Genetic mutation of Tas2r104/Tas2r105/Tas2r114 cluster leads to a loss of taste perception to denatonium benzoate and cucurbitacin B

Bowen Niu ¹Lingling Liu ¹Qian Gao ²Meng-Min Zhu ¹Lixiang Chen ¹Xiu-Hua Peng ¹Boying Qin ¹Xiaohui Zhou ¹Feng Li¹

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作者信息

1. Department of Laboratory Animal Science,Shanghai Public Health Clinical Center,Fudan University,Shanghai,China
2. Department of Biology,College of Life Sciences,Shanghai Normal University,Shanghai,People's Republic of China
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Abstract

Background: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.Methods:To investigate the physiological functions of the bitter taste receptor cluster Tas2r106/Tas2r104/Tas2r105/Tas2r114 in lingual and extraoral tissues,mul-tiple 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 se-quencing 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.Results: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 re-vealed 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 con-tribute to CuB perception.Conclusions: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.

Key words

bitter taste receptor/CRISPR/Cas9/genetic mutation/two-bottle preference test/type 2 taste receptors (Tas2rs)

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基金项目

National Science and Technology Major Project(2017ZX10304402-001-006)

National Science and Technology Major Project(2017ZX10304402-001-012)

Shanghai Science and Technology Commission"R&D Public Service Platform and Institutional Capacity Improvement Project"(21DZ2291300)

Starton Funding from Shanghai Public Health Clinical Center(KY-GW-2019-11)

Starton Funding from Shanghai Public Health Clinical Center(KY-GW-2019-19)

Starton Funding from Shanghai Public Health Clinical Center(KY-GW-2021-39)

出版年

2024

动物模型与实验医学(英文)

中国实验动物学会,中国医学科学院医学实验动物研究所

动物模型与实验医学(英文)

ISSN：2096-5451

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