利用CRISPR/Cas9敲除人源细胞系中LMNA基因的研究

doi:10.16288/j.yczz.18-146

遗传 ›› 2019, Vol. 41 ›› Issue (1): 66-75.doi: 10.16288/j.yczz.18-146

利用CRISPR/Cas9敲除人源细胞系中LMNA基因的研究

刘恒¹,李东明¹,朱兰玉¹,赖乐锦¹,闫婉云¹,陆玉双¹,韦伊¹,黄月琪¹,方媚¹,苏元港¹,杨芳²,舒伟¹

^1. 广西医科大学基础医学院细胞生物学与遗传教研室,南宁 530021
^2. 广西师范大学环境与资源学院,桂林 541004

收稿日期:2018-09-13 修回日期:2018-11-21 出版日期:2019-01-20 发布日期:2018-12-06
作者简介:刘恒,硕士研究生,专业方向：分子生物学与生物化学。E-mail:240627592@QQ.com|李东明,硕士研究生,专业方向：分子生物与生物化学。E-mail: abcd.1456@163.com
基金资助:
国家自然科学基金项目(编号：31660311)和广西医科大学创新创业项目(编号：201710598055)资助

Research on the knockout of LMNA gene by CRISPR/Cas9 system in human cell lines

Heng Liu¹,Dongming Li¹,Lanyu Zhu¹,Lejin Lai¹,Wanyun Yan¹,Yushuang Lu¹,Yi Wei¹,Yueqi Huang¹,Mei Fang¹,Yuangang Su¹,Fang Yang²,Wei Shu¹

^1. Department of Cell Biology and Genetics, Basic Medical College, Guangxi Medical University, Nanning 530021, China
^2. College of Environmental Resources, Guangxi Normal University, Guilin 541004, China

Received:2018-09-13 Revised:2018-11-21 Online:2019-01-20 Published:2018-12-06
Supported by:
[Supported by the National Natural Science Foundation of China (No. 31660311) and Guangxi Medical University Innovation and Entrepreneurship Project (No. 201710598055)]

摘要/Abstract

摘要：

LMNA 基因编码A型和C型核纤层蛋白,参与细胞核核膜的组织,影响基因组稳定性并对细胞分化产生影响。人类肿瘤中LMNA表达异常普遍存在,其突变造成多种核纤层蛋白病,如Emery-Dreifuss 肌营养不良症(Emery-Dreifuss muscular dystrophy, EDMD)、扩张型心肌病(dilated cardiomyopathy, DCM)和儿童早老症(Hutchinson-Gliford progeria syndrome, HGPS)等。为进一步研究LMNA在细胞内的功能,本研究利用CRISPR/Cas9技术对体外培养的293T与HepG2细胞株的LMNA基因进行编辑,获得两株LMNA基因敲除(LMNA KO)的稳定细胞系。与野生型相比,LMNA KO细胞系增殖能力相对减弱,凋亡增加。同时,细胞形态上也发生显著改变,核膜凹凸不平。本研究首次报道了LMNA KO永生细胞系构建和形态研究结果,为后续LMNA基因功能研究和致病突变体研究奠定基础。

关键词: LMNA基因, CRISPR/Cas9, 293T, HepG2, 细胞形态

Abstract:

The LMNA gene encodes the nuclear Lamin A and Lamin C proteins, and is related to nuclear membrane organization, genome stability and cell differentiation. Abnormal expression of LMNA is ubiquitous in human tumors, and its mutation leads to various forms of laminopathies, including Emery-Dreifuss muscular dystrophy (EDMD), dilated cardiomyopathy (DCM), and Hutchinson-Gliford progeria syndrome (HGPS). To further determine the functions of the LMNA gene in cellular physiology, the present study used the CRISPR/Cas9 technique to edit the LMNA gene of 293T and HepG2 cells in vitro, which resulted in two stable LMNA gene knockout (LMNA KO) cell lines. Compared to the respective wild type cells, the LMNA KO cell lines showed decrease in proliferation ability, increase in apoptosis, alteration in cellular morphology and uneven structures in the nucleus membrane. In this study, we report for the first time the results on the construction of LMNA KO immortalized cell lines and characterization of their morphological changes, thereby laying the foundation for the further studies of the LMNA gene functions and pathogenic mutations.

