Method of inactivating a glucocorticoid receptor gene in an isolated cell

A method of selecting isolated human cells comprising a transgene integrated into an endogenous glucocorticoid receptor (GR) gene, the method comprising:

• a) introducing:
• i) one or more polynucleotides encoding a first and second pair of zinc finger nuclease (ZFN);
• and
• ii) a polynucleotide comprising a transgene flanked by homology arms that target a human glucocorticoid receptor (GR) gene into isolated human cells such that the transgene is integrated into an endogenous GR gene,
• wherein
• the first ZFN comprises:
• a zinc finger DNA-binding domain comprising the following recognition helices in the following order:
• (SEQ ID NO:
• 23)
• TSRALTA;
• (SEQ ID NO:
• 17)
• DRANLSR;
• (SEQ ID NO:
• 24)
• RSDNLSE;
• and
• (SEQ ID NO:
• 26)
• ERANRNS;
• and
• the second ZFN comprises:
• a zinc finger DNA-binding domain comprising the following recognition helices in the following order:
• (SEQ ID NO:
• 33)
• DSWNLQV;
• (SEQ ID NO:
• 27)
• RSANLTR;
• (SEQ ID NO:
• 30)
• TSGNLTR;
• and
• (SEQ ID NO:
• 31)
• TSGSLTR;
• or
• the first ZFN comprises:
• a zinc finger DNA-binding domain comprising the following recognition helices in the following order:
• (SEQ ID NO:
• 42)
• RSANLAR;
• (SEQ ID NO:
• 43)
• RSDNLRE;
• (SEQ ID NO:
• 44)
• QSSNLAR;
• and
• (SEQ ID NO:
• 45)
• QSADRTK;
• and
• the second ZFN comprises:
• a zinc finger DNA-binding domain comprising the following recognition helices in the following order:
• (SEQ ID NO:
• 40)
• RQDCLSL;
• (SEQ ID NO:
• 37)
• RNDNRKT;
• (SEQ ID NO:
• 38)
• RSDNLSR;
• and
• (SEQ ID NO:
• 39)
• TNQNRIT;
• and
• b) contacting the isolated human cells obtained in step a) with a corticosteroid such that cells not comprising the integrated transgene are killed and cells comprising the transgene integrated into the endogenous GR gene are selected.