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目的以小鼠白血病L1210细胞系为基础,构建稳定高表达miR-17-92的细胞系。方法采用核转法,将miR-17-92的过表达质粒MSCV-LMP-miR-17-92、对照质粒MSCV-LMP转导入L1210细胞系;利用含嘌呤霉素选择、荧光倒置显微镜下观察、流式细胞术等技术筛选阳性克隆并检测转染效率;并用qPCR技术验证构建的阳性细胞系中的靶标miRNA表达水平,验证miR-17-92相对过表达比例。结果成功将MSCV-LMP-miR-17-92、MSCV-LMP转入L1210细胞系,转染效率较高,利用含嘌呤霉素的培养基筛选核转后的细胞,阳性率达到99%以上,miR-17-92簇中的靶标miRNA表达量持续。结论通过核转仪转染法成功将MSCV-LMP-miR-17-92、MSCV-LMP(对照质粒)转入L1210细胞系,并通过长期药物筛选获得了稳定表达细胞系,为进一步研究该基因在肿瘤中的作用提供了实验平台。
Objective To construct a stable and highly expressed cell line of miR-17-92 based on the mouse leukemia L1210 cell line. Methods The recombinant plasmids MSCV-LMP-miR-17-92 and MSCV-LMP were transfected into L1210 cell line by using nuclear transfer method. The cells were treated with puromycin, Flow cytometry and other techniques to screen positive clones and detect transfection efficiency; and qPCR technology to verify the expression of the target miRNA expression in the positive cell lines to verify the relative expression of miR-17-92 ratio. Results Transfection of MSCV-LMP-miR-17-92 and MSCV-LMP into L1210 cell line was successful. The transfection efficiency was high. The cells transfected with puromycin-containing medium were selected and the positive rate was over 99% Target miRNA expression levels in miR-17-92 clusters persisted. Conclusion MSCV-LMP-miR-17-92 and MSCV-LMP (control plasmid) were successfully transfected into L1210 cell line by nuclear transfection and stable expression cell lines were obtained by long-term drug screening. For further study of this gene The role in the tumor provides an experimental platform.