Characterization of KLRE-1 KO mice. (A) Generation of the EGFP–KLRE-1 knock-in mice. Schematic representation of the portion of the KLRE-1 gene locus (top), targeting construct (middle), and recombinant allele (bottom) including the relevant restriction sites. Probe used for Southern blot analysis is depicted. Exons are represented as black boxes. (B-E) Characterization of the EGFP–KLRE-1 knock-in mice. (B) Southern blot analysis. Southern blot analysis of Pst I-digested DNA from wild-type (WT) (+/+), heterozygous (+/–), or homozygous (–/–) KLRE-1 mice. DNA was hybridized with the probe shown in panel A to discriminate between the WT allele (10.0 kb) and the mutated allele (7.0 kb). (C) RT-PCR analysis for KLRE-1 mRNA. Total RNA prepared from DX5⁺ splenocytes of WT (+/+), heterozygous (+/–), or homozygous (–/–) KLRE-1 mice was subjected to RT-PCR analysis on KLRE-1 and β-actin. PCR on β-actin assured an equal amount of cDNA. (D) Expression of KLRE-1 and GFP in the DX5⁺TCRβ^– splenocytes. The DX5⁺TCRβ^– splenocytes from WT (+/+), heterozygous (+/–), or homozygous (–/–) KLRE-1 mice were stained with the Cy5–anti–KLRE-1 (7E8) mAb. Expression of KLRE-1 (left panel) and EGFP (right panel) is shown as a histogram. (E) Normal development of NK cells in KLRE-1 knock-out (KO) mice. Spleen, bone marrow (BM), and liver mononuclear cells from WT (+/+) and homozygous (–/–) KLRE-1 mice were stained with the PE-DX5 and the Cy5–anti-CD3ϵ. The area representing NK (DX5⁺ CD3ϵ^–) cells is shown with percentage.