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目的利用CRISPR/Cas9系统,敲除小鼠黑色素瘤细胞系的MATP基因,为MATP基因的功能研究奠定基础。方法利用http://crispr.mit.edu/网站,设计针对MATP的特异性引物,并将引物链接到pCAS9/gRNA1载体。将阳性载体转染小鼠黑色素瘤细胞系B16F10,利用无限稀释的方法获得转染后的单克隆细胞株。提取不同细胞株的基因组,通过测序的方法进一步筛选出发生MATP基因切割的细胞株,并利用Western-blot的方法验证MATP的表达情况。结果成功获得了3株MATP基因敲除的细胞株,Western-blot结果表明,该细胞株不表达MATP蛋白。结论利用pCAS9/gRNA1载体,可以实现B16F10细胞系MATP基因的敲除。
Objective To knock out the MATP gene of mouse melanoma cell line by CRISPR / Cas9 system and lay the foundation for the function study of MATP gene. Methods The specific primers for MATP were designed using the http://crispr.mit.edu/ website and the primers were linked to the pCAS9 / gRNA1 vector. The positive vector was transfected into the mouse melanoma cell line B16F10 and the transfectant monoclonal cell line was obtained by using an infinite dilution method. The genomes of different cell lines were extracted, and the cell lines with MATP gene cleavage were further screened by sequencing. The expression of MATP was verified by Western-blot. Results Three MATP knockout cell lines were successfully obtained. The results of Western-blot showed that the cell lines did not express MATP protein. Conclusion The knockout of MATP gene in B16F10 cell line can be achieved by using pCAS9 / gRNA1 vector.