Expression of PV mRNA in the liver and brown adipocytes. RT-PCR: Total RNA was isolated from liver (lanes 1–3) and brown adipocytes (lanes 4–6). RT-PCR was carried out by using the primers N and C as described in Materials and Methods. (A) Diagram: A 689- or 688-bp cDNA fragment was obtained from the mutant RNA and wild-type RNA, respectively. However, only the cDNA from the mutant RNA could be restricted with BamHI to yield two 380- and 309-bp fragments. (B) Northern blot analysis. The probe was the mouse TRβ1 cDNA. Results were from three mice of each genotype, as marked.

Histology of thyroid glands of TRβPV mice. Extensive hyperplasia of the thyroid gland dissected from an 11-week-old female TRβ^PV/PV mouse (C) and a significant increase in follicle size in a TRβ^PV/+ mouse (arrows, B), as compared with the histology of TRβ^+/+ seen in A (×137).

Delayed bone development in TRβPV mice. (A) Lengths of tibias and (B) femurs show significant delayed bone development in TRβ^PV/+ and TRβ^PV/PV mice.

Targeting of the PV mutation onto the TRβ gene locus by homologous recombination. (A) Schematic representation of the PV targeting vector. The 17.5-kb targeting vector contains the PV mutation in exon 10. The locations of the NeoR (flanked by two LoxP sequences) and the HSV-TK genes are as indicated. The directions of transcription of NeoR and HSV-TK are indicated by dotted arrows. (B) Restriction map of the wild-type TRβ gene locus. The expected size of the fragments digested by BamHI and EcoR V, which were detected by internal and external probes (C), respectively, are indicated. (C) Targeting the PV mutation into the TRβ gene locus via homologous recombination (TRβPVNeoR). The expected sizes of the fragments of the targeted locus digested by BamHI and EcoRV, which were detected by the internal and external probes, are indicated. (D) The final targeted locus (TRβPV). The NeoR gene was deleted in vivo by crossing the TRβ^PV/PVNeoR mice with Ella-Cre mice (19). (E) Genotyping of TRβPVNeoR mice. (a) Diagram: Primers used in the PCR analysis of TRβPVNeoR mice. Panel: PCR analysis of TRβPVNeoR mice. The 757-bp fragment from the TRβPVNeoR mice was detected when the pair of primers M5 and M3 was used in PCR. (b) In a separate PCR by using the genomic DNA from the same mice, the wild-type mice yielded a 452-bp fragment (panel) when the pair of primers W5 and W3 was used (diagram). From the two separate runs of PCR, the genotypes of TRβPVNeoR mice were determined. Lanes 2 and 5: wild-type mice; lanes 3 and 8: TRβ^PV/+NeoR mice; lanes 4, 6 and 7: TRβ^PV/PVNeoR mice. (F) Genotype analysis of TRβPV mice. Fragments (452 and 546 bp) obtained by PCR from (a) TRβPV and (b) wild-type mice. (c) The actual genotype analysis.

Elevated levels of total T4 and enlarged thyroid glands in TRβPV mice. (A) Serum levels of total T4 in adult mice. (B) The corresponding sizes of thyroid glands. Each point represents a value for a single mouse and horizontal bars represent the mean values.

Elevated serum TSH levels in TRβPV mice (A) and increases in the number of thyrotrophs (B). (A) Mean TSH levels in TRβ^PV/+ and TRβ^PV/PV mice were 2.1- and 411-fold elevated in 3- to 4-month-old mice, respectively. The horizontal bars represent mean values. (B) TSH-secreting cells were increased in the pituitary of TRβ^PV/PV mice. TSH-positive cells, indicated by arrows, were counted. The number of cells counted in the pituitary of the wild-type TRβ^+/+ (a) and TRβ^PV/PV mice (b) were 866 and 655, respectively.

The expression of T3-target genes in TRβPV mice was altered. Total RNA was isolated from the pituitaries and liver of mice (three for each group of 8-week-old male mice). The intensities of the bands were quantified by using a Molecular Dynamics PhosphorImager. The levels of expression of both genes were normalized by using GAPDH mRNA. The mean values of three experiments are shown and expressed as the fraction (fold) change from the value in the TRβ^+/+ mice.