斑马鱼tectb内耳基因敲除品系的构建及其作用机制

Construction and Mechanism of tectb Inner Ear Gene Knockout Line in Zebrafish

TECTB基因编码的β-盖膜蛋白(β-tectorin)在人类听觉功能中具有重要的调控作用,是内耳器官中覆盖膜的重要组成成分,在声音转导为神经信号的过程中发挥着关键作用。β-tectorin突变会导致人类非综合征性耳聋的发生,而目前关于听觉障碍发生的分子机制仍然有待研究。斑马鱼(Danio rerio)是一种常用的试验动物模型,能够对内耳发育和相关疾病的研究提供重要的价值。为了研究tectb基因在斑马鱼内耳发育中的作用,本研究利用CRISPR/Cas9基因编辑技术,成功构建tectb内耳基因缺失型斑马鱼模型,并对tectb基因缺失的斑马鱼表型进行初步分析。首先,在tectb基因的第三号外显子上设计靶位点以及基因型检测引物,经体外扩增得到向导DNA(sgDNA),再对其进行体外转录,得到向导RNA(sgRNA);然后,将sgRNA与Cas9酶通过显微注射的方式注射到野生型斑马鱼胚胎中,经基因型鉴定筛选出携带突变的嵌合体F0代鱼,培养至性成熟后与野生型斑马鱼杂交得到F1代突变体杂合子;在遗传稳定性鉴定及基因序列测序之后,保留突变类型为移码突变的F1代鱼,培养至成年后,经过自交得到F2代tectb突变体纯合子;最后,通过体式显微镜初步观察及毛细胞染色试验,发现突变体斑马鱼内耳组织结构正常,耳石形态和大小没有明显差异,毛细胞的发育也没有明显缺陷,但是体内器官与组织的发育是否发生变化,需要进行深入研究。这项研究为深入了解内耳发育的分子机制和耳聋等人类疾病提供了有价值的研究模型,借助tectb基因敲除斑马鱼模型,我们可以探究该基因在疾病发生中的具体作用,更好地研究内耳发育中的关键途径,有利于进一步深入研究听力的调控机制。

β-tectorin,encoded by the TECTB gene,plays an important regulatory role in human hearing function,is an important component of the covering membrane in the inner ear organ,and plays a key role in the process of sound transduction into neural signals.The mutation ofβ-tectorin can lead to the occurrence of non-syndromic deafness in humans.However,the molecular mechanism of hearing impairment remains unclear.To study the role of the tectb gene in zebrafish inner ear development,this study successfully constructed a zebrafish model with tectb gene deletion using CRISPR/Cas9 gene editing technology and conducted a preliminary analysis of the phenotype.First,target sites and genotype identification primers were designed on exon 3 of tectb gene.Guide DNA(sgDNA)was amplified in vitro,next guide RNA(sgRNA)was obtained by in vitro transcription.Then,sgRNA and Cas9 enzymes were co-injected into wild-type zebrafish embryos by microinjection.After genotyping,the mutant chimera F0 generation fish were screened;and after being raised to adult,they were crossed with wild-type zebrafish to obtain the mutant heterozygotes of the F1.After genetic stability identification and sequencing,F1-generation fish with frameshift mutation were raised to adulthood,they were crossed to obtain F2 tectb homozygotes.Finally,through observation and hair cell staining experiments,it was found that the inner ear structure of the homozygotes mutant zebrafish appears normal,with no obvious difference in the shape and size of the otolith,and no obvious defect in the development of hair cells.However,whether there were changes in the development of internal organs and tissues should be further studied.This study provides us with a research model to gain insight into the molecular mechanisms of inner ear development and human diseases.With the help of the tectb gene knockout zebrafish model,we can elucidate the role of this gene in diseases and better understand the pathways involved in inner ear development,which is conducive to further research on the regulatory mechanism of hearing.