Journal of Biotechnology 87 (2001) 59 – 65 Analysis of alterations in gene expression after amplification of recombinant genes in CHO cells Johannes Grillari *, Klaus Fortschegger, Reingard M. Grabherr, Otmar Hohenwarter, Renate Kunert, Hermann Katinger Institute of Applied Microbiology, Uni6ersity of Agricultural Sciences, Muthgasse 18, A-1190 Vienna, Austria Received 14 August 2000; received in revised form 1 December 2000; accepted 11 December 2000 Abstract Dihydrofolate reductase (DHFR) based amplification of recombinant genes using increasing concentrations of methotrexate (MTX) is a common method to establish CHO cell lines producing high amounts of the desired protein. Once, cell lines with highly amplified target genes and good expression rates are isolated, further characterization of their transcriptional pattern is intended to clarify the question what other factors are elevated, as a prerequisite or consequence of recombinant protein production. In order to define genes which are upregulated in a cell line that shows high production rates, we have investigated alterations in gene expression which occur beside amplification of the recombinant genes. For this purpose, the suppression subtractive hybridization method was used to create a cDNA library enriched for 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 expected recombinant genes, we have identified 5 transcripts which are upregulated in the recombinant cell line. One sequence has not been found in existing data bases, the others revealed to be genes involved in protein synthesis and regulation of transcription. Furthermore, an alternatively spliced, non-functional form of the DHFR mRNA was detected, suggesting a dramatic increase of the selection pressure exerted by MTX. © 2001 Elsevier Science B.V. All rights reserved. www.elsevier.com/locate/jbiotec 1. Introduction CHO cell lines are a widely used expression system to produce recombinant proteins. In order to achieve high production rates, a dihydrofolate reductase (DHFR) negative CHO cell line (Urlaub and Chasin, 1980) is transfected with an expression vector containing a functional DHFR gene in combination with the gene of interest. Amplification of the inserted genes occurs in re- sponse to addition of increasing amounts of the DHFR antagonist methotrexate (MTX) to the culture medium and clones or subpopulations car- rying multiple copies of the recombinant genes can be selected (reviewed e.g. by Wurm (1990)). * Corresponding author. Tel.: +43-1-360066230; fax: +43- 1-3697615. E-mail address: j.grillari@iam.boku.ac.at (J. Grillari). 0168-1656/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved. PII:S0168-1656(00)00431-4