Figure 1.

Eight-cell stage embryo injection technology facilitates the generation of biallelic GFP cassette knock-in mice. (A) Schematics depicting the targeting strategy for generation of Tbx3-2A-GFP knock-in ESCs. UTR, untranslated region of Tbx3. (B) T7 endonuclease I (T7EI) assay for CRISPR/Cas9 medaite cleavage at the Tbx3 locus in ESCs. The result is shown in the lower panel. (C) Up and bottom pannel show the phase contrast image and corresponding GFP fluorescence image of Tbx3-2A-GFP ESCs. Scale bars, 50 µm. (D) F0 generation of Tbx3-2A-GFP mice generated by eight-cell stage embryo injection (marked by red arrowheads). (E) Summary of generation of Tbx3-2A-GFP mice (F) Identification of F0 generation knock-in mice by Southern blot analysis. WT, wild type; T, targeted mice. (G) Microsatellite analysis of tissues from Tbx3-2A-GFP mice using D1Mit132 primers