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Updated: Jun 17, 2025

CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy

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Published on: March 12, 2018

通过使用CRISPR/RMCE混合系统的循环miR-21诱的有针对性的集成来进行CHO细胞工程

Setare Adibzadeh^1,2, Shahin Amiri^1,2, Farzaneh Barkhordari¹

¹Department of Medical Biotechnology, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran.

Applied microbiology and biotechnology

|August 9, 2024

概括

中国仓鼠卵巢 (CHO) 细胞是生物制药生产的关键,但面临生长限制. 设计这些细胞以表达圆形miR-21诱 (CM21D) 增强了生长和重组蛋白产量.

科学领域:

生物技术是生物技术.
细胞生物学细胞生物学
分子生物学分子生物学

背景情况:

中国仓鼠卵巢 (CHO) 细胞是工业重组蛋白质生产的主要宿主,对制药至关重要.
生物制药的哺乳动物细胞培养受到细胞限制的阻碍,包括生长和生产率低下.
微RNA-21 (miR-21) 影响恶性瘤;然而,其在CHO细胞中的过度表达会降低生长和生产力.

研究的目的:

为了设计一个重组CHO (rCHO) 细胞系来增强重组蛋白质的生产.
研究miR-21海绵对CHO细胞生长和生产力的影响.
开发一种高效的基因工程策略,以改善CHO细胞系.

主要方法:

利用CRISPR介导的精确整合到目标染色体 (CRIS-PITCh) 通过核糖蛋白 (RNP) 输送进行着陆插入.
使用Bxb1重组酶介导磁带交换 (RMCE) 插入一个圆形miR-21诱 (CM21D) 磁带.
评估了CM21D表达,miR-21目标基因调节,细胞生长动力学和复合蛋白产量 (hrsACE2).

主要成果:

CRIS-PITCh和RMCE/Bxb1系统的目标效率提高了1.7倍,减少了目标外影响.
CM21D表达导致miR-21向基因 (Pdcd4,Atp11b) 的显著上调.
工程化rCHO细胞的生长速度增加了1.3倍,hrsACE2产量增加了2.06倍.

结论:

通过CM21D表达进行海绵miR-21有效地提高了CHO细胞生长率和生产力.
CM21D工程方法是改善工业CHO电池线路的一个有希望的策略.
这种方法提供了一个可行的解决方案,以克服生物制药制造中的细胞限制.

关键词:

查查查查查查查查查查查克里斯普尔-Cas9是什么意思这是一个微型RNA.生产力生产力生产力这是RMCE的RMCE.

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