The main challenge in the "post-GWAS" era is to determine the functional meaning of genetic variants and their contribution to disease pathogenesis. Development of suitable mouse models is critical because
disease susceptibility is triggered by complex interactions between genetic, epigenetic, and environmental factors that cannot be modeled by in vitro models.
Thyroglobulin (TG) is a key gene for autoimmune
thyroid disease (AITD) and several single nucleotide polymorphisms (SNPs) in the TG coding region have been associated with AITD. The classical model of experimental
autoimmune thyroiditis (EAT), based on immunization of genetically susceptible mouse strains with purified
TG protein in adjuvant, does not allow testing the impact of TG sequence variants on the development of
autoimmune thyroiditis. Here we describe a protocol for the induction of EAT by immunization of mice susceptible to
thyroiditis with an adenovirus vector carrying full-length human TG
cDNA (Ad-TG EAT). We also provide support protocols for evaluation of
autoimmune thyroiditis including serological assessment of TG
antibodies, in vitro splenocyte proliferation assay and
cytokines secretion, thyroid histology, and evaluation of thyroid lymphocytic infiltration by immunostaining. This protocol for EAT induction allows manipulation of the TG
cDNA to introduce variants associated with AITD, enabling the testing of the functional effects of susceptible variants and their haplotypes on the immunogenicity of TG. Furthermore, the Ad-TG EAT mouse model is a valuable model for studying the interactions of the TG variants with non-genetic factors influencing AITD development (e.g.,
cytokines,
iodine exposure) or with variants of other susceptible genes (e.g., HLA-DRβ1). © 2024 Wiley Periodicals LLC. Basic Protocol: Development of a mouse model of
autoimmune thyroiditis induced by immunization with adenovirus containing full-length
thyroglobulin cDNA Support Protocol 1: Splenocytes isolation Support Protocol 2: T cell stimulation and
carboxyfluorescein diacetate succinimidyl ester (
CFSE) based cell proliferation assay Support Protocol 3:
Cytokine assays: measuring levels of
interferon gamma (IFNγ) and
interleukins IL-2,
IL-4, and
IL-10 in splenocyte supernatants Support Protocol 4: Evaluating thyroid histology and infiltration with immune cells:
hematoxylin-
eosin staining of mice thyroid glands Support Protocol 5: Immunohistochemistry of thyroid tissues: Immunofluorescence protocol of
paraffin-embedded thyroid sections Support Protocol 6:
Anti-thyroglobulin antibody measurement in mice sera by
enzyme-linked
immunosorbent assay (ELISA).