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Exploiting Cell Cycle Phase Specific Expression of Foreign Genes in Perfusion Cultures of Recombinant CHO Cells

利用重组 CHO 细胞灌注培养中外源基因的细胞周期阶段特异性表达

基本信息

批准号：
9504840
负责人：
Dhinakar Kompala
金额：
$ 47.13万
依托单位：
University of Colorado at Boulder
依托单位国家：
美国
项目类别：
Continuing Grant
财政年份：
1995
资助国家：
美国
起止时间：
1995-08-01 至 1999-07-31
项目状态：
已结题

来源：
https://www.nsf.gov/awardsearch/showAward?AWD_ID=9504840&HistoricalAwards=false
关键词：
Exploiting Cell Cycle Phase Specific

项目摘要

9504840 Kompala This project is on research to characterize the dependence of foreign gene expression on cell cycle phase of recombinant mammalian cells, and to exploit this dependency for maximizing the production of heterologous glycoproteins in high cell density perfusion bioreactors. The three major objectives of the proposed research are: (1) to investigate the hypothesized key role of promoters in determining the cell cycle phase in which the foreign proteins are expressed in recombinant Chinese Hamster Ovary (CHO) cells by using the two-parameter flow cytometic analysis, and to identify and characterize a strong G1 phase-specific promoter for maximizing foreign protein expression at low growth rates; (2) to investigate whether the two opposing trends in the relationship between the specific production rate and the cell growth in continuous suspension cultures of recombinant CHO cultures are caused by G1- and S-phase specific expression from the upstream promoters, and to exploit a strong G1 phase specific promoter to maximize the production of a secreted foreign protein in perfusion bioreactors with attached inclined settlers; and (3) to investigate whether the smaller dying apoptotic cells can be removed before their intracellular glycosidases and proteases are released into the medium. ***

9504840 Kompala该项目是关于表征外源基因表达对重组哺乳动物细胞的细胞周期阶段的依赖性的研究，并利用这种依赖性在高细胞密度灌注生物反应器中最大化异源糖蛋白的生产。的三大目标拟开展的研究是：（1）通过使用双参数流式细胞术分析来研究启动子在决定外源蛋白在重组中国卵巢（CHO）细胞中表达的细胞周期时相中的假设关键作用，并确定和表征强 G1期特异性启动子，用于在低生长速率下最大化外源蛋白表达;（2）研究连续悬浮培养中比生产速率与细胞生长之间关系的两种相反趋势是否存在的重组CHO培养物是由来自上游启动子的G1期和S期特异性表达引起的，并且利用强G1期特异性启动子来最大化具有连接的倾斜沉降器的灌注生物反应器中分泌的外源蛋白的生产;和（3）研究是否可以在较小的垂死凋亡细胞的胞内糖苷酶和蛋白酶释放到培养基中之前将其除去。 ***