Generation of Baf250a^E2E3/E2E3 mouse strain. (A) Baf250a targeting strategy. Top, Baf250a locus (NM_001080819_MGI:1935147_Arid1a). Middle, the targeted allele before Cre-mediated excision. Bottom, the recombined Baf250a^E2E3 allele. Exons (■), LoxP sites (▴), primers for PCR-based genotyping (numbered horizontal arrows), and 3′ external genomic probe (□) are shown. Positions of PvuII restriction sites are also denoted. (B) Southern blot analysis of Baf250a modification. DNA isolated from embryonic tissues was digested with PvuII, which cuts outside of the targeted region to generate a 11.6 kb WT and a 7.9 kb Baf250a^E2E3 bands recognized by the 3′ probe depicted in Figure 1A. (C) PCR-based amplifications of Baf250a^E2E3 allele. The primer pair 1 + 3 generates a 210-bp fragment identifying the excised Baf250a^E2E3, and primers 2 + 3 pair generate the 90-bp WT fragment. Positions of primers are denoted in Figure 1A. (D) Quantitative RT-PCR–based analysis of Baf250a^E2E3 mRNA. Note the absence of RT-PCR signal for homozygous Baf250a^E2E3 cells when using primers positioned in exons 2 or 3, and the 172-bp segment amplified with primers in exons 1 and 4. Top, schema of primer positions. Bottom, amplicons generated by the denoted primer pairs. (E) Western blot analysis of BAF250a levels in WT, Baf250a^E2E3, and Baf250a^E2E3/E2E3 embryonic fibroblasts. The BAF250a antibody recognizes the approximately 280 kDa WT, and approximately 250 kDa mutated BAF250a proteins. (F) BAF250a^E2E3 protein interacts with Brg1. Anti-Brg1 antibody coimmunoprecipitates WT and mutated BAF250a.