Gfi1 and Gfi1b expression in HSCs and MPPs and deletion of Gfi1b by gene targeting. (A) Gating scheme for HSCs and MPPs. Bone marrow cells were stained for the indicated markers and were electronically gated for Lin⁻, Sca-1⁺, c-kit⁺ (LSK) cells. The LSK subset was further analyzed for expression of CD150 and CD48 and was subdivided in HSCs (LSK, CD150⁺, CD48⁻), MPP1 (LSK, CD150⁺, CD48⁺), and MPP2 (LSK, CD150⁻, CD48⁺), according to published procedures. Results are representative for at least 3 independent experiments. (B) Activity of the Gfi1b promoter is followed by green fluorescence in cells isolated from Gfi1b:GFP knock-in mice based on the gating scheme indicated under A. Mean fluorescence intensity of GFP (MFI, representing Gfi1b promoter activity) is indicated. Representative for at least 3 independent experiments. The differences in the MFI of GFP of the different subsets were statistically significant (n = 3; P ≤ .05). (C) Activity of the Gfi1 promoter is followed by green fluorescence in cells isolated from Gfi1:GFP knock-in mice based on the gating scheme indicated under A. Mean fluorescence intensity of GFP (MFI, representing Gfi1 promoter activity) is indicated. Representative for at least 3 independent experiments. The differences in the MFI of GFP of the different subsets were statistically significant (n = 3; P ≤ .05). (D) Scheme of the murine Gfi1b locus and the targeting strategy to generate the conditional Gfi1b mouse allele. Exons 2 (which contains the ATG of Gfi1b), 3, and 4 are flanked by loxP sites. Upon activation of a Cre allele, these exons will be excised, thereby abrogating the expression of the Gfi1b protein. (E) Southern blot of DNA obtained from tails of wt (lane 1-2), Gfi1b^fl/+ (lane 3-4), or Gfi1b^fl/fl (lane 5-6) mice. DNA samples were restricted with HindIII. Using the 5′ probe described in D, correct recombination of the locus with the targeting vector is demonstrated by appearance of a 6-kb fragment, whereas the wt restriction fragment has a length of 10.5 kb. (F) Polymerase chain reaction genotyping of DNA from tail tip cells of a MxCre tg Gfi1b^fl/fl mouse (1) and a wt mouse (2). Mice were injected with pIpC, and the detection of a ko allele is the result of contaminating lymphocytes in the tail. (G) Western blot of Abelson transformed pre-B-cell lines established from bone marrow from pIpC-treated Gfi1b^fl/fl and MxCre tg Gfi1b^fl/fl mice. Excision of the Gfi1b locus was stimulated with interferon treatment and abrogated the expression of Gfi1b protein in these cell lines. As loading control, Ponceau staining is shown.