Conditional Pten deletion in DN3 cells allows for T-cell differentiation across the β-selection checkpoint in the absence of Notch signals. (A) Deletion of exons 4 and 5 of the Pten allele in PTEN^{f/f;Lck−cre+} mice is initiated at the DN3 stage of development and completed by the DP stage. DNA from whole thymus of PTEN^{f/f;Lck−cre+} or PTEN^f/f mice, and from sorted DN2, DN3, DN4, and DP thymocyte subsets of PTEN^{f/f;Lck−cre+} mice was extracted and amplified by PCR. Agarose gels with the PCR products corresponding to the deleted and floxed alleles are shown, as indicated. Data are representative of at least 3 independent experiments. (B) Developmental progression of culture-derived PTEN^{f/f;Lck−cre+} DN3 cells cultured for 6 days with OP9-Ctrl cells. Flow cytometric analysis of CD4 and CD8 cell surface expression is shown for CD45⁺ gated cells, whereas panel C shows the corresponding fold expansion and DP cellularity, as indicated. Lin⁻ c-Kit⁺ Sca-1⁺ cells sorted from bone marrow of PTEN^{f/f;Lck−cre+} or PTEN^{+/+;Lck−cre+} mice were cultured with OP9-DL1 cells for 14 days, sorted for DN3a cells, and returned to OP9-Ctrl cells for 6 days. Fold expansion was obtained from the total cellularity divided by the number of cells used at the start of the culture (input), and DP cellularity by multiplication of the total cellularity by the percentage of DP cells present in the cultures. Results are representative of 3 independent experiments.