Rett综合征相关基因MeCP2敲除大鼠模型的构建及分析

doi:10.16288/j.yczz.16-180

遗传 ›› 2016, Vol. 38 ›› Issue (11): 1004-1011.doi: 10.16288/j.yczz.16-180

Rett综合征相关基因MeCP2敲除大鼠模型的构建及分析

翟伟, 胡宏秀, 乐亮, 庄峰峰, 王克柱, 赵英, 王凯, 刘新民, 孙迪安, 王晓英, 匡世焕, 胡克平

北京协和医学院中国医学科学院药用植物研究所,北京 100193

收稿日期:2016-05-18 出版日期:2016-11-20 发布日期:2016-08-26
通讯作者: 胡克平,博士,研究员,研究方向：蛋白质复合物与疾病关系。E-mail: hkpucla@163.com
作者简介:翟伟,硕士研究生,专业方向：生药学。E-mail: gilway@qq.com
基金资助:
国家自然科学基金面上项目(编号：81271255)资助

Generation and analysis of the Rett syndrome-associated MeCP2- null rat model

Wei Zhai, Hongxiu Hu, Liang Le, Fengfeng Zhuang, Kezhu Wang, Ying Zhao, Kai Wang, Xinmin Liu, Di’an Sun, Xiaoying Wang, Shihuan Kuang, Keping Hu

Institute of Medicinal Plant Development, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing 100193, China

Received:2016-05-18 Online:2016-11-20 Published:2016-08-26
Supported by:
[Supported by the National Natural Science Foundation of China(No.81271255)]

摘要/Abstract

摘要： MeCP2(Methyl CpG binding protein 2)基因突变可导致Rett综合征(Rett syndrome, RTT)。目前已报道的MeCP2敲除小鼠表型与RTT病人症状存在显著差异。为探索MeCP2在脑发育中的作用及其导致RTT的机制,本研究利用CRISPR/Cas9技术构建了MeCP2基因敲除大鼠模型。通过构建靶向敲除MeCP2基因的载体,体外将Cas9 mRNA和sgRNA显微注射到SD大鼠受精卵中,在MeCP2基因exon2中造成移码突变,从而获得MeCP2基因敲除大鼠。利用测序和Western blotting方法鉴定MeCP2敲除大鼠,并对其表型和行为学特征进行分析,发现MeCP2敲除大鼠体重降低,存在焦虑倾向和认知缺陷。本研究成功构建了MeCP2基因敲除大鼠模型,其表型类似人类RTT患者的症状,为后续MeCP2功能研究提供了更好的动物模型。

关键词: MeCP2, CRISPR/Cas9, SD大鼠模型, Rett综合征

Abstract: MeCP2 mutations are associated with the Rett syndrome (RTT). Currently, there is an urgent need for new animal models for RTT as the existing MeCP2 knockout mouse models fail to fully mimic the pathogenesis and symptoms of RTT patients. In order to investigate the role of MeCP2 in brain development and RTT pathogenesis, we aimed to set up the MeCP2-null rat model using the CRISPR/Cas9 technology. Firstly we constructed the MeCP2 targeting vector and then microinjected Cas9 mRNA and sgRNA mixtures into fertilized ova of SD rats. The sgRNA was designed to target the exon 2 of MeCP2. Next, knockout rats were confirmed using DNA sequencing and Western blotting. Lastly, phenotypes including growth and behaviors of MeCP2 knockout rats were analyzed. The results indicated that the MeCP2 knockout rats showed body weight loss, anxiety tendency and cognitive deficits. The MeCP2-null rat model established in this study recapitulates the major symptoms of RTT patients and provides an alternative tool for future studies of MeCP2 functions.

Key words: MeCP2, CRISPR/Cas9, SD rat model, Rett syndrome

翟伟, 胡宏秀, 乐亮, 庄峰峰, 王克柱, 赵英, 王凯, 刘新民, 孙迪安, 王晓英, 匡世焕, 胡克平. Rett综合征相关基因MeCP2敲除大鼠模型的构建及分析[J]. 遗传, 2016, 38(11): 1004-1011.

Wei Zhai, Hongxiu Hu, Liang Le, Fengfeng Zhuang, Kezhu Wang, Ying Zhao, Kai Wang, Xinmin Liu, Di’an Sun, Xiaoying Wang, Shihuan Kuang, Keping Hu. Generation and analysis of the Rett syndrome-associated MeCP2- null rat model[J]. Hereditas(Beijing), 2016, 38(11): 1004-1011.

