title: Meganuclease-driven targeted integration in CHO-K1 cells for the fast generation of HTS-compatible cell-based assays.
abstract: The development of cell-based assays for high-throughput screening (HTS) approaches often requires the generation of stable transformant cell lines. However, these cell lines are essentially created by random integration of a gene of interest (GOI) with no control over the level and stability of gene expression. The authors developed a targeted integration system in Chinese hamster ovary (CHO) cells, called the cellular genome positioning system (cGPS), based on the stimulation of homologous gene targeting by meganucleases. Five different GOIs were knocked in at the same locus in cGPS CHO-K1 cells. Further characterization revealed that the cGPS CHO-K1 system is more rapid (2-week protocol), efficient (all selected clones expressed the GOI), reproducible (GOI expression level variation of 12%), and stable over time (no change in GOI expression after 23 weeks of culture) than classical random integration. Moreover, in all cGPS CHO-K1 targeted clones, the recombinant protein was biologically active and its properties similar to the endogenous protein. This fast and robust method opens the door for creating large collections of cell lines of better quality and expressing therapeutically relevant GOIs at physiological levels, thereby enhancing the potential scope of HTS.
authors.affiliation: Cellectis BioResearch, Romainville Cedex, France.
authors.forename: Jean-Pierre
authors.initials: JP
authors.lastname: Cabaniols
authors.forename: Christine
authors.initials: C
authors.lastname: Ouvry
authors.forename: Véronique
authors.initials: V
authors.lastname: Lamamy
authors.forename: Isabelle
authors.initials: I
authors.lastname: Fery
authors.forename: Marie-Laure
authors.initials: ML
authors.lastname: Craplet
authors.forename: Natacha
authors.initials: N
authors.lastname: Moulharat
authors.forename: Sophie-Pénélope
authors.initials: SP
authors.lastname: Guenin
authors.forename: Stéphane
authors.initials: S
authors.lastname: Bedut
authors.forename: Olivier
authors.initials: O
authors.lastname: Nosjean
authors.forename: Gilles
authors.initials: G
authors.lastname: Ferry
authors.forename: Séverine
authors.initials: S
authors.lastname: Devavry
authors.forename: Cécile
authors.initials: C
authors.lastname: Jacqmarcq
authors.forename: Céline
authors.initials: C
authors.lastname: Lebuhotel
authors.forename: Luc
authors.initials: L
authors.lastname: Mathis
authors.forename: Christophe
authors.initials: C
authors.lastname: Delenda
authors.forename: Jean A
authors.initials: JA
authors.lastname: Boutin
authors.forename: Philippe
authors.initials: P
authors.lastname: Duchâteau
authors.forename: Francis
authors.initials: F
authors.lastname: Cogé
authors.forename: Frédéric
authors.initials: F
authors.lastname: Pâques
mesh_terms: D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D002477:Cells / Q000166:cytology / Q000378:metabolism*; D002874:Chromosome Mapping / Q000379:methods; D006224:Cricetinae; D003412:Cricetulus; D003851:Deoxyribonucleases / Q000378:metabolism / Q000502:physiology*; D018390:Gene Targeting / Q000379:methods*; D057166:High-Throughput Screening Assays / Q000379:methods*; D008954:Models, Biological; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D013997:Time Factors; D014162:Transfection
chemical_list: D003851:Deoxyribonucleases