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KMID : 0545120030130050759
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Journal of Microbiology and Biotechnology
2003 Volume.13 No. 5 p.759 ~ p.766
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Development of Recombinant Chinese Hamster Ovary Cell Lines Producing Human Thrombopoietin or Its Analog
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CHUNG, JOO YOUNG
AHN, HAE KYUNG/LIM, SEUNG WOOK/SUNG, YUN HEE/KOH, YEO WOOK/PARK, SEUNG KOOK/LEE, GYUN MIN
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Abstract
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Recombinant Chinese hamster ovary (rCHO) cell lines expressing a high level of human thrombopoietin (hTPO) or its analog, TP033r, were obtained by transfecting expression vectors into dihydrofolate reductase-deficient (dhfr ) CHO cells and subsequent gene amplification in media containing stepwise increments in methotrexate (MTX) level such as 20, 80, and 320 nM. The parental clones with a hTPO expression level >0.40 ㎍/ml (27 out of 1,200 clones) and the parental clones with a TP033r expression level >0.20 ㎍/ml (36 out of 400 clones) were subjected to 20nM MTX. The clones that displayed an increased expression level at 20 nM MTX were subjected to stepwise increasing levels of MTX such as 80 and 320 nM. When subjected to 320 nM MTX, most clones did not display an increased expression level, since the detrimental effect of gene amplification on growth reduction outweighed its beneficial effect of specific TPO productivity (q_(TPO)) enhancement at 320 nM MTX. Accordingly, the highest producer subclones ( 1 -434-80^(＊) for hTPO and 2-3-80" for TP033r), whose q_(TPO) was 2- to 3-fold higher than that of their parental clones selected at 80 nM MTX, were isolated by limiting dilution method and were established as rCHO cell lines. The q_(TPO) of 1-434-80_(＊) and 2-3-80_(＊) was 5.89±0.74 and 1.02±0.23 ㎍/10^(6) cells/day, respectively. Southern and Northern blot analyses showed that the enhanced q_(TPO) of established rCHO cell lines resulted mainly from the increased TPO gene copy number and subsequent increased TPO mRNA level. The hTPO and TP033r produced from the established rCHO cell lines were biologically active in vivo, as demonstrated by their ability to elevate platelet counts in treated mice.
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KEYWORD
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