Development of CHO cell lines producing erythropoietin (EPO) using site-specific recombination = 위치특이적 재조합 효소를 이용한 EPO 생산 CHO 세포주 개발

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To optimize cell line development process, epigenetic approaches - site-specific recombination and S/MAR DNA regulation elements - were employed. The stability of transgene expression as well as expression level was considered from the initial stages of the cell line development to obtain stable high-level erythropoietin (EPO) producers more easily. Screening of chromosomal locus which guarantees stable and high expression of enhanced green fluorescence protein (EGFP) gene and subsequent integration of the EPO gene into the screened locus should easily provide promising stable high-level EPO producers. The integration of the desired gene into the screened locus can be efficiently mediated under the function of site-specific recombinases. Recombinase-mediated cassette exchange (RMCE) strategy was employed for the efficient site-specific recombination among various site-specific recombination strategies since it has been regarded as one of the most powerful tools for gene targeting. For the stable integration of a transgene by the RMCE strategy, spacer mutant recognition target, which is incompatible with wild-type sequence and mediates efficient site-specific recombination, is indispensable. Therefore, a simple and accurate analysis system for the estimation of recombination efficiency in vivo using fluorescence-activated cell sorting (FACS) was designed and was subsequently used to compare the efficiency of recombination related to different spacer mutant. F3 and F5 mutant sequences were used for Flpe-mediated cassette exchange, and m2 and lox2272 mutant sequences were used for Cre-mediated cassette exchange due to their high incompatibilities with wild-type sequences. The incompatibilities with wild-type were almost the same between mutant sequences. However, the recombination efficiencies were different. F3 and m2 could mediate more efficient recombination than F5 and lox2272, respectively. These results are consistent with the fact that the sequence of spa...
Lee, Gyun-Minresearcher이균민researcher
한국과학기술원 : 생명과학과,
Issue Date
268693/325007  / 020035034

학위논문(박사) - 한국과학기술원 : 생명과학과, 2007. 8, [ x, 112 p. ]


Chinese hamster ovary (CHO) cell; cell line development; site-specific recombination; fluorescence-activated cell sorting (FACS); S/MAR element; 세포주 개발; 위치특이적 재조합; Chinese hamster ovary (CHO) cell; cell line development; site-specific recombination; fluorescence-activated cell sorting (FACS); S/MAR element; 세포주 개발; 위치특이적 재조합

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