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. 2021 Aug 25;297(4):101119. doi: 10.1016/j.jbc.2021.101119

Figure 6.

Figure 6

SpyTag/SpyCatcher-assisted reconstitution in the stable integration system.A, schematic representation of the Split-Cre plasmids in this study. All NCre constructs were inserted into the pMSCV-Thy1.1 retroviral vector, and all CCre constructs were inserted into the pMSCV-NGFR retroviral vector. B, schematic representation of the experimental design. NIH3T3 cells were incubated with retroviruses encoding NCre and CCre for 6 h, followed by removal of the medium and replacement with 1 ml of the fresh medium. The infected cells were then analyzed for GFP fluorescence by fluorescence-activated cell sorting (FACS) or transfected with the CAG-loxp-STOP-loxp-TdTomato reporter vector to visualize Cre reconstitution efficiency. C, cells were harvested 24 h after retrovirus infection, and cells coexpressing NCre and CCre (double positive) were detected based on Thy1.1 and NGFR expression. The fluorescence intensity of GFP in double-positive cells is shown. D, cells with stable integration of the NCre and CCre constructs were transfected with the CAG-loxp-STOP-loxp-TdTomato reporter vector and harvested 24 h after transfection. The fluorescence intensity of TdTomato among double-positive cells is shown. ns, not significant. ∗∗∗p < 0.001 (ANOVA with Bonferroni post hoc test).