参考文献

[1] He YQ, Gong XH, Zhang HL, Wang HY, Yang ZM. MeCP2 and neurodevelopmental disorders. Chin Bull Life Sci , 2012, 24(4): 374-379. 何艳琴, 公晓红, 张宏丽, 王红艳, 杨章民. MeCP2与神经发育性疾病. 生命科学, 2012, 24(4): 374-379.
[2] Amir RE, Van den Veyver IB, Wan MM, Tran CQ, Francke U, Zoghbi HY. Rett syndrome is caused by mutations in X-linked MeCP2 , encoding methyl-CpG-binding protein 2. Nat Genet , 1999, 23(2): 185-188.
[3] Bhanushali AA, Mandsaurwala A, Das BR. Homozygous c. 1160C>T (P38L) in the MECP2 gene in a female Rett syndrome patient. J Clin Neurosci , 2016, 25: 127- 129.
[4] Mullaney BC, Johnston MV, Blue ME. Developmental expression of methyl-CpG binding protein 2 is dynamically regulated in the rodent brain. Neuroscience , 2004, 123(4): 939-949.
[5] Kishi N, Macklis JD. MECP2 is progressively expressed in post-migratory neurons and is involved in neuronal maturation rather than cell fate decisions. Mol Cell Neurosci , 2004, 27(3): 306-321.
[6] Shahbazian MD, Antalffy B, Armstrong DL, Zoghbi HY. Insight into Rett syndrome: MeCP2 levels display tissue-and cell-specific differences and correlate with neuronal maturation. Hum Mol Genet , 2002, 11(2): 115-124.
[7] Lewis JD, Meehan RR, Henzel WJ, Maurer-Fogy I, Jeppesen P, Klein F, Bird A. Purification, sequence, and cellular localization of a novel chromosomal protein that binds to methylated DNA. Cell , 1992, 69(6): 905- 914.
[8] Ghosh RP, Horowitz-Scherer RA, Nikitina T, Shlyakhtenko LS, Woodcock CL. MeCP2 binds cooperatively to its substrate and competes with histone H1 for chromatin binding sites. Mol Cell Biol , 2010, 30(19): 4656-4670.
[9] Nan X, Meehan RR, Bird A. Dissection of the methyl-CpG binding domain from the chromosomal protein MeCP2. Nucleic Acids Res , 1993, 21(21): 4886-4892.
[10] Mellén M, Ayata P, Dewell S, Kriaucionis S, Heintz N. MeCP2 binds to 5hmC enriched within active genes and accessible chromatin in the nervous system. Cell , 2012, 151(7): 1417-1430.
[11] Wakefield RID, Smith BO, Nan XS, Free A, Soteriou A, Uhrin D, Bird AP, Barlow PN. The solution structure of the domain from MeCP2 that binds to methylated DNA. J Mol Biol , 1999, 291(5): 1055-1065.
[12] Baker SA, Chen L, Wilkins AD, Yu P, Lichtarge O, Zoghbi HY. An AT-hook domain in MeCP2 determines the clinical course of Rett syndrome and related disorders. Cell , 2013, 152(5): 984-996.
[13] Chahrour M, Jung SY, Shaw C, Zhou XB, Wong STC, Qin J, Zoghbi HY. MeCP2, a key contributor to neurological disease, activates and represses transcription. Science , 2008, 320(5880): 1224-1229.
[14] Ebert DH, Greenberg ME. Activity-dependent neuronal signalling and autism spectrum disorder. Nature , 2013, 493(7432): 327-337.
[15] Tao JF, Hu KP, Chang Q, Wu H, Sherman NE, Martinowich K, Klose RJ, Schanen C, Jaenisch R, Wang WD, Sun YE. Phosphorylation of MeCP2 at Serine 80 regulates its chromatin association and neurological function. Proc Natl Acad Sci USA , 2009, 106(12): 4882-4887.
[16] Young JI, Hong EP, Castle JC, Crespo-Barreto J, Bowman AB, Rose MF, Kang D, Richman R, Johnson JM, Berget S, Zoghbi HY. Regulation of RNA splicing by the methylation-dependent transcriptional repressor methyl-CpG binding protein 2. Proc Natl Acad Sci USA , 2005, 102(49): 17551-17558.
[17] Guy J, Hendrich B, Holmes M, Martin JE, Bird A. A mouse MeCP2-null mutation causes neurological symptoms that mimic Rett syndrome. Nat Genet , 2001, 27(3): 322-326.
[18] Chen RZ, Akbarian S, Tudor M, Jaenisch R. Deficiency of methyl-CpG binding protein-2 in CNS neurons results in a Rett-like phenotype in mice. Nat Genet , 2001, 27(3): 327- 331.
[19] Shahbazian MD, Young JI, Yuva-Paylor LA, Spencer CM, Antalffy BA, Noebels JL, Armstrong DL, Paylor R, Zoghbi HY. Mice with truncated MeCP2 recapitulate many Rett syndrome features and display hyperacetylation of histone H3. Neuron , 2002, 35(2): 243-254.
[20] Liu Z, Zhou X, Zhu Y, Chen ZF, Yu B, Wang Y, Zhang CC, Nie YH, San

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Rett综合征相关基因MeCP2敲除大鼠模型的构建及分析

Generation and analysis of the Rett syndrome-associated MeCP2- null rat model

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