Department of Laboratory Animal Science, Shanghai Public Health Clinical Center, Fudan University, Shanghai, China.
Department of Biology, College of Life Sciences, Shanghai Normal University, Shanghai, People's Republic of China.
Animal Model Exp Med. 2024 Jun;7(3):324-336. doi: 10.1002/ame2.12357. Epub 2023 Dec 28.
Bitter taste receptors (Tas2rs) are generally considered to sense various bitter compounds to escape the intake of toxic substances. Bitter taste receptors have been found to widely express in extraoral tissues and have important physiological functions outside the gustatory system in vivo.
To investigate the physiological functions of the bitter taste receptor cluster Tas2r106/Tas2r104/Tas2r105/Tas2r114 in lingual and extraoral tissues, multiple Tas2rs mutant mice and Gnat3 were produced using CRISPR/Cas9 gene-editing technique. A mixture containing Cas9 and sgRNA mRNAs for Tas2rs and Gnat3 gene was microinjected into the cytoplasm of the zygotes. Then, T7EN1 assays and sequencing were used to screen genetic mutation at the target sites in founder mice. Quantitative real-time polymerase chain reaction (qRT-PCR) and immunostaining were used to study the expression level of taste signaling cascade and bitter taste receptor in taste buds. Perception to taste substance was also studied using two-bottle preference tests.
We successfully produced several Tas2rs and Gnat3 mutant mice using the CRISPR/Cas9 technique. Immunostaining results showed that the expression of GNAT3 and PLCB2 was not altered in Tas2rs mutant mice. But qRT-PCR results revealed the changed expression profile of mTas2rs gene in taste buds of these mutant mice. With two-bottle preference tests, these mutant mice eliminate responses to cycloheximide due to genetic mutation of Tas2r105. In addition, these mutant mice showed a loss of taste perception to quinine dihydrochloride, denatonium benzoate, and cucurbitacin B (CuB). Gnat3-mediated taste receptor and its signal pathway contribute to CuB perception.
These findings implied that these mutant mice would be a valuable means to understand the biological functions of TAS2Rs in extraoral tissues and investigate bitter compound-induced responses mediated by these TAS2Rs in many extraoral tissues.
苦味受体（Tas2rs）通常被认为能够感知各种苦味化合物，以避免摄入有毒物质。现已发现苦味受体在口腔外组织中广泛表达，并且在体内味觉系统之外具有重要的生理功能。
为了研究舌部和口腔外组织中苦味受体簇 Tas2r106/Tas2r104/Tas2r105/Tas2r114 的生理功能，我们利用 CRISPR/Cas9 基因编辑技术构建了多个 Tas2rs 突变小鼠和 Gnat3 基因敲除小鼠。将包含 Cas9 和 sgRNA 信使 RNA 的 Tas2rs 和 Gnat3 基因混合物显微注射到受精卵的细胞质中。然后，使用 T7EN1 检测和测序筛选目标位点的基因突变。利用定量实时聚合酶链反应（qRT-PCR）和免疫染色研究味觉信号转导途径和苦味受体在味蕾中的表达水平。使用双瓶偏好测试研究对味觉物质的感知。
我们成功地利用 CRISPR/Cas9 技术构建了多个 Tas2rs 和 Gnat3 突变小鼠。免疫染色结果显示，在 Tas2rs 突变小鼠中，GNAT3 和 PLCB2 的表达并未改变。但是，qRT-PCR 结果显示，这些突变小鼠的味觉感受器基因 mTas2rs 的表达谱发生了变化。利用双瓶偏好测试，这些突变小鼠由于 Tas2r105 的基因突变而消除了对环已亚硝脲的反应。此外，这些突变小鼠对盐酸奎宁、苯甲地那铵和葫芦素 B（CuB）的味觉感知能力丧失。Gnat3 介导的味觉受体及其信号通路有助于对 CuB 的感知。
这些发现表明，这些突变小鼠将成为一种有价值的手段，可以理解口腔外组织中 Tas2rs 的生物学功能，并研究这些 Tas2rs 介导的苦味化合物在许多口腔外组织中的诱导反应。
