激光生物学报2024，Vol.33Issue(2)：143-150,166,9.DOI:10.3969/j.issn.1007-7146.2024.02.006
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
摘要
Abstract
β-tectorin,encoded by the TECTB gene,plays an important regulatory role in human hearing function,is an impor-tant 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 of β-tectorin can lead to the occurrence of non-syndromic deafness in humans.However,the molec-ular 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 frame-shift 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 re-search 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 in-volved in inner ear development,which is conducive to further research on the regulatory mechanism of hearing.关键词
斑马鱼/tectb/CRISPR/Cas9/内耳发育/毛细胞Key words
zebrafish/tectb/CRISPR/Cas9/inner ear development/hair cells分类
生物学引用本文复制引用
刘玲,朱俊伟,曾婷,谢缤灵,陶桂芳,朱贤宇,谢华平..斑马鱼tectb内耳基因敲除品系的构建及其作用机制[J].激光生物学报,2024,33(2):143-150,166,9.基金项目
国家自然科学基金项目(82170308) （82170308）
湖南省大学生研究性学习和创新性实验计划项目(202310542097). （202310542097）
