Thyroid hormone action in the absence of thyroid hormone receptor DNA-binding in vivo
J. Clin. Invest. Nobuyuki Shibusawa, et al. 112:588 doi:10.1172/JCI18377 [Go to this article.]

Figure 1
Generation of TR-β^GS/GS and TR-β^–/– mice. Schematic strategy of homologous recombination in TR-β^GS/GS (a) and TR-β^–/– mice (b) is illustrated. Diagrams show the WT TR-β locus, the targeting vectors, ES-targeted alleles, and the F1 mutant alleles after the ACN cassette is excised. The mutated exon 3 is shown as shaded boxes. H, Hind III; K, Kpn I; RV, Eco RV; B, Bgl II; X, Xba I. The locations of ES, GS, and KO probes for Southern blot analysis are indicated. The ACN cassette is flanked by loxP sites indicated by black arrowheads. Arrows indicate the positions of PCR primers (5′, match/mismatch, KO5′, KO3′) used for DNA genotyping. The site of restriction fragments obtained by Southern blot analysis is given in kb. Chi, chimeric. (c) Southern blot analysis of DNA from ES clones. WT (11.5 kb) and targeted (8.0 kb) Hind III alleles were detected by a 3′ external ES probe. (d) Southern blot analysis of DNA from F2 mice resulting from a GS125 KI heterozygous intercross compared with chimeric animals. After Eco RV digestion, a 4.8-kb band was detected in targeted allele of chimeric mice using GS probe. After self-excision of ACN cassette, a 3.8-kb band is obtained from the mutant allele. (e) Genotyping of F2 KI offspring by mismatch PCR. The WT allele was detected with WT match primer set and mutant allele was detected only with the mismatch (mutant) primer set. (f) Southern blot analysis of TR-β KO F2 siblings using the KO probe. The mutant allele demonstrated a longer (7.8 kb) band after Kpn I digestion versus the WT allele of 3.8 kb. (g) PCR genotyping of F2 KO mice. A 300-bp shorter band is observed in DNA from the KO versus WT allele.