Abstract
Recombinant CHO cell lines have integrated the expression vectors in various parts of the genome leading to different levels of gene amplification, productivity and stability of protein expression. Identification of insertion sites where gene amplification is possible and the transcription rate is high may lead to systems of site-directed integration and will significantly reduce the process for the generation of stably and highly expressing recombinant cell lines. We have investigated a broad range of recombinant cell lines by FISH analysis and Giemsa–Trypsin banding and analysed their integration loci with regard to the extent of methotrexate pressure, transfection methods, promoters and protein productivities. To summarise, we found that the majority of our high producing recombinant CHO cell lines had integrated the expression construct on a larger chromosome of the genome. Furthermore, except from two cell lines, the exogene was integrated at a single site. The dhfr selection marker was co-localised to the target gene.
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Abbreviations
- DIG:
-
digoxigenin
- GCN:
-
gene copy number
- HGP:
-
highly glycosylated protein
- MCB:
-
master cell bank
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Acknowledgements
This research was kindly funded by ACBT (Austrian Center of Biopharmaceutical Technology), a competence centre supported by the Federal Ministry of Economy and Labour and the federal states of Vienna and Tyrol. The authors are grateful to Annalisa Lasagna for analysis of product concentrations and to Friedemann Hesse for carefully reviewing the manuscript.
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Lattenmayer, C., Loeschel, M., Steinfellner, W. et al. Identification of transgene integration loci of different highly expressing recombinant CHO cell lines by FISH. Cytotechnology 51, 171–182 (2006). https://doi.org/10.1007/s10616-006-9029-0
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DOI: https://doi.org/10.1007/s10616-006-9029-0