Meganuclease-driven targeted integration in CHO-K1 cells for the fast generation of HTS-compatible cell-based assays.
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.
Cabaniols
Jean-Pierre
JP
Cellectis BioResearch, Romainville Cedex, France.
Ouvry
Christine
C
Lamamy
Véronique
V
Fery
Isabelle
I
Craplet
Marie-Laure
ML
Moulharat
Natacha
N
Guenin
Sophie-Pénélope
SP
Bedut
Stéphane
S
Nosjean
Olivier
O
Ferry
Gilles
G
Devavry
Séverine
S
Jacqmarcq
Cécile
C
Lebuhotel
Céline
C
Mathis
Luc
L
Delenda
Christophe
C
Boutin
Jean A
JA
Duchâteau
Philippe
P
Cogé
Francis
F
Pâques
Frédéric
F
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
D003851:Deoxyribonucleases