NELL2a在斑马鱼神经丘的表达及其突变模型构建

Expression of NELL2a in Zebrafish Neuromast and Construction of Its Mutation Model

目的利用CRISPR/Cas9基因编辑技术构建NELL2a基因突变模型,为探究NELL2a在非综合型听神经病谱系障碍(auditory neuropathy spectrum disorder,ANSD)发生发展中的作用奠定基础。方法通过原位杂交技术,证实NELL2a基因在侧线神经丘表达,针对斑马鱼NELL2a基因,设计gRNA靶位点,体外合成gRNA和Cas9 RNA,在胚胎发育的单细胞期,通过显微注射的方法,将其注射入胚胎内。待仔鱼发育至一定时期,通过尾鳍鉴定,进而获得NELL2a F0代,后续通过F0代侧交,获得F1代杂合子,通过F1代自交获得F2代纯合子。通过听觉诱发电位(AEP)检测F2代纯合子的听力状态。结果通过测序证明已经获得NELL2a(ΔGC)纯合子,Nell2a^-/-斑马鱼在600、800、1000和2000 Hz声刺激频率的AEP阈值均显著高于野生型斑马鱼。结论NELL2a表达于斑马鱼侧线神经丘,推测其对神经丘毛细胞的功能起着重要作用,具体机制有待进一步研究。

Objective To construct a NELL2a gene mutation model using CRISPR/Cas9 gene editing technology,and to lay the foundation for exploring the role of NELL2 in the development of non-integrated auditory neuropathy spectrum disorder(ANSD).Methods In situ hybridization was used to confirm the expression of NELL2a gene in the lateral neuromast.The gRNA target site was designed for the zebrafish NELL2a gene,and gRNA and Cas9 RNA were synthesized in vitro.Which was microinjected into the embryo in the single cell phase of embryonic development.After the larvae develop to a certain period of time,the tail fins were identified,and then the NELL2a F0 generation was obtained,and the F1 generation heterozygotes were obtained by F0 generation,and the F2 generation homozygotes were obtained by F1 generation self-crossing.Results NELL2a(ΔGC)homozygotes were obtained by sequencing.Nell2a^-/-zebrafish AEP thresholds were significantly higher than wild-type zebrafish,with sound stimulation frequencies of 600,800,1000 and 2000 Hz.Conclusion NELL2a is expressed in the lateral line neuromast of zebrafish,inditating an important role in the function of hair cells.