Meganuclease-Driven Targeted Integration in CHO-K1 Cells for the Fast Generation of HTS-Compatible Cell-Based Assays

被引：22

作者：

Cabaniols, Jean-Pierre ^{[2
]}

Ouvry, Christine ^{[3
]}

Lamamy, Veronique ^{[3
]}

Fery, Isabelle ^{[3
]}

Craplet, Marie-Laure ^{[4
]}

Moulharat, Natacha ^{[3
]}

Guenin, Sophie-Penelope ^{[3
]}

Bedut, Stephane ^{[4
]}

Nosjean, Olivier ^{[3
]}

Ferry, Gilles ^{[3
]}

Devavry, Severine ^{[3
]}

Jacqmarcq, Cecile ^{[1
]}

Lebuhotel, Celine ^{[1
]}

Mathis, Luc ^{[1
]}

Delenda, Christophe ^{[2
]}

Boutin, Jean A. ^{[3
]}

Duchateau, Philippe ^{[1
]}

Coge, Francis ^{[3
]}

Paques, Frederic ^{[1
]}

机构：

[1] Cellectis SA, F-93235 Romainville, France

[2] Cellectis BioResearch, Romainville, France

[3] Inst Rech Servier, Croissy Sur Seine, France

[4] Inst Rech Servier, Div Pharmacol Cardiovasc, F-92150 Suresnes, France

来源：

JOURNAL OF BIOMOLECULAR SCREENING | 2010年 / 15卷 / 08期

关键词：

meganuclease; homologous gene targeting; protein expression; cell-based assay; high-throughput screening; DOUBLE-STRAND BREAKS; DRUG DISCOVERY; HCN CHANNELS; AUTOTAXIN; TECHNOLOGY; PHOSPHODIESTERASE; IDENTIFICATION; RECOMBINATION; PURIFICATION; EXPRESSION;

D O I：

10.1177/1087057110375115

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

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. (Journal of Biomolecular Screening 2010: 956-967)

引用

页码：956 / 967

页数：12

共 34 条

[1] New selective ligands of human cloned melatonin MT1 and MT2 receptors [J].