Dicer1 is essential for Notch-mediated T-ALL induction. (A) Lin⁻ BM cells from CD45.2⁺ donors (Dicer1^lox/lox or Dicer1^{Δ/Δ CD4-Cre}) were transduced with MigR1-hN1ICD-NGFR RV or MigR1-NGFR RV and transplanted into CD45.1⁺ congenic recipients, and hosts were monitored for T-ALL development by PBL analysis. (B) Dicer1 deletion PCR performed on thymocyte DNA from MIGR1-hN1ICD-NGFR transduced chimeric Dicer1^lox/lox or Dicer1^{Δ/Δ CD4-Cre} mice 8 weeks posttransplantation (p.tr.). wt, wild-type Dicer1, 351 bp; lox, loxP Dicer1 allele, 420 bp; Δ, recombined Dicer1 allele, 550 bp. (C) Kaplan-Meier survival plot of BM chimeras as outlined in panel A. Depicted are 3 experimental cohorts pooled from 2 independent experiments: (1) Dicer1^lox/lox transduced with NGFR control RV particles, Dicer1^lox/loxNGFR; (2) Dicer1^lox/lox transduced with hN1ICD-NGFR RV particles, Dicer1^lox/lox hN1ICD-NGFR; and (iii) Dicer1^Δ/ΔCD4-Cre transduced with hN1ICD-NGFR RV particles, Dicer1^Δ/ΔCD4-Cre hN1ICD-NGFR. The dashed line indicates median survival time (MST; 80 days). (D) T-ALL cell expansion in the periphery assessed by NGFR⁺ cells in total PBLs. FACS analysis on PBLs is shown at the indicated time points p.tr., the percentage of NGFR⁺ cells was measured gating on live cells, and mouse numbers are indicated in brackets. Symbols represent mean ± standard deviation (SD) for mice analyzed per time point. (E) Representative flow cytometric analysis of NGFR⁺ PBLs of MigR1-hN1ICD-NGFR transduced Dicer1^lox/lox (red) or Dicer1^Δ/ΔCD4-Cre (green) BM chimeras at day 31 p.tr. (left panel, histogram and contour plots) and day 39 p.tr. (right panel, histogram and contour plots). The CD4⁺ vs CD8⁺ profile is shown gated on NGFR⁺ cells based on the histograms. (F) Left bar graph indicates the percentage of mice within the Dicer1^lox/lox control cohort with >3% NGFR⁺ cells (T-ALL detectable, ▪) or <3% (T-ALL free, ☐) in total PBLs at 31 days p.tr. (62%; n = 9/13) and 39 days p.tr. (100%; n = 12/12). Dicer1^{Δ/Δ CD4-Cre} chimeras are shown in the right bar graph. At day 31 p.tr., 42% (n = 5/12) of these mice have >3% of NGFR⁺ cells in the PBLs, but 0% (n = 0/10) at day 39 p.tr. (G) Dicer1 deletion PCR on total PBLs of MigR1-hN1ICD-NGFR transduced Dicer1^lox/lox and Dicer1^{Δ/Δ CD4-Cre} chimeras at day 31 (left panel) and day 39 p.tr. (right panel). (H) Expression (qRT-PCR) of oncomiRs miR-19b, miR-20a, miR-26a, miR-92a, and miR-451 is shown at day 31 p.tr. in RNA samples isolated from sorted CD45.2⁺ NGFR⁺ cells from splenocytes of MigR1-hN1ICD-NGFR transduced Dicer1^{Δ/Δ CD4-Cre} BM chimeras (n = 4) and normalized to the expression of sorted NGFR⁺ cells from MigR1-hN1ICD-NGFR transduced Dicer1^lox/lox BM chimeras (n = 5). (I) Dicer1 deletion was assessed in samples from panel H by PCR. Data (D-H) represent 2 independent experiments. *P < .05; **P < .01.