斑马鱼Dio3b基因敲除模型的构建

The establishment of zebrafish Dio3b gene knockout model

目的 通过观察3型甲状腺激素脱碘酶（Dio3b）基因敲除的斑马鱼在心率、自主运动及胚胎发育等方面的变化,探讨斑马鱼Dio3b基因的生物学功能.方法 利用CRISPR-Cas9基因编辑技术敲除斑马鱼Dio3b基因,利用PCR和测序技术鉴定突变位点,确定Dio3b-/-斑马鱼的建立;利用显微镜拍摄并记录Dio3b--和野生型胚胎在受精后48 h的心率变化;利用斑马鱼行为追踪系统观察Dio3b-/-和Dio3b＋/＋胚胎在胚胎出生后5~7 d的运动变化.结果 成功构建了携带8碱基缺失移码突变的斑马鱼Dio3b敲除的斑马鱼模型;在胚胎出生后48 h,Dio3b-/-胚胎的心率相较于野生型明显增加（P＜0.001）;在胚胎出生后5~7 d,Dio3b-/-幼鱼的自主运动明显加快（P＜0.05）.结论 敲除Dio3b基因的斑马鱼存在心率加快、自主运动增加,这类表型与临床甲状腺功能亢进患者相似,说明该模型可以作为局部组织中甲状腺功能亢进的疾病模型,用于研究胚胎期过量的甲状腺激素对发育的影响.

Objective To set up the model of deiodinase （Dio） 3b-/- zebrafish and to observe the effect of which on embryo development. Methods The zebrafish model of Dio3b-/- was set up by CRISPR-Cas9 gene editing technology, PCR and sequencing was used to confirm the efficiency of deletion. The heart rate of embryos at 48 hours post fertilization was counted. The locomotor activity of 5-7 days post fertilization larve was detected using behavior tracking system. Results The model of Dio3b knockout zebrafish was set up successfully. The heart rate of embryos Dio3b-/- increased （P〈 0.001 ） and the locomotor activity of 5-7 days post fertilization larves lacking Dio3b gene increased （P〈0.05） significantly compared with that of wild type control respectively. Conclusion The deletion of zebrafish Dio3b gene resuhs in the phenotype of hyperthyroidism and the model of Dio3b-/- is proper for studying the effect of partial excess thyroid hormone on embryo development.