In many cases, tissue-specific TH action cannot be known from TSH and circulating TH levels alone, and characterization of the latter is challenging. Therefore, in collaboration with the Integrative Neuroendocrinology Research Group and the Medical Gene Technology Unit of our Institute, we have developed and patented the THAI (Thyroid Hormone Action Indicator) mouse model (European Patent Office EP3223606; National Intellectual Property Office HU230964; United States Patent and Trademark Office 10,694,724 B2). Each cell of the mouse expresses a TH-sensitive luciferase reporter gene detectable at both RNA and protein levels, and thus provides a unique opportunity for direct, specific and quantitative determination of local TH signalling even in living animals.
Patents:
- Szellemi Tulajdon Nemzeti Hivatala HU230964. Pajzsmirigyhormon hatás mérésére szolgáló transzgenikus indikátor egér és rekombináns DNS-konstrukció. 2019
- European Patent Office EP3223606. Transgenic mouse for the assessment of thyroid hormone (TH) action. 2019
- United States Patent and Trademark Office 10,694,724 B2. Transgenic mouse for the assessment of thyroid hormone (TH) action. 2020
The in vivo imaging laboratory at our institute allows us to study local TH action in living animals using the THAI mouse model. The device is used to measure the bioluminescent light generated by the THAI mouse luciferase reporter in an anaesthetised animal. This measurement is also used to study the effects of agents that potentially interfere with TH effects, such as plastic additives considered as endocrine disruptors.
Although determining the expression level of D2 is a common way to characterise the intracellular activation of TH, this can often be inaccurate due to the complex posttranslational regulation of the enzyme. Uniquely in Hungary, our laboratory routinely measures D2 activity in animal and human samples using 125I isotope-labelled substrate. We are also part of the European Thyroid Association consortium for standardizing the measurement of TH metabolism.
Our group routinely uses immortalised cell cultures to reduce animal sacrifices and to study precise molecular mechanisms. We also use primary neuronal astrocyte, neuronal and tanycytic cell cultures to study the mechanisms of cortical and hypothalamic TH signalling.
The main tool for our transfection experiments on both primary and immortalised cell cultures is our proprietary library of almost a thousand DNA constructs. These constructs form the basis of many of our transgenic animal models (e.g. the THAI mouse) aswell.
The primary effect of TH is on gene activity, so it is of paramount importance to be able to quantify gene expression. For this purpose, we use standardised Taqman quantitative PCR technique and both manual and automated capillary Western blot methods. These approaches allow us to investigate the direct effect of TH and its consequences at the protein level, even in microdissected brain regions such as hypothalamic nuclei.
