PDF(Pubmed)
Abstract：
During the development of traditional Chinese hamster ovary (CHO) cell lines, target genes randomly integrate into the genome upon entering the nucleus, resulting in unpredictable productivity of cell clones. The characterization and screening of high-yielding cell lines is a time-consuming and expensive process. Site-specific integration is recognized as an effective approach for overcoming random integration and improving production stability. We have designed a multifunctional expression cassette, called CDbox, which can be manipulated by the site-specific recombination systems Cre/lox and Dre/rox. The CDbox expression cassette was inserted at the Hipp11(H11) locus hotspot in the CHO-K1 genome using CRISPR/Cas9 technology, and a compliant CHO-CDbox cell platform was screened and obtained. The CHO-CDbox cell platform was transformed into a pool of EGFP-expressing cells using Cre/lox recombinase-mediated cassette exchange (RMCE) in only 2 weeks, and this expression remained stable for at least 75 generations without the need for drug stress. Subsequently, we used the Dre/rox system to directly eliminate the EGFP gene. In addition, two practical applications of the CHO-CDbox cell platform were presented. The first was the quick construction of the Pembrolizumab antibody stable expression strain, while the second was a protocol for the integration of surface-displayed and secreted antibodies on CHO cells. The previous research on site-specific integration of CHO cells has always focused on the single functionality of insertion of target genes. This newly developed CHO cell platform is expected to offer expanded applicability for protein production and gene function studies.
摘要：
在中国传统仓鼠卵巢（CHO）细胞系的发育过程中，目标基因在进入细胞核后随机整合到基因组中，导致不可预测的细胞克隆生产力。高产细胞系的表征和筛选是耗时且昂贵的过程。特定站点集成被认为是克服随机集成和提高生产稳定性的有效方法。我们设计了一个多功能表达盒，叫做CDbox,其可以通过位点特异性重组系统Cre/lox和Dre/rox进行操作。使用CRISPR/Cas9技术将CDbox表达盒插入CHO-K1基因组中的Hipp11（H11）基因座热点，筛选并获得符合CHO-CDbox的细胞平台。使用Cre/lox重组酶介导的盒交换（RMCE）在仅2周内将CHO-CDbox细胞平台转化为表达EGFP的细胞池，并且这种表达在不需要药物应激的情况下保持稳定至少75代。随后，我们使用Dre/rox系统直接消除EGFP基因。此外,介绍了CHO-CDbox电池平台的两种实际应用。首先是Pembrolizumab抗体稳定表达菌株的快速构建，而第二种是将表面展示和分泌的抗体整合到CHO细胞上的方案。以往关于CHO细胞位点特异性整合的研究一直集中在靶基因插入的单一功能性上。这种新开发的CHO细胞平台有望为蛋白质生产和基因功能研究提供扩展的适用性。
