Fig. 2. Targeting strategy to generate the Tbx2^tm1Pa allele. (A) Shown are the Tbx2 cDNA and the endogenous Tbx2 genomic locus, where black indicates untranslated regions and gray represents the T-box coding sequence. Two hundred and seven base pairs of exon 1 and all of exon 2 were targeted for deletion with a construct containing a neomycin-thymidine kinase selection cassette (neo-TK) flanked with loxP sites and a negative selection diphtheria toxin element (DTA) attached at the 5' end. A targeted line was electroporated in vitro with a Cre recombinase gene to excise the selection cassette and generate the final Tbx2^tm1Pa allele with a 2.2 kb deletion. (B) Southern blot analysis, with the 5' internal probe (5' int) indicated in A, confirming the presence of the Tbx2^tm1Pa allele in genomic XhoI digests of yolk sac DNA. The wild-type fragment is 5.1 kb, the mutant fragment is 2.8 kb. (C) PCR with the three primers indicated in A amplifies a 180 bp wild-type product and an 88 bp mutant product from yolk sac DNA. E, EcoRI; EV, EcoRV; N, NotI; X, XhoI.