Generation of high-expressing cells by methotrexate amplification of destabilized dihydrofolate reductase selection...

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.