Extended Data Fig. 4: Generation and characterization of Runx transgenic mouse strains.
(a) A scheme showing the structure of the Rosa26 allele with DNA fragments inserted for the inducible transgenic expression of Runx proteins. (b) Immunoblots showing expression of Flag-tagged transgenic Runx proteins in total thymocyte of non-transgenic (NTG), R26DP-R3, R26DP-R3-WRPW, R26DP-R1, R26DP-R1-WRPE, R26DP-R1-WRPF, and R26DP-R1-WRPW mice. Antibodies against Runx1 and Runx3 were used to detect both endogenous and transgenic Runx1 and Runx3 proteins. Gapdh was used for a loading control. Two independent experiments were performed. (c) Dot plots showing CD24 and TCRβ expression in total thymocytes and contour plots showing CD69 and TCRβ expression in CD24+ thymocytes. Graphs at the right show absolute numbers of CD24+CD69+ and CD24−TCRβ+ cells of mice with the indicated genotype. NTG (n = 14), R26DP-R1-WRPW (n = 6) and R26DP-R3-WRPW (n = 5). Mean ± SD. *: p < 0.05, **: p < 0.01 (one-way ANOVA with Tukey’s multiple comparison) (d) Dot Plots showing CD4 and CD8α expression in mature thymocytes of Cd4ΔS/+ and R26DP-R1-WRPW: Cd4ΔS/+ mice. The right graph shows the frequency of CD4SP and DP in mature thymocytes and the numbers of mature CD4SP and DP thymocytes. Cont (n = 5) and R26DP-R1-WRPW (n = 5). Mean ± SD. **: p < 0.01 (unpaired Student’s t-test, two-sided) (e) DNA sequences of PCR products of the R26lsl-R1-WRPE allele generated by CRISPR/Cas9 genome editing on the R26lsl-R3 allele and the R26lsl-R1-WRPF allele generated by gene targeting in ES cells. (f) DNA sequences of PCR products of ICR background Rx3E and Rx3F F0 founder generated by CRISPR/Cas9 genome editing.
