基础医学与临床 ›› 2021, Vol. 41 ›› Issue (3): 318-324.

• 研究论文 • 上一篇    下一篇

利用CRISPR-PITCH系统构建表达CSDE1-EGFP的B16细胞株

刘孟雨1,周雅博2,黄波2,吕家迪3   

  1. 1. 中国医学科学院基础医学研究所 北京协和医学院基础学院
    2. 中国医学科学院基础医学研究所
    3. 北京协和医学院基础学院
  • 收稿日期:2020-12-03 修回日期:2020-12-24 出版日期:2021-03-05 发布日期:2021-03-01
  • 通讯作者: 吕家迪 E-mail:lvjiadi666@sina.com
  • 基金资助:
    国家自然科学基金

Construction of B16 cell strain expressing CSDE1-EGFP using CRISPR-PITCH system

  • Received:2020-12-03 Revised:2020-12-24 Online:2021-03-05 Published:2021-03-01
  • Contact: Jia-di JiaLV E-mail:lvjiadi666@sina.com
  • Supported by:
    National Natural Science Foundation of China

摘要: 目的 用新型CRISPR-PITCH系统构建表达CSDE1-EGFP的B16细胞系,分选出低表达CSDE1与高表达CSDE1的B16细胞系,并进行功能探讨。方法 用微同源介导的末端连接方法将EGFP编码序列插入到CSDE1基因最后一个外显子上;用流式细胞术、PCR、免疫荧光及活细胞成像来检测CSDE1-EGFP是否插入成功,检验EGFP的荧光强弱与CSDE1的表达高低是否一致;用Transwell实验、小鼠荷瘤模型实验观察低表达CSDE1与高表达CSDE1的B16细胞系之间的功能差异。结果 分选出低表达CSDE1与高表达CSDE1的B16细胞系,检测出EGFP表达强弱与CSDE1表达高低一致。观察到CSDE1蛋白表达量不同的B16细胞系在功能上有所差异。结论 以新型CRISPR-PITCH系统为研究CSDE1在其他肿瘤细胞内的动态定位,以及因表达量不同而产生的功能差异提供快速有效的直观方法。

关键词: 关键词:CRIS-PITCH系统, 增强型绿色荧光蛋白, 含E1的冷休克域, 微同源末端连接

Abstract: Objective To construct the melanoma cell line expressing endotopic CSDE1-EGFP by using the new CRISPR-PITCH system, then to select the cell line of low expression CSDE1 and high expression CSDE1. Methods The EGFP coding sequence was inserted into the last exon of the CSDE1 gene using a micro-homology-mediated end joining method ; Use flow cytometry sorting, PCR, immunofluorescence and live cell imaging to detect whether CSDE1-EGFP is inserted successfully, and whether the fluorescence intensity of EGFP is consistent with the expression level of CSDE1; Use transwell experiment and mouse tumor-bearing experiment were used to observe the functional differences between the B16 cell lines with low and high expression of CSDE1. Results The B16 cell line of low expression CSDE1 and high expression CSDE1 were selected, and the strength of EGFP expression was detected to be consistent with CSDE1 expression. It was observed that melanoma cells with different expression of CSDE1 protein have different functions. Conclusion The new CRISPR-PITCH system provides a fast and effective intuitive method for studying the dynamic positioning of CSDE1 in other tumor cells and the functional differences due to different expressions.

Key words: Key words: CRIS-PITCH system, EGFP, CSDE1, MMEJ

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