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Exp Clin Endocrinol Diabetes 2022; 130(02): 134-140
DOI: 10.1055/a-1522-8535
Article

Design and Characterization of a Fluorescent Reporter Enabling Live-cell Monitoring of MCT8 Expression

Adina Sophie Graffunder
1   Institute of Experimental Pediatric Endocrinology, Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin and Berlin Institute of Health
,
Sarah Paisdzior
1   Institute of Experimental Pediatric Endocrinology, Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin and Berlin Institute of Health
,
Robert Opitz
1   Institute of Experimental Pediatric Endocrinology, Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin and Berlin Institute of Health
,
Kostja Renko
2   German Federal Institute for Risk Assessment (BfR), German Centre for the Protection of Laboratory Animals (Bf3R), Berlin, Germany
,
Peter Kühnen
1   Institute of Experimental Pediatric Endocrinology, Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin and Berlin Institute of Health
,
Heike Biebermann
1   Institute of Experimental Pediatric Endocrinology, Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin and Berlin Institute of Health
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Abstract

The monocarboxylate transporter 8 (MCT8) is a specific thyroid hormone transporter and plays an essential role in fetal development. Inactivating mutations in the MCT8 encoding gene SLC16A2 (solute carrier family 16, member 2) lead to the Allan-Herndon-Dudley syndrome, a condition presenting with severe endocrinological and neurological phenotypes. However, the cellular distribution pattern and dynamic expression profile are still not well known for early human neural development.

Objective Development and characterization of fluorescent MCT8 reporters that would permit live-cell monitoring of MCT8 protein expression in vitro in human induced pluripotent stem cell (hiPSC)-derived cell culture models.

Methods A tetracysteine (TC) motif was introduced into the human MCT8 sequence at four different positions as binding sites for fluorescent biarsenical dyes. Human Embryonic Kidney 293 cells were transfected and stained with fluorescein-arsenical hairpin-binder (FlAsH). Counterstaining with specific MCT8 antibody was performed. Triiodothyronine (T3) uptake was indirectly measured with a T3 responsive luciferase-based reporter gene assay in Madin-Darby Canine Kidney 1 cells for functional characterization.

Results FlAsH staining and antibody counterstaining of all four constructs showed cell membrane expression of all MCT8 constructs. The construct with the tag after the first start codon demonstrated comparable T3 uptake to the MCT8 wildtype.

Conclusion Our data indicate that introduction of a TC-tag directly after the first start codon generates a MCT8 reporter with suitable characteristics for live-cell monitoring of MCT8 expression. One promising future application will be generation of stable hiPSC MCT8 reporter lines to characterize MCT8 expression patterns during in vitro neuronal development.

Key words

thyroid hormone transport - MCT8 - FlAsH - TC-tag - expression - functional characterization

Supplementary Material



Publication History

Received: 16 November 2020
Received: 18 May 2021

Accepted: 28 May 2021

Article published online:
05 August 2021

© 2021. Thieme. All rights reserved.

Georg Thieme Verlag KG
Rüdigerstraße 14, 70469 Stuttgart, Germany

 

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