Analysis of alterations in gene expression after amplification of recombinant genes in CHO cells.
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.
Grillari
J
J
Institute of Applied Microbiology, University of Agricultural Sciences, Muthgasse 18, A-1190, Vienna, Austria. j.grillari@iam.boku.ac.at
Fortschegger
K
K
Grabherr
R M
RM
Hohenwarter
O
O
Kunert
R
R
Katinger
H
H
D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000378:metabolism; D001219:Aspartate Aminotransferases / Q000235:genetics; D016466:CHO Cells; D006224:Cricetinae; D020869:Gene Expression Profiling; D006801:Humans; D017403:In Situ Hybridization / Q000379:methods; D008250:Lysine-tRNA Ligase / Q000235:genetics; D008969:Molecular Sequence Data; D011506:Proteins / Q000235:genetics; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics* / Q000378:metabolism; D012269:Ribosomal Proteins / Q000235:genetics; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic; D015854:Up-Regulation
D000911:Antibodies, Monoclonal; C433743:KL10 protein, Cricetulus griseus; D011506:Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; D012269:Ribosomal Proteins; C031249:ribosomal protein L6; D013762:Tetrahydrofolate Dehydrogenase; D001219:Aspartate Aminotransferases; D008250:Lysine-tRNA Ligase