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该项目正在研究中，以表征外国基因表达对重组哺乳动物细胞的细胞周期阶段的依赖性，并利用这种依赖性以最大程度地提高高细胞密度灌注生物反应器中异源糖蛋白的产生。拟议研究的三个主要目标是：（1）调查启动子在确定重组中国仓鼠卵巢（CHO）细胞中表达外蛋白在细胞周期阶段中的主要作用，并使用两参数流式流式细胞分析，以及确定和表征强大的G1阶段启动子来降低异源蛋白质的增长和表征和表征最大化的外国蛋白质的较低的蛋白质，以达到最大化的蛋白质的增长率；（2）调查特异性生产率和细胞生长之间的两个相反趋势是否是由上游启动子的G1-和S阶段特异性表达引起的连续悬架培养物，并在上游启动子中表达特定的表达，并利用强大的G1相位特异性启动子来最大程度地提高与附着的垂直于附及器的灌注型外国蛋白质的产生。（3）研究是否可以在细胞内糖苷酶和蛋白酶释放到培养基中之前去除较小的垂死凋亡细胞。 ***