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

Paper ID Volume ID Publish Year Pages File Format Full-Text
24235 43506 2010 8 PDF Available
Title
Generation of stable cell lines by site-specific integration of transgenes into engineered Chinese hamster ovary strains using an FLP-FRT system
Abstract

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.

Keywords
Recombinant protein; Site-specific integration; Transcriptionally active sites; Chinese hamster ovary (CHO)
First Page Preview
Generation of stable cell lines by site-specific integration of transgenes into engineered Chinese hamster ovary strains using an FLP-FRT system
Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 147, Issue 2, 17 May 2010, Pages 122–129
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering