title: Amplified expression constructs for human tissue-type plasminogen activator in Chinese hamster ovary cells: instability in the absence of selective pressure.
abstract: By linking an expression cassette for human tissue-type plasminogen activator (t-PA) to an amplifiable marker gene, its introduction into Chinese hamster ovary dhfr- cells and subsequent amplification with methotrexate, we have generated cell lines that overproduce the heterologous protein and contain 300-1100 copies of the expression constructs integrated into the hamster genome. We present a detailed investigation of the fate of amplified sequences in the presence and absence of selective pressure by parallel examination of three producer cell lines with respect to relevant parameters. These include the determination of t-PA production upon continuous propagation in culture, the genomic organization of the integrated expression constructs by Southern blotting, and the localization of homogeneously staining regions by in-situ hybridization with biotinylated probes and visualization by interference reflection microscopy. We conclude that in the three cell lines examined, the decrease in production of t-PA in the absence of methotrexate selection is accompanied by decreases in the number of integrated expression constructs and the size of the amplified regions, whereas all these parameters are stable when selective pressure is maintained. The instability is probably due to the head-to-tail mode of integration of the expression constructs in the hamster genome, which increases the frequency of homologous recombination between the integrated plasmids in recombination-proficient cells in the absence of selective pressure.
mesh_terms: D000818:Animals; D002874:Chromosome Mapping; D002999:Clone Cells; D006224:Cricetinae; D004274:DNA, Recombinant; D005784:Gene Amplification; D005786:Gene Expression Regulation; D006801:Humans; D007163:Immunosorbent Techniques; D008727:Methotrexate / Q000494:pharmacology; D009693:Nucleic Acid Hybridization; D010957:Plasmids; D012641:Selection, Genetic; D010959:Tissue Plasminogen Activator / Q000235:genetics*; D014162:Transfection
chemical_list: D004274:DNA, Recombinant; D010959:Tissue Plasminogen Activator; D008727:Methotrexate