonal cell population in which ~70% of cells express both genes. The subsequent one-step gene amplification of the genome-integrated genetic cassettes under the selective pressure of increased concentrations of methotrexate can increase the expression of both integrated genes up to 8.2% eGFP and 9.9% mCherry of total protein. This approach can be used for the development of cell lines for the production of functional heterodimeric proteins, e.g. polypeptide hormones and therapeutic antibodies. |
| Databáze: | OpenAIRE |
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