应用CRISPR/Cas9技术制备MrgD基因敲除小鼠模型

doi:10.3969/j.issn.1006-7795.2018.04.007

首都医科大学学报 ›› 2018, Vol. 39 ›› Issue (4): 517-521.doi: 10.3969/j.issn.1006-7795.2018.04.007

• 糖尿病合并症的基础与临床研究 • 上一篇下一篇

应用CRISPR/Cas9技术制备MrgD基因敲除小鼠模型

曹曦^1,2, 宋丽妮^1,2, 张怡尘^1,2, 李奇^1,2, 杨金奎^1,2

1. 首都医科大学附属北京同仁医院内分泌科, 北京 100730;
2. 糖尿病防治研究北京市重点实验室, 北京 100730

收稿日期:2018-06-04 出版日期:2018-07-21 发布日期:2018-07-21
通讯作者: 杨金奎 E-mail:jkyang@ccmu.edu.cn
基金资助:
国家自然科学基金（81670774，81471014），北京市自然科学基金（7162047）。

Establishment of MrgD knockout mice model with CRISPR/Cas9 gene targeting technology

Cao Xi^1,2, Song Lini^1,2, Zhang Yichen^1,2, Li Qi^1,2, Yang Jinkui^1,2

1. Department of Endocrinology, Beijing Tongren Hospital, Capital Medical University, Beijing 100730, China;
2. Beijing Key Laboratory of Diabetes Research and Care, Beijing 100730, China

Received:2018-06-04 Online:2018-07-21 Published:2018-07-21
Supported by:
This study was supported by National Natural Science Foundation of China (81670774, 81471014), Natural Science Foundation of Beijing(7162047).

摘要/Abstract

摘要： 目的利用CRISPR/Cas9基因编辑技术建立MrgD基因敲除的小鼠模型，为研究MrgD基因在糖、脂代谢中的作用提供实验工具。方法根据MrgD基因第一外显子碱基序列，设计针对MrgD基因的sgRNA（single guide RNA），构建sgRNA表达质粒，利用T7RNA聚合酶体外转录sgRNA和Cas9后，将Cas9 mRNA和sgRNA对C57BL/6J小鼠的受精卵进行显微注射。使用基因测序检测MrgD基因碱基的突变情况。结果获得了MrgD基因突变的F2代纯合子小鼠，即MrgD基因缺失173bp的小鼠。并且，初步研究显示MrgD基因纯合突变小鼠在普通饲料喂养下糖、脂代谢正常。结论成功构建了MrgD基因敲除小鼠动物模型，为探讨MrgD基因在糖尿病发生发展中的作用提供研究平台。

关键词: 基因敲除, CRISPR/Cas9, MrgD基因

Abstract: Objective To establish the MrgD gene knockout mice model by using CRISPR/Cas9 gene editing technique so as to provide the tool to study the regulating role of MrgD in glucose and lipid metabolism. Methods Single guide RNA (sgRNA) plasmids against the exon 1 of MrgD were designed and constructed. Then the sgRNA and Cas9 was transcribed by T7 RNA polymerase in vitro. Cas9 mRNA and sgRNA were mixed and microinjected into fertilized eggs of C57BL/6J mice. Gene sequencing was used to detect the mutations of MrgD. Results Finally the F2 generation of homozygous MrgD deficient mice (MrgD-173/-173) were gained. Preliminary studies showed that MrgD gene homozygous mutant mice may not have disorders of glucose and lipid metabolism. ConclusionA mouse model with MrgD deficiency has been successfully established which shall lay a foundation for future investigation of MrgD.

Key words: gene knockout, CRISPR/Cas9, MrgD gene

中图分类号:

Q75
R587.1

曹曦, 宋丽妮, 张怡尘, 李奇, 杨金奎. 应用CRISPR/Cas9技术制备MrgD基因敲除小鼠模型[J]. 首都医科大学学报, 2018, 39(4): 517-521.

Cao Xi, Song Lini, Zhang Yichen, Li Qi, Yang Jinkui. Establishment of MrgD knockout mice model with CRISPR/Cas9 gene targeting technology[J]. Journal of Capital Medical University, 2018, 39(4): 517-521.

