High-fidelity large-diversity monoclonal mammalian cell libraries by cell cycle arrested recombinase-mediated cassette exchange.

ABSTRACT: Mammalian cells carrying defined genetic variations have shown great potentials in both fundamental research and therapeutic development. However, their full use was limited by lack of a robust method to construct large monoclonal high-quality combinatorial libraries. This study developed cell cycle arrested recombinase-mediated cassette exchange (aRMCE), able to provide monoclonality, precise genomic integration and uniform transgene expression. Via optimized nocodazole-mediated mitotic arrest, 20% target gene replacement efficiency was achieved without antibiotic selection, and the improved aRMCE efficiency was applicable to a variety of tested cell clones, transgene targets and transfection methods. As a demonstration of this versatile method, we performed directed evolution of fragment

SUBMITTER: Chen C 

PROVIDER: S-EPMC10711435 | biostudies-literature | 2023 Dec

REPOSITORIES: biostudies-literature