For the biomanufacturing of protein biologics, establishing stable cell lines with high transgene transcription is critical for high productivity. Modern genome engineering tools can direct transgene insertion to a specified genomic locus and can potentially become a valuable tool for cell line generation. In this study, the authors survey transgene integration sites and their transcriptional activity to identify characteristics of desirable regions. A lentivirus containing destabilized Green Fluorescent Protein (dGFP) is used to infect Chinese hamster ovary cells at a low multiplicity of infection, and cells with high or low GFP fluorescence are isolated. RNA sequencing and Assay for Transposase Accessible Chromatin using sequencing data shows integration sites with high GFP expression are in larger regions of high transcriptional activity and accessibility, but not necessarily within highly transcribed genes. This method is used to obtain high Immunoglobulin G (IgG) expressing cell lines with a single copy of the transgene integrated into transcriptionally active and accessible genomic regions. Dual recombinase-mediated cassette exchange is then employed to swap the IgG transgene for erythropoietin or tumor necrosis factor receptor-Fc. This work thus highlights a strategy to identify desirable sites for transgene integration and to streamline the development of new product producing cell lines.
Bibliographical noteFunding Information:
The authors thank Atsushi Inoue and Professor Scott McIvor for valuable discussion and Professor Yuri Voziyanov for the Flp-2A-Cre vector and his advice. Computational resources were provided by the Minnesota Supercomputing Institute. This work was supported in part by the Minnesota Futures Grant. SAO and MGM were supported in part by the NIGMS Biotechnology Training Program (T32GM008347-22). TSL was supported in part by the Vietnam Education Foundation (VEF).
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- bioprocess engineering
- cell culture
- cho cells
- protein expression
- recombinant proteins