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Generation of High-Expressing Cells by Methotrexate Amplification of Destabilized Dihydrofolate Reductase Selection Marker

  • Say Kong NgEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 801)

Abstract

A method combining the use of a destabilized dihydrofolate reductase (DHFR) selection marker with methotrexate (MTX) amplification to generate high-expressing cells is described here. The selection marker expression is weakened with the use of the murine ornithine decarboxylase PEST region and AU-rich element to target the DHFR protein and mRNA, respectively, for degradation in the cell. Cells that produce higher levels of DHFR protein, and the adjoining recombinant protein gene, can compensate for the more rapid turnover of the DHFR protein and survive the selection process. This effect can complement MTX amplification to reduce the amount of MTX and shorten the time needed to generate a high-expressing clone. The gene of interest is first inserted into an expression vector that contains a destabilized DHFR selection marker. The resulting expression vector is then linearized and transfected into suspension CHO-DG44 cells. Selection is performed by culturing the cells in a selection medium lacking hypoxanthine and thymidine. Low concentrations of MTX are then used to amplify the transfected genes for increased protein expression. A single cell cloning protocol is also described. This can be used after each stage of MTX amplification to isolate high-expressing clones that are also consistent producers over longer culture periods.

Key words

CHO Mammalian cell Recombinant protein expression Dihydrofolate reductase Methotrexate amplification MTX Murine ornithine decarboxylase PEST AU-rich element Stable transfection 

Notes

Acknowledgments

The author would like to thank Professor Daniel I.C. Wang and Professor Miranda M.G.S. Yap for their guidance and advice, S.F. Lim for providing the rhIFNγ construct, and support from Bioprocessing Technology Institute (Biomedical Sciences Institutes, A*STAR) and Singapore-MIT Alliance.

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Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  1. 1.Bioprocessing Technology Institute, Agency for ScienceTechnology and Research (A*STAR)SingaporeSingapore

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