Generation of ΔSAM–SLP-76 knockin mice. (A) Illustration of the ΔSAM–SLP-76 knockin targeting strategy. The first 6 slp-76 exons are indicated. The initiation codon (*) is at exon 1. The SAM domain spans from exons 1 to 4. Part of exon 1, along with exons 2 and 3, is replaced by PGK-Neo, which is later deleted upon crossing with β-actin Cre transgenic mice. ▴, Represent the Loxp sites. P1 and P2 represent PCR primers used for ES clone screening. P′ represents a probe used in Southern blotting analysis to confirm correctly targeted ES clones. X = XbaΙ, the restriction endonuclease used to digest genomic DNA of ES cells for Southern blotting. (B) Southern blotting analysis of genomic DNA from 5 ES cell clones (lanes 1-5) and wild-type ES cells (lane 6). (C) RT-PCR products from SLP-76^m/+ and SLP-76^m/m thymocytes using 2 primers flanking the deleted region. (D) SLP-76 protein expression. DP thymocytes from SLP-76^+/+, SLP-76^m/+, and SLP-76^m/m littermates were FACS sorted and subsequently lysed. Postnuclear lysates were subjected to Western blotting with anti-SLP-76. Anti-PLC-γ1 blot is used as a loading control. (E) Intracellular staining of SLP-76 in CD4⁺ splenic T cells from SLP-76^m/+ (solid line) and SLP-76^m/m (dotted line) littermates. The gray area represents staining control using B220⁺ lymphocytes.