Robust cell line development using meganucleases.
Cell line development for protein production or for the screening of drug targets requires the reproducible and stable expression of transgenes. Such cell lines can be engineered with meganucleases, sequence-specific endonucleases that recognize large DNA target sites. These proteins are powerful tools for genome engineering because they can increase homologous gene targeting by several orders of magnitude in the vicinity of their cleavage site. Here, we describe in details the use of meganucleases for gene targeting in Chinese hamster ovary-K1 cells, with a special emphasis on a gene insertion procedure using a promoter-less marker gene for selection. We have also monitored the expression of genes inserted by meganucleases-induced recombination, and show that expression is reproducible among different targeted clones, and stable over a 4 mo period. These experiments were conducted with the natural yeast I-SceI meganuclease, but the general design and process can also be applied to engineered meganucleases.
Cabaniols
Jean-Pierre
JP
CELLECTIS S.A., Romainville, France.
Pâques
Frédéric
F
D000818:Animals; D015139:Blotting, Southern; D015704:CD4 Antigens / Q000235:genetics; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D015252:Deoxyribonucleases, Type II Site-Specific; D004351:Drug Resistance / Q000235:genetics; D015870:Gene Expression; D018390:Gene Targeting / Q000379:methods*; D017930:Genes, Reporter; D005818:Genetic Engineering / Q000379:methods; D016254:Mutagenesis, Insertional; D015345:Oligonucleotide Probes; D011995:Recombination, Genetic; D029701:Saccharomyces cerevisiae Proteins; D014162:Transfection; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000235:genetics
D015704:CD4 Antigens; D015345:Oligonucleotide Probes; D029701:Saccharomyces cerevisiae Proteins; C037725:SCEI protein, S cerevisiae; D015252:Deoxyribonucleases, Type II Site-Specific; D001616:beta-Galactosidase