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J Biotechnol. 2010 May 17;147(2):122-9. doi: 10.1016/j.jbiotec.2010.03.020. Epub 2010 Apr 3.

Generation of stable cell lines by site-specific integration of transgenes into engineered Chinese hamster ovary strains using an FLP-FRT system.

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  • 1Laboratory for Protein Engineering, Institute of Biotechnology, 20 Dongdajie Street, Fengtai District, Beijing 100071, China.


Random integration linking genomic amplification is widely used to generate desired cell lines for stable and high-level expressing recombinant proteins. But this technique is laborious, and the expression level is unpredictable due to position effects. After reading many reports on gene amplification, we hypothesized that there should be several loci in the genome of Chinese hamster ovary (CHO) cells that allow not only high-level, but also stable gene expression. Based on this hypothesis, we constructed a plasmid pMCEscan, which introduces a site-specific recombinase-recognition sequence, FRT, for gene targeting into those sites. Another targeting vector, pcDNA5/FRT, has an FRT sequence fused to a promoterless hygromycin-resistance gene that can be expressed only when correct gene targeting occurs. Using the pMCEscan, which is a novel and stringent selection system used to create a few high protein-producing clones, we constructed engineered CHO strains that can be used for high-level production of foreign proteins by gene targeting. We selected 28 CHO strains that expressed a high-level of reporter genes and carried one copy of the pMCEscan in their chromosomes, and we treated these strains with methotrexate (MTX) to evaluate dihydrofolate reductase (DHFR)-mediated gene amplification. Nine clones showed high-level tissue plasminogen activator (tPA) production without amplification. We then targeted other genes (tPA, secreted alkaline phosphatase (SEAP), erythropoietin (EPO)) to test the basal expression ability of nine CHO strains. CHO strains 8-1 and 8-11 consistently expressed high basal levels of the recombinant genes. Using this cell-vector system, we obtained the tPA stable high producers by gene targeting and gene amplification. This system allows for rapid generation of recombinant proteins without cloning and greatly simplifies selection of cell lines for the production of potential therapeutic proteins.

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