Figure 5.

SpyTag/SpyCatcher-assisted reconstitution of Split-GFP-Cre.A, comparison of the level of Cre recombination after transient transfection of different complementary pairs of Cre fragments. HEK 293T cells were transiently transfected with plasmids encoding ΔN17Cre or the components of the Split-GFP-Cre or Split-Spy-GCre system. All genes were first inserted into pMSCV-Thy1.1 expression reporter vector. The cells were cotransfected with the CAG-loxp-STOP-loxp-TdTomato reporter vector to visualize Cre reconstitution efficiency. The cells were harvested 20 h after transfection, and cells with different Cre expression levels as indicated by the Thy1.1 expression level were analyzed by flow cytometry. B, the fluorescence intensity of TdTomato and average frequencies of TdTomato⁺ cells among cells expressing different Cre levels (Thy1.1 low, medium, and high) are shown. The data shown are typical results from two experiments. The small horizontal bars indicate the mean ± SEM. ∗p < 0.05 and ∗∗∗p < 0.001 (Student’s t test and ANOVA with Bonferroni post hoc test). HEK 293T, human embryonic kidney cancer cells 293T; ns, not significant.