The combined expression of the Rag1-GFP and λ5 promoter controlled hCD25 gene identifies distinct LIN⁻FLT3^highIL-7R⁺SCA1^lowKIT^low early lymphoid progenitor populations. (A) Representative FACS plot showing the expression of the Rag1-GFP reporter gene in LIN^−/lowSCA1^highKIT^high bone marrow cells. (B) Representative FACS plots where LIN⁻FLT3^highIL-7R⁺KIT^lowSCA1^low cells are divided into B220⁻ (CLP) and B220⁺ (FrA). The CLPs and the FrA cells are further subdivided based on expression of the Rag1-GFP reporter (Rag1^high or Rag1^low) and on expression of the λ5 promoter controlled human CD25 gene (λ5Tg). The dashed line box represents the total percentage of λ5Tg reporter-positive cells in the CLP/FrA compartment. Representative FACS plots shown are from KIT-enriched BM. Numbers indicate percentage of cells in parental gate. LIN includes: GR1, CD11B, CD3ϵ, TER119, NK1.1, CD11C, LY6C, and CD19. (C) Generation of different cell subsets from Rag1^low and Rag1^high CLPs when cocultured on OP9 stoma cells. Percentages for indicated cells types of total live, GR1, and CD11B⁻ cells within the forward and side scatter gate containing hematopoietic cells (percentage of GR1/CD11B/7AAD⁻ cells) are indicated The data are collected from 2 experiments and represent an average (SEM shown). (D) Representative FACS plots from OP9 stroma cocultures at indicated time points. Gates indicate CD19⁻hCD25⁻, CD19⁻hCD25⁺, and CD19⁺ populations, respectively.