Development of recombinant Chinese hamster ovary cell lines producing human thrombopoietin or its analog

摘要: Recombinant Chinese hamster ovary (rCHO) cell lines expressing a high level of human thrombopoietin (hTPO) or its analog, TPO33r, were obtained by transfecting expression vectors into dihydrofolate reductase-deficient (dhfr - ) CHO cells and subsequent gene amplification in media containing stepwise increments methotrexate (MTX) such as 20, 80, 320 nM. The parental clones with hTPO >0.40 μg/ml (27 out 1,200 clones) the TPO33r >0.20 (36 400 subjected to 20 nM MTX. that displayed an increased at MTX increasing levels 80 When MTX, most did not display level, since detrimental effect on growth reduction outweighed beneficial specific TPO productivity (q enhancement Accordingly, highest producer subclones (1-434-80* for 2-3-80* TPO33r), whose q was 2- 3-fold higher than their selected isolated limiting dilution method established rCHO lines. 1-434-80* 5.89± 0.74 1.02±0.23 μg/10 6 cells/day, respectively. Southern Northern blot analyses showed enhanced resulted mainly from copy number mRNA level. produced biologically active vivo, demonstrated ability elevate platelet counts treated mice.