Cell cycle-dependent DHFR and t-PA production in cotransfected, MTX-amplified CHO cells revealed by dual-laser flow cytometry

Experimental Cell Research
M Kubbies, H Stockinger

Abstract

The cell cycle-dependent regulation of the cellular dihydrofolate reductase content (DHFR) and tissue plasminogen activator (t-PA) production and secretion in plasmid-amplified cells was investigated in the DHFR-negative CHO cells transfected with the plasmid pSV-tPA.dhfr. This plasmid, carrying the dhfr and t-PA gene under control of different promotors, was amplified by serial passages in 5 microM methotrexate (MTX) for dhfr gene amplification. The intracellular amount of DHFR was quantitated in viable cells by MTX-FITC labeling and flow cytometric analysis of the FITC fluorescence. In comparison with the original CHO cells, the pSVtPA.dhfr-amplified cells showed a greater than 230-fold increase in MTX-FITC fluorescence. Using dual laser flow cytometry (uv: vital cell cycle with Hoechst 33342; 488 nm: DHFR with MTX-FITC), we show a maximum increase in the intracellular DHFR content during G1 and/or at G1/S transition (100 to 157%), followed by a continuous increase to 200% during S and G2/M. To determine t-PA production CHO cells were sorted from G1-, early/late S-, and G2/M-phase. After 1-, 2-, and 4-h incubation periods, t-PA production was quantitated using a sensitive t-PA ELISA technique. We found that t-PA production an...Continue Reading

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Citations

May 5, 1997·Biotechnology and Bioengineering·K UchiyamaS Shioya
Jul 19, 2008·Biotechnology and Bioengineering·M FusseneggerJ E Bailey
Oct 23, 2001·Biotechnology and Bioengineering·S Frykman, F Srienc
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Oct 24, 2003·Biotechnology and Bioengineering·Simone M SchatzFriedrich Scheiflinger
Feb 14, 2019·Journal of Clinical Medicine·Rabia Mukhtar RanaKeun Woo Lee

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