摘要
目的制备人Waardenburg综合征点突变SOX10 p.R106W小鼠模型,比较分析该突变在小鼠、小型猪及人中诱发的听觉功能表型的异同。方法通过比对人、猪和小鼠的SOX10编码蛋白的氨基酸序列,找到与人SOX10 p.R106W突变所对应的小鼠Sox10基因同源位点,通过CRISPR/Cas9介导的基因编辑引入突变,构建Sox10 c.316A>T(Sox10 p.R106W)点突变小鼠。采用听觉脑干反应测试、耳蜗组织切片等方法评估突变小鼠听力表型。对耳蜗组织进行mRNA转录组测序,对差异表达基因进行基因本体(gene ontology,GO)富集分析,借助String数据库分析差异表达基因与Sox10之间的关联性。结果获得了携带有Sox10 c.316A>T突变的基因编辑小鼠,该突变小鼠表现为腹部白斑,杂合稳定遗传。与野生型小鼠比较,Sox10 c.316A>T杂合突变小鼠对声波刺激的敏感性及内耳形态结构均没有明显的差异。转录组分析发现突变小鼠耳蜗中323个基因表达上调,283个基因表达下调,但大部分基因表达差异较小。对差异表达基因GO富集分析发现上调基因与免疫相关,下调基因与神经功能有关。利用String数据库分析显示,表达下调的基因中有5个与Sox10基因有关联,分别是神经丝重多肽(neurofilament heavy polypeptide,Nefh)、脂肪酸2-羟化酶(fatty acid 2-hydroxylase,Fa2h)、缝隙连接蛋白B1(gap junction protein beta 1,Gjb1)、神经生长因子受体(nerve growth factor receptor,Ngfr)、锌指蛋白536(zinc finger protein 536,Zfp536),均与内耳发育或功能无关。结论应用CRISPR/Cas9系统成功构建Sox10 p.R106W基因编辑小鼠,其听力表型无显著改变,与该突变在人和小型猪中诱发的疾病表型差异较大。
ObjectiveTo compare the otological phenotypic characteristics induced by Waardenburg syndrome mutation SOX10 p.R106W in mouse,miniature pig and human,and to construct a mouse model harboring orthologous human SOX10 p.R106W mutation.MethodsBy comparing the amino acid sequences of SOX10 proteins from human,miniature pig and mouse,the mouse orthologous site corresponding to human SOX10 p.R106W mutation was identified,and the nucleic acid mutation Sox10 c.316A>T coding Sox10 p.R106W was precisely introduced into the orthologous site of mouse Sox10 coding sequence by CRISPR/Cas9-induced homologous recombination.Characteristics of otological phenotypes were assessed by auditory brainstem response test and cochlear tissue histology.mRNA sequencing was conducted on cochlear tissues,and differential expressed genes(DEGs)were accepted gene ontology(GO)analysis.The correlation between DEGs and Sox10 was analyzed through String database.ResultsThe gene-edited mice harboring Sox10 c.316A>T(Sox10 p.R106W)mutation were successfully generated.The skin of gene-edited mice exhibited white spots and stable heterozygous inheritance.Sox10 c.316A>T/+mutant mice showed no notable difference in cochlea histological structure as well as response to acoustic stimulation,compared with wild type(WT)littermates.Transcriptomic analysis revealed that Sox10 c.316A>T/+mutant mice had 323 upregulated genes in concomitant with 283 downregulated genes.GO analysis showed that upregulated genes were associated with immunity while downregulated genes were associated with neurological functions.Five genes,neurofilament heavy polypeptide(Nefh)、fatty acid 2-hydroxylase(Fa2h)、gap junction protein beta 1(Gjb1)、nerve growth factor receptor(Ngfr)and zinc finger protein 536(Zfp536)were correlated with Sox10,but had no relationship with cochlear development and function.ConclusionSox10 p.R106W mutant mouse model is successfully established using CRISPR/Cas9 system.Compared with WT mice,the gene-edited mice have no difference in otological phenotypes,while there is significant difference in disease phenotype induced by these mutation in human and miniature pig.
作者
王露露
谢飞
赵清远
徐凯
刘传宏
贺秋月
郭科男
孙宇
王勇
WANG Lulu;XIE Fei;ZHAO Qingyuan;XU Kai;LIU Chuanhong;HE Qiuyue;GUO Kenan;SUN Yu;WANG Yong(Department of Laboratory Animal Science,College of Basic Medical Sciences,Army Medical University(Third Military Medical University),Chongqing,400038;Department of Otorhinolaryngology,Affiliated Union Hospital of Tongji Medical College,Huazhong University of Science and Technology,Wuhan,Hubei Province,430022,China)
出处
《陆军军医大学学报》
CAS
CSCD
北大核心
2023年第24期2512-2520,共9页
Journal of Army Medical University
基金
国家重点研发计划(2021YFF0702301)。
