| Title: | Transcriptome study and identification of potential marker genes related to the stable expression of recombinant proteins in CHO clones |
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| Authors: | ID Blejec, Andrej (Author)
ID Blas, Marjanca (Author)
ID Nikolić, Petra (Author)
ID Gaser, Dominik (Author)
ID Gruden, Kristina (Author)
ID Belič, Aleš (Author)
ID Baebler, Špela (Author)
ID Jamnikar, Uroš (Author)
ID Francky, Andrej (Author)
ID Laux, Holger (Author) |
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| Files: |  URL - Presentation file, visit http://dx.doi.org/10.1186/s12896-015-0218-9
 PDF - Presentation file, download (1,20 MB)
MD5: 62F70A28E2906261619890C2521FB735
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| Language: | English |
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| Typology: | 1.01 - Original Scientific Article |
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| Organization: |  NIB - National Institute of Biology
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| Abstract: | Background
Chinese hamster ovary (CHO) cells have become the host of choice for the production of recombinant proteins, due to their capacity for correct protein folding, assembly, and posttranslational modifications. The most widely used system for recombinant proteins is the gene amplification procedure that uses the CHO-Dhfr expression system. However, CHO cells are known to have a very unstable karyotype. This is due to chromosome rearrangements that can arise from translocations and homologous recombination, especially when cells with the CHO-Dhfr expression system are treated with methotrexate hydrate. The present method used in the industry for testing clones for their long-term stability of recombinant protein production is empirical, and it involves their cultivation over extended periods of time prior to the selection of the most suitable clone for further bioprocess development. The aim of the present study was the identification of marker genes that can predict stable expression of recombinant genes in particular clones early in the development stage.
Results
The transcriptome profiles of CHO clones with stable and unstable recombinant protein production were investigated over 10-weeks of cultivation, using a DNA microarray. We identified 14 genes that were differentially expressed between the stable and unstable clones already at 2 weeks from the beginning of the cultivation. Their expression was validated by reverse-transcription quantitative real-time PCR (RT-qPCR). Furthermore, the k-nearest neighbour algorithm approach shows that the combination of the gene expression patterns of only five of these 14 genes is sufficient to predict stable recombinant protein production in clones in the early phases of cell-line development.
Conclusions
The exact molecular mechanisms that cause unstable recombinant protein production are not fully understood. However, the expression profiles of some genes in clones with stable and unstable recombinant protein production allow prediction of such instability early in the cell-line development stage. We have thus developed a proof-of-concept for a novel approach to eliminate unstable clones in the CHO-Dhfr expression system, which saves time and labour-intensive work in cell-line development. |
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| Keywords: | CHO cell line, stable recombinant protein production, gene expression, RT-qPCR, DNA microarray, mMarker genes |
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| Publication status: | Published |
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| Publication version: | Version of Record |
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| Publication date: | 23.10.2015 |
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| Year of publishing: | 2015 |
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| Number of pages: | str. 1-10 |
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| Numbering: | Vol. 15, no. 98 |
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| PID: | 20.500.12556/DiRROS-5777  |
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| ISSN: | 1472-6750 |
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| UDC: | 577 |
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| DOI: | 10.1186/s12896-015-0218-9 |
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| COBISS.SI-ID: | 3715663 |
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| Publication date in DiRROS: | 29.07.2024 |
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| Views: | 340 |
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| Downloads: | 329 |
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