4.0 Article

Development of recombinase-based targeted integration systems for production of exogenous proteins using transposon-mediated landing pads

期刊

CURRENT RESEARCH IN BIOTECHNOLOGY
卷 3, 期 -, 页码 269-280

出版社

ELSEVIER
DOI: 10.1016/j.crbiot.2021.10.001

关键词

Targeted integration; Recombination; Antibody; CHO; Recombinase mediated cassette exchange

向作者/读者索取更多资源

Novel targeted integration systems were developed to achieve high-level expression of recombinant proteins in CHO cells by inserting genes at specific genomic locations, leading to improved expression and productivity. The use of post-integration enrichment significantly increased recombinant protein titer and specific productivity, while maintaining specificity and reproducibility in establishing various cell lines expressing different recombinant protein therapeutics.
Current methods for stably expressing recombinant protein therapeutics in CHO cells often rely on random or semi-random genomic integration events which result in a widely heterogeneous cell population. Consequently, a significant portion of cell line development efforts involves extensive pool and clone screening to identify clones with high expression, growth, and product quality. In this study, we developed two targeted integration systems that express high levels of recombinant protein in CHO cells. We first generated two clonal cell lines stably expressing enhanced green fluorescent protein (eGFP) reporter landing pads in genomic hot spots. We then demonstrated functional integration of several donor vectors encoding both monoclonal antibodies and a Fc-fusion molecule using Cre or PhiC31 recombinase mediated integration. TLA and PCR characterization of integrated cell lines showed correct targeting of landing pads. Post-integration enrichment for fully saturated landing pads using GCV increased recombinant protein titer by 2-2.5-fold and specific productivity by similar to 3.4-fold with observed potential off-target random integration detected only following GCV enrichment. By targeting predefined genomic locations that are known to support high expression of an exogenous protein, several cell lines expressing different recombinant protein therapeutics can easily be established with a high degree of specificity and reproducibility.

作者

我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。

评论

主要评分

4.0
评分不足

次要评分

新颖性
-
重要性
-
科学严谨性
-
评价这篇论文

推荐

暂无数据
暂无数据