Analysis of alterations in gene expression after amplification of recombinant genes in CHO cells

Dihydrofolate reductase (DHFR) based amplification of recombinant genes usi ng increasing concentrations of methotrexate (MTX) is a common method to es tablish CHO cell lines producing high amounts of the desired protein. Once, cell lines with highly amplified target genes and good expression rates ar e isolated, further characterization of their transcriptional pattern is in tended to clarify the question what other factors are elevated, as a prereq uisite or consequence of recombinant protein production. In order to define genes which are upregulated in a cell line that shows high production rate s, we have investigated alterations in gene expression which occur beside a mplification of the recombinant genes. For this purpose. the suppression su btractive hybridization method was used to create a cDNA library enriched f ur differentially expressed sequences in the recombinant antibody producing CHO cell line versus the original counterpart. Differential expression was confirmed by Northern blotting and Northern ELISA. In addition to the expe cted recombinant genes, we have identified 5 transcripts which are upregula ted in the recombinant cell line. One sequence has not been found in existi ng data bases, the others revealed to be genes involved in protein synthesi s and regulation of transcription. Furthermore, an alternatively spliced, n on-functional form of the DHFR mRNA was detected, suggesting a dramatic inc rease of the selection pressure exerted by MTX. (C) 2001 Elsevier Science B .V. All rights reserved.