Fig. 1. (A-D) Generation of Tbx20 targeted allele. (A) Two LoxP sites were induced into Tbx20 exon 2 where the T-box domain starts. (B) Southern blot of ES cell DNA digested with NcoI and hybridized with a genomic fragment external to the targeting construct with wild-type band 5.5 kb and recombinant band 4.3 kb. Recombinant ES cells were then transfected with a Cre plasmid in order to remove PGKNeo, HSV-TK cassettes. Tbx20-null mice were obtained by crossing mice with Tbx20 floxed allele to protamine-Cre mice. (C) RT-PCR using whole heart RNA obtained from adult wild-type (lane 1) and heterozygous mice (lane 2) with primers located in exon 1 (P-RT-5') and exon 3 (P-RT-3') showed the wild-type band 677 bp and mutant band 427 bp. Excision of exon 2 of Tbx20 created a new immediate stop codon within exon 3. (D) Genotypes determined by PCR of one littermate embryos from heterozygous cross. The wild-type allele band was 310 bp and the mutant allele band was 650 bp. (E-L) Whole-mount (E,F,I,J) and histological (G,H,K,L) views of Tbx20-null mice and littermate controls at E8.5 and E9.5. The left, middle and right columns give right, frontal and left views, respectively. Hearts of Tbx20-null mice are severely hypoplastic relative to control littermates at both stages. Arrows indicate the heart region.