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
- Authors: Kovnir S.V.1, Orlova N.A.1, Khodak Y.А.1, Kondrashova M.P.1, Gabibov A.G.2, Skryabin K.G.1, Vorobiev A.I.3, Vorobiev I.I.1,2
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Affiliations:
- Institute of Bioengineering, Federal Research Centre “Fundamentals of Biotechnology”, Russian Academy of Sciences
- M. M. Shemyakin and Yu. A. Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences
- Hematology Research Center, Ministry of Health of the Russian Federation
- Issue: Vol 163, No 2 (2017)
- Pages: 245-249
- Section: Genetics
- URL: https://journals.rcsi.science/0007-4888/article/view/238791
- DOI: https://doi.org/10.1007/s10517-017-3776-0
- ID: 238791
Cite item
Abstract
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.
About the authors
S. V. Kovnir
Institute of Bioengineering, Federal Research Centre “Fundamentals of Biotechnology”, Russian Academy of Sciences
Email: ptichman@gmail.com
Russian Federation, Moscow
N. A. Orlova
Institute of Bioengineering, Federal Research Centre “Fundamentals of Biotechnology”, Russian Academy of Sciences
Email: ptichman@gmail.com
Russian Federation, Moscow
Yu. А. Khodak
Institute of Bioengineering, Federal Research Centre “Fundamentals of Biotechnology”, Russian Academy of Sciences
Email: ptichman@gmail.com
Russian Federation, Moscow
M. P. Kondrashova
Institute of Bioengineering, Federal Research Centre “Fundamentals of Biotechnology”, Russian Academy of Sciences
Email: ptichman@gmail.com
Russian Federation, Moscow
A. G. Gabibov
M. M. Shemyakin and Yu. A. Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences
Email: ptichman@gmail.com
Russian Federation, Moscow
K. G. Skryabin
Institute of Bioengineering, Federal Research Centre “Fundamentals of Biotechnology”, Russian Academy of Sciences
Email: ptichman@gmail.com
Russian Federation, Moscow
A. I. Vorobiev
Hematology Research Center, Ministry of Health of the Russian Federation
Email: ptichman@gmail.com
Russian Federation, Moscow
I. I. Vorobiev
Institute of Bioengineering, Federal Research Centre “Fundamentals of Biotechnology”, Russian Academy of Sciences; M. M. Shemyakin and Yu. A. Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences
Author for correspondence.
Email: ptichman@gmail.com
Russian Federation, Moscow; Moscow
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