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.

中文翻译：

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能够对 MCT8 表达进行活细胞监测的荧光报告基因的设计和表征

单羧酸转运蛋白 8 (MCT8) 是一种特定的甲状腺激素转运蛋白，在胎儿发育中起着至关重要的作用。MCT8 编码基因 SLC16A2（溶质携带者家族 16，成员 2）中的失活突变导致 Allan-Herndon-Dudley 综合征，这是一种具有严重内分泌和神经表型的病症。然而，早期人类神经发育的细胞分布模式和动态表达谱仍然不为人所知。目的 开发和表征荧光 MCT8 报告基因，可在人诱导多能干细胞 (hiPSC) 衍生的细胞培养模型中对 MCT8 蛋白的体外表达进行活细胞监测。方法 将四半胱氨酸 (TC) 基序引入人 MCT8 序列的四个不同位置作为荧光双砷染料的结合位点。人胚肾 293 细胞被转染并用荧光素-砷发夹结合剂 (FLAsH) 染色。使用特定的 MCT8 抗体进行复染。在 Madin-Darby Canine Kidney 1 细胞中，使用基于 T3 响应荧光素酶的报告基因检测间接测量三碘甲腺原氨酸 (T3) 摄取，以进行功能表征。结果所有四种构建体的FLASH染色和抗体复染显示所有MCT8构建体的细胞膜表达。在第一个起始密码子之后带有标签的构建体表现出与 MCT8 野生型相当的 T3 吸收。结论我们的数据表明，在第一个起始密码子之后直接引入 TC 标签会生成一个 MCT8 报告基因，该报告基因具有适合 MCT8 表达的活细胞监测的特征。一个有前途的未来应用将是生成稳定的 hiPSC MCT8 报告系，以表征体外神经元发育过程中的 MCT8 表达模式。