在CHO-K1细胞中使用CRISPR/Cas9修饰的捐赠体进行高效的特定位点的集成
Mohammad Hassan Kheirandish1,2, Behnaz Rahmani2,3, Hossein Zarei Jaliani4
1Medical Biotechnology Department, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran.
Molecular biology reports
|May 27, 2023
概括
这项研究使用新的方法增强了CRISPR介导的基因集成在CHO细胞中,提高了重组蛋白质开发的效率和生产率. 这些策略加快了稳定,高表达细胞系的生成.
科学领域:
- 生物技术是生物技术.
- 分子生物学分子生物学
- 细胞线工程 细胞线工程
背景情况:
- 传统的重组CHO (rCHO) 细胞系的发展依赖于随机集成,导致漫长的克隆选择过程.
- CRISPR/Cas9为同质克隆提供了特定站点的集成和更快的选择,但需要高效的集成率和稳定的表达站点.
- 在优化CRISPR介导的整合效率和识别适合的基因组位点以维持rCHO细胞中转基因表达方面仍然存在挑战.
研究的目的:
- 使用CRISPR/Cas9.9提高GFP记者在CHO-K1细胞中的染色体3 (Chr3) 伪 ATP位点的整合率.
- 评估基于PCR的供体线性化和mSA-生物素结合的有效性,以增强特定站点的整合.
- 通过使用表达盒准 Chr3 伪attP 位点来评估生成的重组细胞系的生产力.
主要方法:
- 用CRISPR/Cas9对CHO-K1细胞中Chr3伪-attP位点进行特定位点基因向.
- 利用两种策略来提高敲门效率:基于PCR的供体线性化和mSA-生物素绑定来增加本地供体度.
- 使用定量PCR量化整合效率和评估单副本整合.
主要成果:
- 与传统的CRISPR向相比,捐赠者线性化和绑定方法的敲进效率分别提高了1.6倍和2.4倍.
- 定量PCR证实,84%和73%的目标克隆实现了单拷贝集成与各自的方法.
- 通过绑定将hrsACE2表达盒定向到Chr3伪attP位点,与随机集成细胞系相比,生产率增加了2倍.
结论:
- 开发并验证了可靠的策略,以提高CHO细胞中CRISPR介导的基因整合效率.
- 确定了 Chr3 伪attP 位点,作为 rCHO 细胞系发育中持续转基因表达的有希望的基因.
- 这些发现为加速开发用于生物制药生产的高性能rCHO细胞系提供了途径.
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