Thyroid-specific expression of IFN-γ in transgenic mice. (A) Organization of thyr-IFN-γ transgene. The thyroglobulin promoter, IFN-γ cDNA, and growth hormone splice donor and acceptor sequences are indicated by rectangles. The primers used for reverse transcriptase–PCR are indicated by arrows. (B) Thyroidal expression of IFN-γ in normal C57BL/6 mice (N) and in the three transgenic lines (A, B, and C), as assessed by Northern blot analysis. (C) Expression of IFN-γ in thyroids and other tissues: water control (lane 1); genomic DNA from line A transgenic (lane 2); genomic DNA from normal C57BL/6 (lane 3); thyroid RNA from line A transgenic with (lane 4) and without (lane 5) reverse transcriptase; thyroid RNA from line B transgenic (lane 6), line C transgenic (lane 7), and normal C57BL/6 (lane 8); and RNA from liver (lane 9), lymph nodes (lane 10), salivary glands (lane 11), spleen (lane 12), brain (lane 13), and testicles (lane 14). Sizes of DNA standards, in base pairs, are indicated on the right. (D) Immunohistochemical analysis of the expression of IFN-γ by thyroid cells. (E) Control slide without addition of the primary antibody.

Histological analysis of the thyroid glands in thyr-IFN-γ transgenic and control mice. Hematoxylin/eosin staining of thyroid glands from line A transgenic mice at ×10 magnification (A), ×20 magnification (B), and ×40 magnification (C). A normal thyroid from C57BL6 mouse is shown for comparison (D).

Ability of transgenic IFN-γ to modulate NIS expression. Western blot analysis was performed by using 100 μg of total proteins extracted from the thyroids of normal C57BL6 mice (N) and the three transgenic lines (A, B, and C). Blots were sequentially hybridized with antibodies for NIS and actin. The ratio of binding of each probe to actin was calculated to correct for loading.

Ability of transgenic IFN-γ to induce in the thyroid cells the expression of MHC class I, MHC class II, and CIITA genes. Northern blot analyses were performed by using 20 μg of total RNA extracted from thyroids of normal C57BL6 mice (N) and the three transgenic lines (A, B, and C). Blots were sequentially hybridized with probes for MHC class I, MHC class II, CIITA, and β-actin. The ratio of binding of each probe to β-actin was calculated to correct for the amount of loading.

Influence of transgenic IFN-γ on the expression of thyroid-specific/restricted genes. Northern analyses were performed by using 20 μg of total RNA extracted from thyroids of normal C57BL6 mice (N) and the three transgenic lines (A, B, and C). Blots were sequentially hybridized with probes for TG, TPO, TSH-R, NIS, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The ratio of binding of each probe to β-actin was calculated to correct for the amount of loading.

The effect of IFN-γ on NIS expression is dose and time dependent. FRTL-5 cells in six hormones were washed with serum-free medium and exposed to different doses of IFN-γ for 2 or 6 days. In the 6-day experiments, the culture medium with IFN-γ was replaced every other day. At the end of the experiments, Northern blot analyses were performed by using 20 μg total of RNA. (A) Blots were sequentially hybridized with probes for NIS, MHC class II (as control), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (to correct for loading). (B) The ratio of binding of each probe to GAPDH was calculated, and this value was set at 1 for MHC class II. NIS values were compared with the corresponding MHC class II control and expressed as relative mRNA levels. Data are the mean ± SD of three different experiments, each performed in duplicate. Single or double asterisks represent a significant difference with P < 0.05 or < 0.01, respectively, by two-way ANOVA.