Coamplification and coexpression of human tissue-type plasminogen activator and murine dihydrofolate reductase sequences in Chinese hamster ovary cells.

Abstract

Expression of human tissue-type plasminogen activator (t-PA) at high levels has been achieved in Chinese hamster ovary (CHO) cells by cotransfection and subsequent coampiffication of the transfected sequences. Expression vectors containing the t-PA cDNA gene and a dihydrofolate reductase (DHFR) cDNA gene were cotransfected into CHO DHFR-deficient cells. Transformants expressing DHFR were selected by growth in media lacking nucleosides and contained low numbers of t-PA genes and DHFR genes. Stepwise selection of the DHFR+ transformants in increasing concentrations of methotrexate generated cells which had amplified both DHFR genes and t-PA genes over 100-fold. These cell lines expressed elevated levels of enzymatically active t-PA. To optimize both t-PA sequence amplification and t-PA expression, various modifications of the original procedure were used. These included alterations to the DHFR expression vector, optimization of the molar ratio of t-PA to DHFR sequences in the cotransfection, and modification of the methotrexate resistance selection procedure. The structure of the amplified DNA, its chromosomal location, and its stability during growth in the absence of methotrexate are reported.

A study of monoclonal antibody‐producing CHO cell lines: What makes a stable high producer?

“…The specific productivities of clones 2.9 and 2.12 increased only to a level of about 5 pg cell À1 day À1 during amplification, whereas clone 2.11 reached a specific productivity of 36 pg cell À1 day À1 , corresponding to 32-fold increase as compared to the non-amplified clone, at the 500 nM MTX amplification stage whereupon it decreased again. A similar decline in recombinant protein expression at higher selective pressure during stepwise MTX selection has been reported previously (Fann et al, 2000;Kaufman et al, 1985;Kim et al, 2001;Pendse et al, 1992). For example, a 12.5-fold increase in specific productivity to 8 pg cell À1 day À1 with increasing MTX level of up to 320 nM was observed when gradually gene-amplifying a CHO parental clone expressing a humanized antibody against S surface antigen of Hepatitis B virus.…”

Identification of transgene integration loci of different highly expressing recombinant CHO cell lines by FISH

“…Whereas association of amplified DNA with HSRs is often observed in highly and stably producing cell lines (Kaufman et al 1985), amplified genes localised to DMs are often lost, especially in the absence of selective pressure (Kaufman et al 1983;Wahl et al 1982).…”

“…Furthermore, signals on small derivative chromosomes or fragments are reported (Pallavicini et al 1990), and small chromosomes might disappear (Kaufman et al 1985). Yoshikawa et al (2000a, b) investigated the location of the insertion site on the chromosome and analysed a recombinant cell line adapted to different concentrations of Methotrexate (MTX) and distinguished three different types of recombinant cell lines regarding the insertion site of the transgene: telomeric type cells had integrated the exogenous target together with the selection marker into the telomeric region, while the two other groups showed integration to other chromosomal regions or did not show any signal in FISH analysis.…”

Expression of human pancreatic polypeptide precursors from a dicistronic mRNA in mammalian cells

“…In COS cells [30] they also demonstrated expression from a tricistronic transcript which included the three abovementioned cDNAs. Human plasminogen activator cDNA (tPA) has also been expressed [31] from a dicistronic construct in CHO DUKX-Bll cells, and we have obtained expression of human clotting factor IX (FIX), E. coli chloramphenicol acetyltransferase (CAT) and mouse DHFR from di-and tricistronic vectors transfected into BHK cells (Berkner et al, in preparation). The neomycin gene (neo) from Tn5 [32] can be used as well to obtain stable transformants from polycistronic transcripts.…”