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

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

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)]

Abstract

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

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.

References

[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

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