Key words: LMNA gene, CRISPR/Cas9, 293T, HepG2, cell morphology

刘恒, 李东明, 朱兰玉, 赖乐锦, 闫婉云, 陆玉双, 韦伊, 黄月琪, 方媚, 苏元港, 杨芳, 舒伟. 利用CRISPR/Cas9敲除人源细胞系中LMNA基因的研究[J]. 遗传, 2019, 41(1): 66-75.

Heng Liu, Dongming Li, Lanyu Zhu, Lejin Lai, Wanyun Yan, Yushuang Lu, Yi Wei, Yueqi Huang, Mei Fang, Yuangang Su, Fang Yang, Wei Shu. Research on the knockout of LMNA gene by CRISPR/Cas9 system in human cell lines[J]. Hereditas(Beijing), 2019, 41(1): 66-75.

参考文献

[1]	Burke B, Stewart CL . Functional architecture of the cell's nucleus in development, aging, and disease. Curr Top Dev Biol, 2014,109:1-52. [DOI]
[2]	Gruenbaum Y, Foisner R . Lamins: Nuclear intermediate filament proteins with fundamental functions in nuclear mechanics and genome regulation. Annu Rev Biochem, 2015,84:131-164. [DOI]
[3]	Gruenbaum Y, Medalia O . Lamins: The structure and protein complexes. Curr Opin Cell Biol, 2015,32:7-12. [DOI]
[4]	Swift J, Ivanovska IL, Buxboim A, Harada T, Dingal PCDP, Pinter J, Pajerowski JD, Spinler KR, Shin JW, Tewari M, Rehfeldt F, Speicher DW, Discher DE . Nuclear lamin-A scales with tissue stiffness and enhances matrix-directed differentiation. Science, 2013,341(6149):1240104. [DOI]
[5]	Davidson PM, Denais C, Bakshi MC, Lammerding J . Nuclear deformability constitutes a rate-limiting step during cell migration in 3-D environments. Cell Mol Bioeng, 2014,7(3):293-306. [DOI]
[6]	Kohsaka S, Saito T, Akaike K, Suehara Y, Hayashi T, Takagi T, Kaneko K, Ueno T, Kojima S, Kohashi KI, Mano H, Oda Y, Yao T . Pediatric soft tissue tumor of the upper arm with LMNA-NTRK1 fusion. Hum Pathol, 2018,72:167-173. [DOI]
[7]	Sakthivel KM, Sehgal P . A novel role of lamins from genetic disease to cancer biomarkers. Oncol Rev, 2016,10(2):309. [DOI]
[8]	Butin-Israeli V, Adam SA, Goldman AE, Goldman RD . Nuclear lamin functions and disease. Trends Genet, 2012,28(9):464-471. [DOI]
[9]	Worman HJ, Fong LG, Muchir A, Young SG . Laminopathies and the long strange trip from basic cell biology to therapy. J Clin Invest, 2009,119(7):1825-1836. [DOI]
[10]	Gordon LB, Rothman FG, López-Otín C, Misteli T . Progeria: A paradigm for translational medicine. Cell, 2014,156(3):400-407. [DOI]
[11]	Gonzalo S, Kreienkamp R . DNA repair defects and genome instability in Hutchinson-Gilford Progeria Syndrome. Curr Opin Cell Biol, 2015,34:75-83. [DOI]
[12]	Vidak S, Foisner R . Molecular insights into the premature aging disease progeria. Histochem Cell Biol, 2016,145(4):401-417. [DOI]
[13]	Rodríguez S, Eríksson M . Low and high expressing alleles of the LMNA gene: Implications for laminopathy disease development. PLoS One, 2011,6(9):e25472. [DOI]
[14]	Vigouroux C, Guénantin AC, Vatier C, Capel E, Le Dour C, Afonso P, Bidault G, Béréziat V, Lascols O, Capeau J, Briand N, Jéru I . Lipodystrophic syndromes due to LMNA mutations: Recent developments on biomolecular aspects, pathophysiological hypotheses and therapeutic perspectives. Nucleus, 2018,9(1):251-264. [DOI]
[15]	Cenni V, D'Apice MR, Ga

编辑推荐

Metrics

www.chinagene.cn
备案号：京ICP备09063187号-4
总访问:,今日访问:,当前在线:

利用CRISPR/Cas9敲除人源细胞系中LMNA基因的研究

Research on the knockout of LMNA gene by CRISPR/Cas9 system in human cell lines

RichHTML

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

编辑推荐

Metrics