Identification of a New Integration Site and Study on Site-Specific Integration in CHO-K1 Cells

• Main Authors: Hong Liu (Author), Wei Zhang (Author), Liping Xie (Author), Youjia Hu (Author)
• Format: Book
• Published: MDPI AG, 2023-12-01T00:00:00Z.
• Subjects: article
• Online Access: Connect to this object online.
• Notes: 10.3390/ph17010008; 1424-8247
• Keywords: CHO-K1; site-specific integration; gene copy number; CRISPR/Cas9; etanercept; Medicine; R; Pharmacy and materia medica; RS1-441
• Source: Pharmaceuticals, Vol 17, Iss 1, p 8 (2023)

Site-specific integration is an important approach used to address the problem of unstable cell lines in industry. In this study, we observed a reduction in the gene copy number and antibody production in a CHOK1 cell line BA03 capable of high antibody expression. We identified a new integration site named locus 7 in the intron region of the <i>parva</i> gene through sequencing, FISH, and genome walking. We demonstrate that the integration of the exogenous gene at this locus does not affect the transcription of the <i>parva</i> and, therefore, has a minimal impact on cell growth. We designed sgRNA and donor vectors to integrate the etanercept-coding gene into locus 7 and obtained a cell line, SSI-4. We performed a passaged stability study on SSI-4 and proved the possibility of the stable, site-specific integration of exogenous genes at this locus in terms of integration site, copy number, expression level, and cell growth. In summary, our study has identified a new integration site suitable for site-specific integration, which lays the foundation for the subsequent development of site-specific integration cell lines.