参考文献

[1] 傅继梁. 基因工程小鼠(精)[M]. 上海:上海科技出版社, 2006.
[2] Li W, Teng F, Li T, et al. Simultaneous generation and germline transmission of multiple gene mutations in rat using CRISPR-Cas systems[J]. Nat Biotechnol, 2013, 31(8):684-686.
[3] Hsu P D, Lander E S, Zhang F. Development and applications of CRISPR-Cas9 for genome engineering[J]. Cell, 2014, 157(6):1262-1278.
[4] Platt R J, Chen S, Zhou Y, et al. CRISPR-Cas9 knockin mice for genome editing and cancer modeling[J]. Cell, 2014, 159(2):440-455.
[5] Burgess D J. Technology:a CRISPR genome-editing tool[J]. Nat Rev Genet, 2013, 14(2):80.
[6] Dong X, Han S, Zylka M J, et al. A diverse family of GPCRs expressed in specific subsets of nociceptive sensory neurons[J]. Cell, 2001, 106(5):619-632.
[7] Lembo P M, Grazzini E, Groblewski T, et al. Proenkephalin a gene products activate a new family of sensory neuron——specific GPCRs[J]. Nat Neurosci, 2002, 5(3):201-209.
[8] Shinohara T, Harada M, Ogi K, et al. Identification of a G protein-coupled receptor specifically responsive to beta-alanine[J]. J Biol Chem, 2004, 279(22):23559-23564.
[9] Santos R A, Simoes e silva A C, Maric C, et al. Angiotensin-(1-7) is an endogenous ligand for the G protein-coupled receptor Mas[J]. Proc Nat Acad Sci U S A, 2003, 100(14):8258-8263.
[10] Gembardt F, Grajewski S, Vahl M, et al. Angiotensin metabolites can stimulate receptors of the Mas-related genes family[J]. Mol Cell Biochem, 2008, 319(1-2):115-123.
[11] 康怡,张宝和,徐洪涛,等.北京市部分中老年男性高胰岛素血症患者代谢紊乱及胰岛功能评估[J].首都医科大学学报,2016,37(3):385-390.
[12] 史婷婷, 杨芳远, 曹曦,等. 血管紧张素转化酶2减少胰岛细胞的氧化应激及凋亡[J]. 首都医科大学学报, 2017, 38(2):151-155.
[13] Cao X, Yang F, Shi T, et al. Angiotensin-converting enzyme 2/angiotensin-(1-7)/Mas axis activates Akt signaling to ameliorate hepatic steatosis[J]. Sci Rep, 2016, 6:21592.
[14] 寻英, 秦映芬, 徐梦婕,等. ACE2-Ang-(1-7)-Mas轴对糖代谢作用的研究进展[J]. 广东医学, 2015(1):150-153.
[15] Hrenak J, Paulis L, Simko F. Angiotensin A/alamandine/MrgD axis:another clue to understanding cardiovascular pathophysiology[J]. Int J Mol Sci, 2016, 17(7):pii:E1098.
[16] Nishimura S, Uno M, Kaneta Y, et al. MRGD, a MAS-related G-protein coupled receptor, promotes tumorigenisis and is highly expressed in lung cancer[J]. PLoS One, 2012, 7(6):e38618.
[17] Crozier R A, Ajit S K, Kaftan E J, et al. MrgD activation inhibits KCNQ/M-currents and contributes to enhanced neuronal excitability[J]. J Neurosci, 2007, 27(16):4492-4496.

应用CRISPR/Cas9技术制备MrgD基因敲除小鼠模型

Establishment of MrgD knockout mice model with CRISPR/Cas9 gene targeting technology

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 8

编辑推荐

Metrics

本文评价

[1]	陈建宏, 林欣, 冯金秋, 丁鹏鹏, 王苗苗, 林琳, 刘红, 吴静. 肝细胞条件性Eva1a/Tmem166基因敲除小鼠的表型分析[J]. 首都医科大学学报, 2021, 42(4): 568-574.
[2]	熊枫然, 卢晶, 张英超, 谢荣荣, 赵儒轩, 李奇, 杨金奎. Kcnh6点突变单基因糖尿病家系的小鼠模型构建及表型分析[J]. 首都医科大学学报, 2021, 42(4): 575-581.
[3]	谷思宇, 杨晓梅, 贺俊崎. CRISPR/Cas9基因编辑技术:基因剪刀—重写生命密码的工具——2020年诺贝尔化学奖简介[J]. 首都医科大学学报, 2020, 41(6): 1014-1018.
[4]	柳鑫, 徐有青, 崔纯莹, 赵志刚. GPIHBP1基因敲除小鼠在严重高三酰甘油血症急性胰腺炎导致肺损伤研究中的应用[J]. 首都医科大学学报, 2020, 41(4): 564-569.
[5]	李奇, 卢晶, 朱晓蓉, 熊枫然, 杨金奎. Unc13基因胰岛β细胞条件性敲除小鼠模型的构建和鉴定[J]. 首都医科大学学报, 2020, 41(1): 14-20.
[6]	卢晶, 吴谦, 谢荣荣, 仇海燕, 程呈, 杨金奎. β位淀粉样前体蛋白裂解酶2基因敲除斑马鱼动物模型的构建及其初步应用[J]. 首都医科大学学报, 2015, 36(6): 839-843.
[7]	程呈, 吴谦, 卢晶, 仇海燕, 李倩, 杨金奎. 基于CRISPR/Cas9研究RFX6对斑马鱼胰腺发育的影响[J]. 首都医科大学学报, 2015, 36(6): 844-847.
[8]	丁磊;高宏;朱煜冰;李文斌. Claudin-7 潜在的抑癌基因[J]. 首都医科大学学报, 2012, 33(5): 569-574.