Expression of C3G-F enhances Notch-dependent β-selection in vitro bypassing pre-TCR. (A) Fetal thymocytes (E17) from B6 or SPA-1^−/− mice were infected with empty MIG or MIG containing C3G-F and cultured on TSt4/DLL1 cells in the absence (○) or presence (●) of DAPT (1 μg/mL). The cells were harvested on day 1, day 4, and day 7, and the GFP⁺ cell numbers were determined (left). The experiments were repeated twice, and similar results were obtained. On day 4, aliquots of the cells were harvested and 2-color stained with anti-CD4 and anti-CD8 antibodies; the profiles in a GFP⁺ gate are indicated (right). (B) Fetal thymocytes (E17) from Rag2^−/− mice were infected with empty MIG or MIG containing C3G-F, and then cultured on TSt4/DLL1 cells in the absence (○) or presence (●) of DAPT (1 μg/mL) without the addition of anti-CD3ϵ antibody. The cells were harvested on day 1, day 4, and day 7, and the GFP⁺ cell numbers were determined (left). Similar results were obtained in 2 independent experiments. The cells were cultured in the presence of TSt4 or TSt4/DLL1 cells and stained with anti-CD4 and anti-CD8 antibodies on day 4; the profiles in a GFP⁺ gate are indicated (right).