Floxed reporter genes: Flow-cytometric selection of clonable cells expressing high levels of a target gene after tamoxifen-regulated Cre–loxP recombination

Tamoxifen treatment allows MerCreMer fusion recombinase to localize to the nucleus where MerCreMer can excise a floxed inhibitory DNA segment, thereby activating the expression of a downstream gene. This excision is irreversible, and it is therefore difficult to predict which non-activated clones will express the gene at high levels after recombination. We transfected a vector using HLA-A2.1 as floxed inhibitory DNA element and its expression level as surrogate marker predicting future expression of the attenuated downstream target gene. The target gene encoded an EGFP-linked fusion protein. In the unsorted population, 6% of the cells expressed the transfected target gene after recombination and less than 10-fold higher than the population before recombination. However after flow-cytometric selection for high HLA-A2.1 expression, 47% of the cells expressed the target gene after recombination and at levels 37-fold higher than the sorted population before recombination. 58% of the clones were capable of expressing the fusion protein and some over 200-fold above background of untransfected cells and greater than 20-fold higher levels of expression than before recombination. We describe an efficient method to select for clones expressing high levels of a target gene after tamoxifen regulated Cre–loxP recombination. Other floxed reporter genes should be equally useful.