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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志  2017, Vol. 37 Issue (11): 94-100    DOI: 10.13523/j.cb.20171113
技术与方法     
应用CRISPR/Cas9技术构建miR-362敲除的95-D肺癌细胞株
罗丹1, 王莉娟2, 孙秀璇2, 张征2, 陈志南1
1. 北京交通大学理学院生命科学与生物工程研究院 北京 100044;
2. 空军军医大学(第四医科大学)细胞工程研究中心 西安 710032
Construction of miR-362 Knockdown 95-D Cell Line by CRISPR/Cas9 Technology
LUO Dan1, WANG Li-juan2, SUN Xiu-xuan2, ZHANG Zheng2, CHEN Zhi-nan1
1. College of Life Science and Bioengineering, School of Science, Beijing Jiaotong University, Beijing 100044, China;
2. Cell Engineering Research Center, The Fourth Military Medical University, Xi'an 710032, China
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摘要: 目的:运用CRISPR/Cas9基因编辑技术,建立miR-362基因敲除的95-D肺癌细胞株,并研究miR-362在肿瘤中的调控作用。方法:针对人源miR-362基因序列设计gRNA,构建px330-gRNA载体;T7E1 assay确定gRNA的有效性。分别扩增miR-362上下游同源臂序列构建donor载体。利用脂质体将CRISPR系统和donor载体共转至人肺癌细胞系95-D,通过同源重组方法将筛选标志基因整合至基因组中,通过流式分选以及qPCR方法检测筛选出的细胞中miR-362表达水平。利用Transwell分析miR-362对细胞运动能力的影响。结果:与95-D细胞相比,95-D-KnockDown细胞中miR-362表达水平显著降低,且迁移侵袭能力分别下降了53.1%(P=0.000 6)和48.3%(P=0.000 2)。结论:利用CRISPR/Cas9系统成功构建了miR-362基因敲除的95-D细胞,miR-362可促进细胞的运动能力,为后续研究miR-362在肿瘤中的作用机制和功能奠定了基础。
关键词: miR-36295-D迁移CRISPR/Cas9侵袭    
Abstract: Objective:to construct miR-362 knockdown 95-D cells by using CRISPR/Cas9 genome engineering technology, and study the function of miR-362 in cancer.Methods:gRNA sequences targeting the miR-362 gene were selected. Px330-gRNA recombination plasmids were constructed and the validity were evaluated by T7E1 assay. Left and right arms of miR-362 were amplified from genomic DNA by PCR, and sequentially cloned into the donor vector. The CRISPR/Cas9 system and donor vector were co-transfection into 95-D cells, and the selection system that allows for marker genes were integrated into the genome through homologous recombination (HR). The expression of miR-362 of 95-D-KnockDown cells sorted through FACS was detected by qPCR, and the migration and invasion were determined by Transwell assay. Results:Compared with 95-D cells, the expression of miR-362 in 95-D-KD cells was significantly down-regulated, and down-regulation of miR-362 expression can suppress the cell migration and invasion capacity of 95-D-KD cells. Conclusion:The miR-362 knockdown lung cancer cell line(95-D-KD cells)had been successfully constructed by using CRISPR/Cas9 system, which lays the foundation for further study of the mechanism and function of miR-362 in cancers.
Key words: 95-D    miR-362    CRISPR/Cas9    Invasion    Migration
收稿日期: 2017-06-14 出版日期: 2017-11-15
ZTFLH:  Q2  
基金资助: 国家自然科学基金资助项目(81572817)
通讯作者: 陈志南     E-mail: znchen@fmmu.edu.cn
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引用本文:

罗丹, 王莉娟, 孙秀璇, 张征, 陈志南. 应用CRISPR/Cas9技术构建miR-362敲除的95-D肺癌细胞株[J]. 中国生物工程杂志, 2017, 37(11): 94-100.

LUO Dan, WANG Li-juan, SUN Xiu-xuan, ZHANG Zheng, CHEN Zhi-nan. Construction of miR-362 Knockdown 95-D Cell Line by CRISPR/Cas9 Technology. China Biotechnology, 2017, 37(11): 94-100.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20171113        https://manu60.magtech.com.cn/biotech/CN/Y2017/V37/I11/94

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