High level expression of human proteins in murine hybridoma cells: induction by methotrexate in the absence of gene amplification.

We have developed an inducible system for high level expression of heterologous genes in murine hybridoma cells. The rapid induction by methotrexate (MTX) does not involve gene amplification and is controlled at the level of mRNA accumulation. Transfection was achieved by protoplast fusion with an expression vector containing the cDNA of interest and a marker gene encoding dihydrofolate reductase. The initial clones, selected at 100 nM MTX, produced high levels of the protein of interest and contained about 100-400 copies of the integrated plasmid DNA. They could adapt to a 100- to 1000-fold stepwise increase in MTX concentration in a few weeks, during which the expression of the gene of interest but not its copy number, increased several-fold. Furthermore, the induction is freely reversible. If cells were propagated in MTX-free media, the expression level decreased, but the cells could be reinduced to their original high level of expression by adding MTX back to the media. A several-fold increase in the mRNA levels of the dihydrofolate reductase and the gene of interest could be detected after induction for 18 h.