The Construction of AEG-1-Knockout U251 Cell Line by CRISPR/Cas9 Technology and Study of The Effect of AEG-1 on the Metastasis in U251 Cells

doi:10.13523/j.cb.20181005

China Biotechnology

2018, Vol. 38

Issue (10): 38-47 DOI: 10.13523/j.cb.20181005

Orginal Article

The Construction of AEG-1-Knockout U251 Cell Line by CRISPR/Cas9 Technology and Study of The Effect of AEG-1 on the Metastasis in U251 Cells

Yu-rui SHENG¹,Bin LI¹,Bin WANG^1,²,Di ZUO²,Lin MA²,Xiao-fan REN²,Le GUO^1,^3,^**(

),Kun-mei LIU^2,^**(

)

1 Ningxia Medical University Clinical Medical College , Yinchuan 750004, China
2 Ningxia Key Laboratory of Cerebrocranial Diseases, Ningxia Medical University, Yinchuan 750004, China
3 Ningxia Clinical Microbiology Key Laboratory, Yinchuan 750003, China

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Abstract

Astrocyte elevated gene-1 (AEG-1) was overexpressed in a diverse array of cancers and played an important role in the development and progression of cancer. The study aimed to construct the AEG-1-knockout U251 cell line by CRISPR/Cas9 technology and to explore the effect of AEG-1 on the metastasis in U251 Cells. Firstly, the designed sgRNA targeted to AEG-1 was synthesized, and was cloned into the pX459 plasmid to obtain the AEG-1-pX459 recombinant vector. The recombinant vector was transfected into human glioma U251 cells, and the activity of sgRNA was identified by TA cloning sequencing. Then, the U251 cells transferred with the recombinant vector were screened by puromycin to get the AEG-1-knockout cell line. The efficiency of gene knockout was detected by Western blot assay. Finally, the migration ability of the AEG-1-knockout cell line was evaluated by the methods of Transwell and Scratch experiment. The results showed that the AEG-1-pX459 recombinant vector was successfully constructed, and the sgRNA activity was confirmed by TA cloning sequencing. The AEG-1-knockout U251 cell line was successfully established. Western blot assay analysis showed that the knockout efficiency high to 98%. The Transwell and Scratch experiment results illustrated that the migration ability of the AEG-1-knockout cell line reduced obviously.

Key words： Astrocyte elevated gene-1(AEG-1) CRISPR/Cas9 Gene knockout U251 cells

Received: 10 May 2018 Published: 09 November 2018

ZTFLH:

Q813

Corresponding Authors: Le GUO,Kun-mei LIU E-mail: guoletian1982@163.com;lkm198507@126.com

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	Yu-rui SHENG
	Bin LI
	Bin WANG
	Di ZUO
	Lin MA
	Xiao-fan REN
	Le GUO
	Kun-mei LIU

Cite this article:

Yu-rui SHENG,Bin LI,Bin WANG,Di ZUO,Lin MA,Xiao-fan REN,Le GUO,Kun-mei LIU. The Construction of AEG-1-Knockout U251 Cell Line by CRISPR/Cas9 Technology and Study of The Effect of AEG-1 on the Metastasis in U251 Cells. China Biotechnology, 2018, 38(10): 38-47.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20181005 OR https://manu60.magtech.com.cn/biotech/Y2018/V38/I10/38

Fig.1 Schematic of pX459 plasmid and Bbs I endonuclease site (a) Schematic of pX459 plasmid, sgRNA scaffold is located under U6 promoter and contains 2 Bbs I endonuclease sites (b) Bbs I endonuclease site, which can recognize GAAGAC sequence and cut from the 2^nd base after that sequence and the 6^th base from the complimentary strand to form the specific cohesive terminus

Table 1 AEG-1 sgRNA Oligo

Table 2 PCR primers for short-chain PCR and used for TA cloning analysis

Fig.2 Identification of pX459-AEG-1 (a) Enzyme digestion results illustrated that all the plasmids had donor DNA insert (b) DNA sequencing of pX459-AEG-1, the results demonstrated that all the plasmids had donor DNA insert with correct ORF

Fig.3 TA cloning analysis to identify Indel mutations (a) The mutations of pX459-AEG-1-2 (b) The mutations of pX459-AEG-1-3

Fig.4 Evaluation of AEG-1 gene expression in stable AEG-1 gene knock out cell lines (a) Western blot verified the protein expression level of AEG-1 (b) GraphPad prism software analyzed the results of Western blot. The efficiency of pX459-AEG-1-3 was the best. The knockout efficiency reached 98% , ^**** P<0.001 vs control (Cas9) group

Fig.5 Transwell chamber experiments test the invasive ability of the AEG-1 knockout cell lines

Fig.6 Scratch experiments test the migration ability of the AEG-1 knockout cell lines (a) Scratch experiments verified the wound healing distance of AEG-1 (b) GraphPad prism software analyzed the results of scratch experiments ^**** P<0.001 vs control (Cas9) group


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