Biotechnology Bulletin ›› 2023, Vol. 39 ›› Issue (2): 283-291.doi: 10.13560/j.cnki.biotech.bull.1985.2022-0863

Previous Articles     Next Articles

Establishment and Application of Multigene Engineering Transformation Strategy for CHO Cells

CHENG Jing-wen1(), CAO Lei1, ZHANG Yan-min1, YE Qian1, CHEN Min2, TAN Wen-song1, ZHAO Liang1,2()   

  1. 1. State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237
    2. Shanghai Bioengine Sci-Tech Co., Ltd., Shanghai 201203
  • Received:2022-07-14 Online:2023-02-26 Published:2023-03-07

Abstract:

In recent years, CHO(Chinese hamster ovary)cell engineering transformation mainly changes a single function of cells by knock-in or knock-out genes, but the knock-in and knock-out genes often cannot simultaneously play the corresponding functions in a single experimental operation, which limits the application of multigene synchronous transformation. In this study, Bcl-2, the anti-apoptotic gene in the apoptosis pathway, and FUT8, the key enzyme gene of fucose synthesis in the protein fucosylation pathway, were selected as the knock-in and knock-out genes. CRISPR/Cas9 technology was used to establish the site-specific synchronous knock-in and knock-out gene editing strategy. The monoclonal cell line obtained by using this strategy overexpressed Bcl-2 protein and lost FUT8 protease function. After subculture, it was found that the gene expressions of the cell lines obtained by the established site-specific synchronous knock-in and knock-out gene editing strategy were stable within 60 d. Compared with the original wild-type cells, the cell line demonstrated higher tolerance to serum deprivation and stronger resistance to death. This reveals that the gene editing strategy based on site-specific synchronous knock-in and knock-out is feasible and can be used to construct CHO engineering cell lines for recombinant protein production.

Key words: CHO cells, CRISPR/Cas9, gene editing, site-specific synchronization, cell line construction