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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2018, Vol. 38 Issue (10): 38-47    DOI: 10.13523/j.cb.20181005
技术与方法     
利用CRISPR/Cas9技术构建AEG-1基因敲除U251细胞系并探讨其转移行为的特点 *
盛玉瑞1,李斌1,王斌1,2,左娣2,马琳2,任晓璠2,郭乐1,3,**(),刘昆梅2,**()
1 宁夏医科大学临床医学院 银川 750004
2 宁夏医科大学宁夏颅脑疾病重点实验室 银川 750004
3 宁夏临床微生物重点实验室 宁夏医科大学总医院 银川 750003
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 WANG1,2,Di ZUO2,Lin MA2,Xiao-fan REN2,Le GUO1,3,**(),Kun-mei LIU2,**()
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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摘要：

星形胶质细胞上调基因-1(astrocyte elevated gene-1,AEG-1)在多种肿瘤中过表达,参与肿瘤的形成、转移等过程。本实验利用CRISPR/Cas9技术敲除AEG-1基因并研究其在胶质瘤细胞转移过程中的作用。首先设计构建sgRNA/Cas9二合一表达载体并转染到人胶质瘤U251细胞中,通过TA克隆测序鉴定sgRNA的活性;然后筛选建立稳定的AEG-1敲除U251细胞系,并利用Western blot实验检测AEG-1的敲除效率;最后利用Transwell小室、划痕实验评价AEG-1敲除后对肿瘤细胞迁移能力的影响。结果显示,成功构建靶向敲除AEG-1基因的sgRNA/Cas9二合一表达载体,所构建的载体与实验设计相一致,通过TA克隆测序鉴定sgRNA有活性;成功建立稳定的AEG-1敲除U251细胞系,Western blot实验结果表明敲除效率高达98%;Transwell小室实验、划痕实验结果表明AEG-1敲除U251细胞系的转移能力明显降低。
关键词： 星形胶质细胞上调基因-1(AEG-1);  CRISPR/Cas9;  基因敲除;  U251细胞    
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
收稿日期: 2018-05-10 出版日期: 2018-11-09
ZTFLH:  Q813  
基金资助: * 国家自然科学基金(31660267);宁夏回族自治区2016年大学生创新(201610752012);教育部“春晖计划”合作科研(Z2016060);宁夏高等学校科学技术研究(NGY201591);宁夏回族自治区“十三五”重大科技(2016BZ07)
通讯作者: 郭乐,刘昆梅     E-mail: guoletian1982@163.com;lkm198507@126.com
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盛玉瑞
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刘昆梅

引用本文:

盛玉瑞,李斌,王斌,左娣,马琳,任晓璠,郭乐,刘昆梅. 利用CRISPR/Cas9技术构建AEG-1基因敲除U251细胞系并探讨其转移行为的特点 *[J]. 中国生物工程杂志, 2018, 38(10): 38-47.

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.

链接本文:

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

图1  pX459质粒图及Bbs I酶切位点
名称
Name		 序列
Sequence
AEG-1- sgRNA-1 5'-CACCGACTTCAACAGTCCGCCCATT-3'
3'-CTGAAGTTGTCAGGCGGGTAACAAA-5'
AEG-1- sgRNA-2 5'-CACCGCAAAACAGTTCACGCCATGA-3'
3'-CGTTTTGTCAAGTGCGGTACTCAAA-5'
AEG-1- sgRNA-3 5'- CACCGACAGCAGCGTAAACGTGATA-3'
3'- CTGTCGTCGCATTTGCATATCAAA-5'
表1  AEG-1 sgRNA核酸片段
名称
Name		 序列
Sequence		 PCR产物大小
The size of PCR product
Short-chain primer of AEG-1-1 5'-TCCTGCGTATAAATCTTTG-3'
3'-TTTACCACCCATCCACTA-5'		 324
Short-chain primer of AEG-1-2 5'-TAAAGCAGTGCAAAACAG -3'
3'-TTCCAGGAGACAAAGACA-5'		 325
Short-chain primer of AEG-1-3 5'-GGTGCTGACTGATTCTGG -3'
3'-CTGCTGGTATTCGGTAAA-5'		 293
表2  用于TA克隆的短链PCR引物
图2  pX459-AEG-1鉴定结果
图3  TA克隆分析检测Indel突变
图4  AEG-1基因在AEG-1基因敲除细胞系中的表达水平
图5  Transwell小室实验检测AEG-1基因敲除细胞系的侵袭能力
图6  划痕实验检测AEG-1基因敲除细胞系的迁移能力
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