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克里斯普尔删除miR-27影响CHO细胞中的重组蛋白质生产

Kevin Kellner¹, Nga T Lao², Niall Barron^3,4

¹National Institute for Cellular Biotechnology, Dublin City University, Dublin, Ireland.

Methods in molecular biology (Clifton, N.J.)

|June 26, 2024

概括

克里斯普尔/Cas9有效地准CHO细胞中的微RNA,产生miR-27b淘汰克隆. 这种枯竭增强了生物处理中的细胞活力,改善了重组蛋白质的生产.

科学领域:

生物技术是生物技术.
分子生物学分子生物学
细胞生物学细胞生物学

背景情况:

微RNAs (miRNAs) 是基因表达的关键调节者,影响细胞表型.
目前用于miRNA枯竭的方法需要引入额外的遗传物质.
在宿主细胞中,CRISPR/Cas9提供了针对性miRNA淘汰的替代方案.

研究的目的:

开发和验证一种基于CRISPR/Cas9的协议,用于在中国仓鼠卵巢 (CHO) 细胞中准miRNA.
研究miR-27a/b在CHO细胞生长和重组蛋白质生产率中的作用.
评估miR-27b耗尽对CHO细胞生物处理性能的影响.

主要方法:

利用CRISPR/Cas9基因编辑生成缺乏特定miRNA表达的CHO细胞系.
由于其在低温条件下的上调,因此有针对性的miR-27a/b.
采用了indel分析,TOPO-TA克隆和桑格测序进行验证.
在批量和食批量培养中评估细胞活力.

主要成果:

在CHO细胞中达到≥80%的miRNA向效率.
鉴定了由CRISPR/Cas9活动产生的各种因子.
成功生成了具有不可检测的成熟miR-27b表达的克隆.
在 miR-27b 耗尽后,在晚期培养物中证明了细胞活性的改善.

结论:

克里斯普尔/卡斯9是一种有效的工具,用于在CHO细胞中向miRNA淘汰.
在生物处理过程中,miR-27b的减少对CHO细胞活力产生了积极的影响.
向miR-27b是一个有希望的策略,用于增强CHO细胞中的重组蛋白质生产.

关键词:

这就是CRISPR/Cas9的作用.细胞线工程细胞线工程中国仓鼠的卵巢细胞.微RNA的枯竭导致了微RNA的枯竭.重组蛋白质生产的重组蛋白质生产

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