• 主题
高级搜索  >
历史搜索 清空历史
首页> 外文期刊>Biotechnology Progress >Limitations to the Development of Humanized Antibody Producing Chinese Hamster Ovary Cells Using Glutamine Synthetase-Mediated Gene Amplification
【24h】

Limitations to the Development of Humanized Antibody Producing Chinese Hamster Ovary Cells Using Glutamine Synthetase-Mediated Gene Amplification

机译：谷氨酰胺合成酶介导的基因扩增对人源化抗体生产中国仓鼠卵巢细胞发展的限制

获取原文
获取原文并翻译 | 示例
           
页面导航
  • 摘要
  • 著录项
  • 相似文献
  • 相关主题

摘要

Recombinant Chinese hamster ovary (CHO)cells expressing a humanized antibody were obtained by transfection of an antibody expression vector (pKC-GS-HC-huS)into CHO-K1 cells and subsequent glutamine synthetase (GS)-mediated gene amplification in media containing different concentrations of methionine sulfoximine (MSX).Concentrations consisted of 25,200,500,and 1000 mu M of MSX.The highest producer (HP)subclones were isolated from each MSX level by the limiting dilution method and were characterized with respect to antibody production.No positive relationship was observed between specific antibody productivity (q_(Ab))and MSX concentration.Furthermore,it was found that the antibody production stability of these subclones was very poor even in the presence of selection pressure.During long-term cultures in the presence of the corresponding concentrations of MSX,q_(Ab)of all HP subclones significantly decreased for the first six passages and thereafter stabilized.Southern and slot blot analyses showed that the loss of antibody gene copies was only partially responsible for the decreased q_(Ab).Fluorescence in situ hybridization (FISH)analysis revealed some cytogenetic features indicative of antibody production instability.Unstable chromosomal structures including dicentrics,rings,and extremely long chromosomes were observed.Amplified sequences enclosed in nuclear projections were often observed.The telomeric repeat sequence,which may be involved in the stabilization of amplified arrays,was found to be absent at the ends of most marker chromosomes.Furthermore,FISH analysis revealed that the overall chromosome content was duplicated in some HP subclones.When metaphase of 12 high producing parental clones was examined,the frequency of occurrence of the polyploidy was 25%.Taken together,the data obtained here suggests that instability could be a concern in the development of CHO cells with GS-mediated gene amplification.
机译：表达人源化抗体的重组中国仓鼠卵巢（CHO）细胞是通过将抗体表达载体（pKC-GS-HC-huS）转染到CHO-K1细胞中并随后在含有不同氨基酸的培养基中谷氨酰胺合成酶（GS）介导的基因扩增而获得的浓度由25,200,500和1000μM的MSX组成。通过极限稀释法从每个MSX级别中分离出最高生产者（HP）亚克隆，并针对抗体生产进行了表征。在特异性抗体生产率（q_（Ab））和MSX浓度之间观察到。此外，发现即使在选择压力下，这些亚克隆的抗体生产稳定性也很差。在最初的六次传代中，所有HP亚克隆的相应MSX，q_（Ab）浓度均显着降低，然后稳定下来。裂解结果显示，抗体基因拷贝的丢失仅是q_（Ab）降低的部分原因。荧光原位杂交（FISH）分析显示了一些细胞遗传学特征，表明抗体产生不稳定。不稳定的染色体结构包括双着丝粒，环和极长的观察到染色体，经常观察到被核投影包围的扩增序列。发现大多数标记染色体的末端不存在可能与扩增阵列稳定有关的端粒重复序列。此外，FISH分析表明在某些HP亚克隆中，总染色体含量重复。当检查12个高产亲本克隆的中期时，多倍体的发生频率为25％。综上所述，此处获得的数据表明，不稳定性可能是胚胎发育的一个问题。 CHO细胞具有GS介导的基因扩增。

著录项

相似文献

  • 外文文献
  • 中文文献
  • 专利
获取原文

客服邮箱：kefu@zhangqiaokeyan.com

京公网安备：11010802029741号 ICP备案号：京ICP备15016152号-6 六维联合信息科技 (北京) 有限公司©版权所有

  • 客服微信

  • 服务号