Figure 4.

The ETP population in mice after Notch1 ablation contains Notch1-deficient T cell lineage precursors. (A) In the absence of Notch1, only very few ETPs can be isolated for the detection of T cell lineage precursors. The FACS plots are gated on Lin⁻ CD44⁺ CD25⁻ DN1 thymocytes derived from Notch1^lox/lox//CCR9^EGFP/+ and Notch1^lox/lox//MxCre//CCR9^EGFP/+ mice 2 wk after pIpC injection. Percentages are indicated. (B) Single TMPs were sorted from Notch1^lox/lox//CCR9^EGFP/+ and Notch1^lox/lox//MxCre//CCR9^EGFP/+ mice 4 wk after pIpC injection on OP9-DL1 stromal layers and cultured for 12–14 d. Cells falling into the right half of the ETP gate shown in A were considered TMPs. DN3/4 thymocytes developed on OP9-DL1 stroma even in the absence of Notch1, as previously described (reference 20). Percentages are indicated. (C) Genomic PCR was performed on DNA purified from the OP9-DL1 culture of DN3/4 thymocytes generated from a single TMP of a Notch1^lox/lox//CCR9^EGFP/+ and a Notch1^lox/lox//MxCre//CCR9^EGFP/+ mouse 4 wk after pIpC injection. Two sets of primers were used, with one detecting the wild-type (N1 wild-type) and the floxed (N1^lox allele) Notch1 allele (left lane) and the other detecting the deleted allele of Notch1 (Δ N1; right lane). Because the added hematopoietic cells did not carry a wild-type Notch1 allele, this band is thought to be derived from OP9-DL1 stroma. PCR fragment sizes in base pairs are given in parenthesis.