Abstract
Dihydrofolate reductase (DHFR)-mediated gene amplification has been widely used to establish high-producing mammalian cell lines [1-3]. However, since gene amplification is an infrequent event, in that many rounds of methotrexate (MTX) selection to amplify the transgene and screening of over several hundred individual clones are required to obtain cells with high gene copy numbers [4]. Consequently, the process for DHFR-mediated gene amplification is a time-consuming and laborious step for cell line construction. Here, we present a novel concept to accelerate gene amplification through cell cycle checkpoint engineering [5]. In our knowledge, there is no previous report which focused on controlling cell cycle checkpoint to enhance the efficiency of DHFR gene amplification system.
Highlights
Dihydrofolate reductase (DHFR)-mediated gene amplification has been widely used to establish high-producing mammalian cell lines [1-3]
Analysis of green fluorescent protein (GFP) expression level during gene amplification process The ratio of GFP-expressing cells was evaluated by flow cytometry analysis during the gene amplification process at 100, 250, and 500-nM MTX concentrations
The volumetric productivity of ATR knockdown cells was an average of 0.035 mg L−1 day−1, which was approximately three times higher than the average of 0.013 mg L−1 day−1 of the mock cells, suggesting that ATR knockdown generated the pool of higher-producing cells during the gene amplification process
Summary
Dihydrofolate reductase (DHFR)-mediated gene amplification has been widely used to establish high-producing mammalian cell lines [1-3]. Since gene amplification is an infrequent event, in that many rounds of methotrexate (MTX) selection to amplify the transgene and screening of over several hundred individual clones are required to obtain cells with high gene copy numbers [4]. The process for DHFR-mediated gene amplification is a time-consuming and laborious step for cell line construction. We present a novel concept to accelerate gene amplification through cell cycle checkpoint engineering [5]. There is no previous report which focused on controlling cell cycle checkpoint to enhance the efficiency of DHFR gene amplification system
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