CD11b expression on Joker leukocytes, genetic mapping, and identification of the Joker mutation. (A) CD27 and CD11b expression on Joker (Jkr) NK cells on the Jinx (Unc13d^j/j) background. Splenic NK cells were defined as NK1.1⁺CD3ϵ⁻ cells. The level of CD11b and CD27 expression on NK cells, as well as the distribution of each NK-cell subset in quadrants, are comparable in C57BL/6J mice (data not shown) and Unc13d^j/j/Jkr^+/+ mutants. Each dot plot represents data for 1 mouse of 5 tested for each genotype. (B) Geometric mean fluorescence intensity (Geo MFI) of CD11b expression on NK cells of Joker heterozygous (Jkr^+/−) and homozygous mutants (Jkr^−/−) on the Unc13d^j/j background. (C) Percentages of CD11b⁺ NK cells in the blood of Joker homozygous and heterozygous mutants on the Unc13d^j/j background. (D) The genetic linkage of Joker was performed on 16 mice using a panel of 128 informative markers. The Joker mutation was confined between D10mit31 and D10mit230 on chromosome 10. (E) Genomic sequence from a part of intron 6 of Itgb2 reveals an A → T transversion. Introns of eukaryotic genes have 2 distinct nucleotides at either end. At the 3′ end, the nucleotides are AG and constitute the acceptor splice site of the intron (depicted). The A → T transversion in intron 6 of Itgb2^Jkr affects the A nucleotide of the AG acceptor site, impairing its recognition by the splicing machinery. In Joker mice, the donor site of intron 6 interacts with the acceptor site of intron 7, leading to the complete splicing of intron 6, exon 7, and intron 7 from the mRNA. (F) Effect of the Joker mutation at the mRNA level. The Joker cDNA lacks the 242 bp of exon 7. BsgrI cuts the WT cDNA once in exon 7 of 2456-bp cDNA amplification fragment, leading to 875-bp and 1581-bp bands. In the 242-bp deletion of exon 7 in Joker cDNA, the nucleotides are insensitive to BsgrI digestion and no WT transcript is detectable.