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lepr和mc4r基因突变斑马鱼的制备及表型分析

费飞, 孙少阳, 姚玉霄, 王旭^*

复旦大学基础医学院生物化学与分子生物学系，代谢与分子医学教育部重点实验室，上海 200032

摘要

肥胖已经成为全球性的公共卫生问题，严重影响人类的身体健康和生活品质。本文运用CRISPR/Cas9技术，构建了肥胖相关基因——瘦素受体(leptin receptor, lepr)和黑素皮质素受体4 (melanocortin-4 receptor, mc4r)的斑马鱼突变体，并对其进行形态和功能分析。结果显示，lepr−/−和mc4r−/−斑马鱼在胚胎和幼鱼期没有明显异常，但在成年期，lepr−/−和mc4r−/−斑马鱼相比对照进食增多、体重增加、体脂含量升高，呈现肥胖表型。血糖测定结果显示，饱食喂养条件可以诱导lepr−/−和mc4r−/−成年斑马鱼出现糖耐量受损的现象。进一步地，运用real time RT-PCR对76个能量代谢相关基因在lepr−/−和mc4r−/−斑马鱼肝脏中转录表达水平进行检测，并与Lepob/ob小鼠肝脏cDNA microarray的数据比较，发现胰岛素/胰岛素样生长因子信号转导通路(insulin/IGF signaling pathway, ISS)相关的基因在lepr−/−斑马鱼和Lepob/ob小鼠肝脏中表达变化具有较高的正相关性，提示肥胖调控网络在进化中维持了一定的功能保守性。

关键词： 肥胖; 斑马鱼; 瘦素受体 ; 黑素皮质素受体4 ; CRISPR/Cas9技术

分类号：Q344;Q75

Generation and phenotype analysis of zebrafish mutations of obesity-related genes lepr and mc4r

FEI Fei, SUN Shao-Yang, YAO Yu-Xiao, WANG Xu^*

Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences； Key Laboratory of Metabolism and Molecular Medicine of Ministry of Education, Fudan University, Shanghai 200032, China

Abstract

Obesity has become a severe public health problem across the world, and seriously affects the health and life quality of human beings. Here we generated lepr and mc4r mutant zebrafish via the CRISPR/Cas9 technique, and performed morphological and functional characterizations of those mutants. We observed that there was no significant phenotypic difference between homozygous mutants and wild-type controls before 2.5 months post-fertilization (mpf). However, the adult lepr−/− and mc4r−/− individuals displayed increased food intake, heavier weight, and higher body fat percentage, the characteristics of obesity phenotypes. Blood glucose test showed that overfeeding induced significantly impaired glucose tolerance in adult lepr−/− and mc4r−/− zebrafish. Furthermore, we analyzed 76 energy metabolism-related transcripts in lepr−/− and mc4r−/− zebrafish livers by using real-time RT-PCR, and compared the results with the published microarray data of Lepob/ob mouse livers, and found that the changes in the expression of insulin/IGF signaling (IIS) pathway genes in lepr−/− zebrafish and Lepob/ob mouse were positively correlated, suggesting that the IIS pathway maintains functional conservation between zebrafish and mammals during the evolution of the obesity-regulating molecule network.

Key words: obesity; zebrafish; lepr ; mc4r ; CRISPR/Cas9 technology

收稿日期：2016-08-31　　录用日期：2016-09-13

通讯作者：王旭　　E-mail: wangxu2013@fudan.edu.cn

引用本文：

费飞, 孙少阳, 姚玉霄, 王旭. lepr和mc4r基因突变斑马鱼的制备及表型分析[J]. 生理学报 2017; 69 (1): 61-69.

FEI Fei, SUN Shao-Yang, YAO Yu-Xiao, WANG Xu. Generation and phenotype analysis of zebrafish mutations of obesity-related genes lepr and mc4r. Acta Physiol Sin 2017; 69 (1): 61-69 (in Chinese with English abstract).