Figure 1. Inducible cassette exchange recombination in A2Lox.cre mES cells.

(A) The incoming plasmid is shown above the ICE target locus, which is integrated at a unique site in the genome (5′ of HPRT in the mouse ES cells). At the ICE target locus, cre is flanked by heterologous loxP sites, and is downstream of a doxycycline (tetracycline)-responsive promoter (TRE). Arrows indicate recombination between homologous loxP sites. The heterologous loxP sites on the incoming plasmid are in the opposite orientation relative to their cognates on the chromosome, therefore recombination catalyzes the integration of the entire plasmid. Cre is exchanged for the incoming gene of interest, rendering that gene doxycycline-inducible in the derivative recombinants. Selection is enabled by correction of the Δneo gene which acquires a PGK promoter, Kozak translational consensus and ATG.

(B) FACS analysis of A2Lox.cre ES cells following recombination in the absence of selection. Cells were either not treated with doxycycline (left) or given a 24-hour pulse of doxycycline (right) prior to nucleofection. After recovery, cells were re-induced with doxycycline to visualize recombinants.

(C) Measured frequency of recombination. n=3, p < 0.0049.

(D) FACS analysis of a derivative inducible-GFP (iGFP) cell line and control (E14) exposed to doxycycline.