Meganuclease-driven targeted integration in CHO-K1 cells for the fast generation of HTS-compatible cell-based assays

Journal of Biomolecular Screening
Jean-Pierre CabaniolsFrédéric Pâques

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, there...Continue Reading

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Citations

Dec 14, 2011·Applied Microbiology and Biotechnology·Jee Yon KimGyun Min Lee
Nov 8, 2011·BMC Evolutionary Biology·Adi BarzelLilach Hadany
Oct 26, 2011·Future Medicinal Chemistry·Béla Gyurcsik, Anikó Czene
Mar 5, 2013·Annual Review of Plant Biology·Daniel F Voytas
Dec 29, 2011·European Journal of Pharmacology·Séverine DevavryJean A Boutin
Jan 17, 2013·Biotechnology and Bioengineering·Payel DattaSusan T Sharfstein
Mar 12, 2013·Biotechnology and Bioengineering·Justin EyquemJulianne Smith
Jan 31, 2012·Critical Reviews in Biochemistry and Molecular Biology·Jesús PrietoGuillermo Montoya
Feb 22, 2011·Journal of Biomolecular Screening·Roland SchuchtTobias May
May 3, 2018·Nucleic Acids Research·Adam J BogdanoveBarry L Stoddard

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Methods Mentioned

BETA
transfection
electrophoresis
flow cytometry
fluorescence-activated cell sorting

Software Mentioned

GraphPad Prism
pClamp
cGPS
Prism
GraphPad

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