Fig. 3: Generation of PU.1 knock-in mice and phenotypic analysis of their hematopoietic compartments.

a Knock-in strategy. The point mutations (Q202L mutation shown here) were introduced into exon 5 by site-directed mutagenesis. Location of restriction sites within the targeted region and corresponding fragment sizes of digested DNA are shown. Cre-mediated recombination selectively excises the region between the loxP sites, comprising the neomycin selection cassette. Location of the DNA probe to screen for successful gene targeting is shown. b Left panel: Southern blot analysis of ES cell DNA after restriction with BamHI and SpeI shows wild-type (WT) and targeted (KI) alleles with their respective sizes, as marked in a. Right panel: verification of the point mutations by sequencing. The base pair change leading to the Q202L amino acid mutation was confirmed as indicated. Three independent experiments were performed with representative images shown. c The fetal liver cells of KI mice have self-renew ability. Total fetal liver cells of both WT and KI E14.5–16.5 mice were seed in M3434 methylcellulose containing SCF, IL-3, IL-6, and EPO. The total colony number was counted and same number of cells were replated in fresh medium every 7 days. Three independent experiments were performed. d–f Flow cytometry analysis of E14.5–16.5 fetal liver cells. The relative contribution of the listed cell populations to total live (DAPI⁻) fetal liver cells is shown as the mean of values from quadruplicates ±SD. Representative FACS profiles are shown for wild-type (WT), heterozygous (Het) and homozygous PU.1 Q202L knock-in cells (KI). Statistical p-values are control vs KI are as follows—for7 days, p < 0.0001; 14 days, p < 0.0001; 21 days, p < 0.0001; 28 days, p = 0.0004. d Analysis of mature B cell (B220⁺), T cell (CD3⁺), and myeloid (Gr-1⁺ Mac-1⁺) compartments. Statistical values are as follows—for B220 + cells—Het vs WT, p = 0.020; KI vs WT p = 0.002; KI vs Het, p < 0.0001. For CD3⁺ cells—Het vs WT, p = 0.058; KI vs WT p = 0.0026; KI vs Het, p = 0.002. For Gr1⁺Mac1⁺ cells—Het vs WT, p = 0.020; KI vs WT p = 0.0003; KI vs Het, p < 0.0001. e Analysis of hematopoietic lin⁻ c-kit⁺ Sca-1⁻ progenitor cell compartment. Distinct populations are marked in boxes: MEP (CD34⁻ CD16/32⁻), CMP (CD34⁺ CD16/32^low), GMP (CD34⁺ CD16/32^hi). f Analysis of hematopoietic LSK (c-kit⁺ Sca-1⁺ lin⁻) cells. Distinct populations are marked: ST-HSC (CD34⁺ Flt3⁻/CD48⁻ CD150⁻), LT-HSC (CD34⁻ Flt3⁻/ CD48⁻ CD150⁺), MPP (CD34⁻ Flt3⁺/CD48⁺). Statistical p-values are as follows—for MPP cells—Het vs WT, p = 0.41; KI vs WT p = 0.0067; KI vs Het, p = 0.049. For ST-HSC cells—Het vs WT, p = 0.77; KI vs WT p = 0.016; KI vs Het, p = 0.094. For LT-HSC cells—Het vs WT, p = 0.37; KI vs WT p = 0.079; KI vs Het, p = 0.041. All comparisons tested using two-tailed t-test with Welch’s Correction. ns p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. The experiments for d, f were validated through three independent experiments. The experiment for e was validated through two independent experiments.