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目的 :运用CRISPR/Cas9基因编辑技术,建立IL-15基因敲除的MGC-803胃癌细胞株。方法 :针对IL-15基因作用的功能域,设计了靶向IL-15基因Exon2的gRNA。构建PX458-gRNA重组质粒并转化感受态细胞Stbl3,筛选出重组子后进行测序,通过测序确认了所设计的gRNA的有效性。并进一步通过流式细胞分选以及ELISA法检测筛选出的MGC-803胃癌细胞株IL-15基因的表达水平。结果 :测序结果显示敲除质粒构建成功,与阴性对照组相比,转染PX458-IL-15-g RNA质粒组IL-15的表达水平明显低于阴性对照组,差异具有统计学意义(P<0.001)。结论:利用CRISPR-Cas9系统成功构建了IL-15基因敲除的MGC-803细胞,为后续研究IL-15在肿瘤中的作用机制和功能奠定了基础。
OBJECTIVE: To establish MGC-803 gastric cancer cell line with IL-15 knockout by using CRISPR / Cas9 gene editing technology. Methods: In response to the functional domain of IL-15 gene, gRNA targeting IL-15 gene Exon2 was designed. The recombinant plasmid pG458-gRNA was constructed and transformed into competent cells Stbl3. The recombinant plasmids were selected and sequenced. The effectiveness of the designed gRNAs was confirmed by sequencing. The expression of IL-15 gene in MGC-803 gastric cancer cell line was further detected by flow cytometry and ELISA. Results: The sequencing results showed that the knock-out plasmid was successfully constructed. Compared with the negative control group, the expression level of IL-15 in the transfected PX458-IL-15-g RNA plasmid group was significantly lower than that in the negative control group (P <0.001). Conclusion: IL-15 gene knockout MGC-803 cells were successfully constructed by CRISPR-Cas9 system, which laid the foundation for further study on the mechanism and function of IL-15 in tumor.