利用CRISPR/PITCH系统构建表达CSDE1-EGFP的B16细胞株

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

目的用新型CRISPR/PITCH系统构建表达CSDE1-EGFP的小鼠黑色素瘤B16细胞株,分选出低表达CSDE1与高表达CSDE1的B16细胞株,并进行功能探讨。方法用微同源介导的末端连接(MMEJ)方法将增强型绿色荧光蛋白(EGFP)编码序列插入到CSDE1基因最后一个外显子上;用流式细胞测量术、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小室法、小鼠荷瘤模型实验观察低表达CSDE1与高表达CSDE1的B16细胞株之间的功能差异。结果分选出低表达CSDE1与高表达CSDE1的B16细胞株,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细胞株在功能上有所差异。结论为研究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,新型CRISPR/PITCH系统提供了快速有效的直观方法。

Objective To construct a melanoma cell strain expressing endotopic CSDE1-EGFP using new CRISPR-PITCH system,then to select the cell strain of low expression CSDE1 and high expression CSDE1.MethodsThe EGFP coding sequence was inserted into the last exon of the CSDE1 gene using a micro-homology-mediated end joining(MMEJ)method;Using flow cytometry sorting,PCR,immunofluorescence and live cell imaging to detect whether CSDE1-EGFP was inserted successfully,and whether the fluorescence intensity of EGFP was consistent with the expression level of CSDE1;The Transwell experiment and mouse tumor-bearing experiment were used to observe the functional differences between the B16 cell strain with low and high expression of CSDE1.Results The B16 cell strains of low expression CSDE1 and high expression CSDE1 were selected,and the strength of EGFP expression was found to be consistent with CSDE1 expression.The melanoma cells with different expression level of CSDE1 protein had different functions.Conclusions The new CRISPR-PITCH system provides a fast and effective intuitive method for studying the dynamic positioning of CSDE1 in other tumor cells and thefunctional differences are resulted from different expressions.