Vav‐1 gene‐targeting strategy. Vav‐1 gene‐targeting strategy. (A) The promoterless targeting vector contained a bicistronic selection cassette encoding GFP and Neor (open boxes). Two targeting plasmids were generated for sequential disruption of both Vav‐1 alleles. The 5′‐flanking region in the first construct spanned exons 2–4 (∼1.1 kb) of the human Vav‐1 gene, while the second vector contained a 5′‐homologous region derived from exons 5–7 (∼2.4 kb) of Vav‐1. Both targeting vectors contained a 3′‐homologous region spanning exons 24–27 (∼7 kb) from the Vav‐1 gene. The respective targeted Vav‐1 alleles are depicted in the lower portion of the figure, along with the DNA probe used for Southern blot analyses of the Vav‐1 gene loci. Primers used for clone screening and RT–PCR are indicated with arrowheads. The ApaI (A) restriction sites used for Southern analysis are indicated in the figure, along with the predicted size of the genomic restriction fragments from the targeted Vav‐1 alleles. (B) Southern blot analysis of genomic DNA isolated from cells containing wild‐type Vav‐1 (+/+), a Vav‐1+/− heterozygous clone (+/−) and three Vav‐1−/− nullizygotes. Note the presence of a third, non‐disrupted Vav‐1‐related genomic sequence in each of the Vav‐1−/− clones. (C) Vav‐1 protein expression. Detergent‐soluble proteins were immunoblotted with α‐Vav‐1 antibodies, followed by α‐ZAP‐70 antibodies as a control for protein loading. (D) RT–PCR analysis. The amplification products were obtained with primers f and r3 (see A). The predicted PCR product from the wild‐type Vav‐1 cDNA is 2 kb. Youjia Cao et al. EMBO J. 2002;21:4809-4819 © as stated in the article, figure or figure legend