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

陈二方;邱阳;王人凤;刘珍珍;石力;查定军;邱建华;

1:中国人民解放军空军军医大学第一附属医院耳鼻咽喉头颈外科

摘要
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、1 000和2 000 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。
关键词
NELL2a;斑马鱼;基因编辑;突变模型
基金项目(Foundation):
国家自然科学基金海外重大合作项目(81120108008)
作者
陈二方;邱阳;王人凤;刘珍珍;石力;查定军;邱建华;
参考文献

1 Oyasu M,Kuroda S,Nakashita M,et al.Immunocytochemical localization of a neuron-specific thrombospondin-1-like protein,NELL2:light and electron microscopic studies in the rat brain[J].Molecular Brain Research,2000,76:151.

2 赵昱,石力,王剑,等.常染色体显性遗传性听神经病基因突变筛查[J].中华耳科学杂志,2014,12:267.

3 王锦玲,石力,薛飞,等.听神经病听力学特征及病损部位分析[J].听力学及言语疾病杂志,2007,14:89.

4 Jao LE,Wente SR,Chen W.Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system[J].Proc Natl Acad Sci USA,2013,110:13904.

5 Da L,Zhanxiang W,An X,et al.Efficient gene targeting in zebrafish mediated by a zebrafish-codon-optimized cas9 and evaluation of off-targeting effect[J].Journal of Genetics and Genomics,2014,41:41.

6 张峰华,王厚鹏,黄思雨,等.两种密码子优化的Cas9编码基因在斑马鱼胚胎中基因敲除效率的比较[J].遗传,2016,38:144.

7 Chang N,Sun C,Gao L,et al.Genome editing with RNA-guided Cas9 nuclease in zebrafish embryos[J].Cell Res,2013,23:465.

8 Thisse C,Thisse B.High-resolution in situ hybridization to whole-mount zebrafish embryos[J].Nat Protoc,2008,3:59.

9 潘豪来,王际平,于栋帧,等.斑马鱼听觉诱发电位检测系统的建立和应用[J].听力学及言语疾病杂志,2017,25:269.

10 Manchaiah VK,Zhao F,Danesh AA,et al.The genetic basis of auditory neuropathy spectrum disorder (ANSD)[J].International Journal of Pediatric Otorhinolaryngology,2011,75:151.

11 Ha CM,Choi J,Choi EJ,et al.NELL2,a neuron-specific EGF-like protein,is selectively expressed in glutamatergic neurons and contributes to the glutamatergic control of GnRH neurons at puberty[J].Neuroendocrinology,2008,88:199.

12 Kim DG,Hwang EM,Yoo JC,et al.Identification and characterization of a truncated isoform of NELL2[J].Biochem Biophys Res Commun,2010,391:529.

13 Montgomery J,Cartion G,Voigt R,et al.Sensory processing of water currents by fishes[J].Biological Sciences,2000,355:1325.

14 Metcalfe WK,Kimmel CB,Schabtach E.Anatomy of the posterior lateral line system in young larvae of the zebrafish[J].J Comp Neurol,1985,233:377.

15 Romero-Carvajal A,Acedo JN,Jiang L,et al.Regeneration of sensory hair cells requires localized interactions between the Notch and wnt pathways[J].Developmental Cell,2015,34:267.

16 Raible DW,Kruse GJ.Organization of the lateral line system in embryonic zebrafish[J].J Comp Neurol,2000,421:189.

17 Kim H,Ha CM,Choi J,et al.Ontogeny and the possible function of a novel epidermal growth factor-like repeat domain-containing protein,NELL2,in the rat brain[J].J Neurochem,2002,83:1389.

18 周莎莎.NELL2及其在促性腺激素释放激素分泌中的作用[J].国际儿科学杂志,2011,38:162.