Development of recombinant chinese hamster ovary cell lines producing human thrombopoietin analogs = 인간 혈소판증식인자 유도체를 생산하는 재조합 CHO 세포주의 개발

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Recombinant Chinese hamster ovary (CHO) cell lines expressing a high-level of human thrombopoietin were developed by transfecting expression vectors into dihydrofolate reductase (DHFR)-deficient CHO cells and subsequent gene amplification in medium containing stepwise increments in methotrexate (MTX) level such as 20, 80, 320 nM. Out of 1800 initial transfectants, 24 parental clones producing a high-level of TPO were selected and subjected to increasing levels of MTX. The expression analysis of 24 clone lineages during gene amplification revealed that the degree of enhancement in expression level varied significantly among clones. However, we found out that the degree of enhancement of the expression level of clones in the increment of MTX from 0 to 20 nM was proportional to that in the stepwise increment of MTX from 0 to 80 nM. Since most clones produced less TPO above 80 nM MTX, the highest producing and stable subclones were isolated at 80 nM MTX. The maximum TPO concentration and $q_{TPO}$ of hTPO in the established subclones with the highest expression level at 80 nM MTX level in batch culture were 10.3 ㎍/ml and $6.1 ㎍/10^6/cells/day$, respectively. The established subclones at 80 nM MTX had $q_{TPO}$ 10 folds higher than the parental clones. Analysis of a clone lineage showed that the reduction of the expression level at 320 nM was mainly due to the decreased growth rate, not due to the reduction of $q_{TPO}$. Southern and Northern blot analyses showed that the enhanced $q_{TPO}$ resulted mainly from the increased TPO gene copy number and subsequent increased mRNA level. Furthermore, TPOs produced from the established clones were biologically active in vivo as demonstrated by its ability to elevate platelet count in treated mice. To facilitate the establishment of rCHO cell lines with dhfr-mediated gene amplification, a primary selection method based on morphology of parental CHO clones has been developed. Morphology of parental clones that were made b...
Lee, Gyun-Minresearcher이균민researcher
한국과학기술원 : 생물과학과,
Issue Date
177215/325007 / 000985354

학위논문(박사) - 한국과학기술원 : 생물과학과, 2002.8, [ ix, 102 p. ]


Thrombopoietin; 혈소판증식인자

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