Figure 4.

Introduction of a 15-kb inducible gene expression cassette in the AAVS1 locus by single-step ObLiGaRe. (A) Strategy to introduce an inducible gene expression cassette. ZFN cutting sites are the same as in Figure 1. The expression of rtTA is controlled by the constitutive CAG promoter, while GFP is under the doxycycline responsive promoter (tetO) (Gossen and Bujard 1992). The STOP sign indicates a transcription termination cassette. Int and neo represent the probes for Southern blot. p1-p2 and p3-p4 are PCR primers used to amplify the 5′ and 3′ junctions. K indicates KpnI. (B) Southern blot of eight positive clones is shown with both internal (int) and vector-specific probe (neo). (LP) Expected ligation product. The sequences for 5′ and 3′ junctions of the eight clones are reported in the table on the right with dotted lines indicating deletions. (C) GFP fluorescence is detected by fluorescence microscopy in cells from clone 6 without (−) and with (+) 48 h treatment of 1 μg/mL doxycycline (dox).