A mouse model for conditional ITK-SYK expression. (A) Schematic representation of the gene-targeting strategy. A targeting vector that carries the human ITK-SYK cDNA together with an IRES eGFP sequence preceded by a loxP-flanked NEO-STOP cassette was constructed and used to generate Rosa26^{loxSTOPlox-ITK-SYK} mice as described in Materials and methods. The recombinant Rosa26 locus and the ITK-SYK–expressing locus upon Cre-mediated deletion of the NEO-STOP cassette are indicated. SA, splice acceptor site; pA, polyA sequence; 1–3, Rosa26 exon 1–3; probe, flanking probe for Southern blot analysis. (B) Southern blot analysis. Genomic DNA from a wild-type (WT) and a successfully targeted Rosa26^{loxSTOPlox-ITK-SYK} embryonic stem cell clone was digested with XbaI and Southern blotted with the flanking probe indicated in A. Sizes of the wild-type and recombinant (Rec) fragments are indicated. (C) Conditional expression of ITK-SYK in T and B cells in vivo. ROSA26^{loxSTOPlox-ITK-SYK} mice were crossed to CD4-Cre or CD19-Cre transgenic mice for T or B cell–specific ITK-SYK expression. Peripheral lymphocyte suspensions of double transgenic 5-wk-old ITK-SYK^CD4-Cre or ITK-SYK^CD19-Cre mice were stained against TCR-β or B220. eGFP fluorescence indicative of ITK-SYK expression in the TCR-β⁺ T cells or B220⁺ B cells of the respective animals was analyzed using FACS. Data shown are representative of >40 mice per genotype analyzed. (D) Western blot analysis of conditional ITK-SYK expression. Individual mature T and B cell populations from 5-wk-old Rosa26^{loxSTOPlox-ITK-SYK}, ITK-SYK^CD4-Cre, and ITK-SYK^CD19-Cre mice were sorted with magnetic beads and subjected to Western blot analysis with an anti-ITK antibody. Bands of wild-type ITK and ITK-SYK are indicated. Western blotting for β-actin demonstrated equal protein loading. Data shown are representative of five independent experiments.