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首页> 外文期刊>Radiology and Oncology >Evaluation of shRNA-mediated gene silencing by electroporation in LPB fibrosarcoma cells
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Evaluation of shRNA-mediated gene silencing by electroporation in LPB fibrosarcoma cells

机译：电穿孔法检测LPB纤维肉瘤细胞中shRNA介导的基因沉默

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Background. Silencing oncogenes or other genes that contribute to tumor malignancy and progression offers a promising approach to treating cancer. Specific and efficient silencing of gene expression can be achieved by RNA interference (RNAi) technology using small interfering RNA (siRNA) or short hairpin RNA (shRNA). However, a major challenge in RNAi technology is effective delivery of interfering molecules into target cells. The aim of our study was to evaluate electroporation as a perspective method for efficient in vitro transfection of LPB fibrosarcoma cells with plasmid DNA expressing shRNA.Methods. Induction of shRNA-mediated gene silencing by electroporation was determined by fluorescence microscopy, flow cytometry and western blot analysis. The effect of electroporation conditions on cell survival and proliferation was determined by clonogenic assay.Results and conclusions. Our results demonstrated that electroporation is a feasible and effective method for delivery of plasmid DNA expressing shRNA into cancer cells in vitro. Electrotransfection of murine LPB fibrosarcoma cells, continuously expressing green fluorescence protein - GFP (LPBGFP), with plasmid DNA encoding shRNA-GFP, reduced GFP expression, which was determined on the protein level, as well as by measurement of GFP fluorescence intensity. A pronounced reduction in GFP expression level was detected from the second to the fifth day after treatment. Moreover, the method is easy to perform and showed low cell damaging effects, which are the most important and preferential factors for further in vivo studies.
机译：背景。沉默致癌基因或其他有助于肿瘤恶性肿瘤和进展的基因为治疗癌症提供了一种有希望的方法。通过使用小干扰RNA（siRNA）或短发夹RNA（shRNA）的RNA干扰（RNAi）技术，可以实现基因表达的特异性和有效沉默。然而，RNAi技术的主要挑战是将干扰分子有效递送到靶细胞中。我们的研究目的是评估电穿孔作为一种有效的体外表达表达shRNA质粒DNA的LPB纤维肉瘤细胞转染的透视方法。通过荧光显微镜，流式细胞术和蛋白质印迹分析确定通过电穿孔诱导shRNA介导的基因沉默。通过克隆形成试验确定了电穿孔条件对细胞存活和增殖的影响。结果与结论。我们的结果表明，电穿孔是一种将表达shRNA的质粒DNA体外递送至癌细胞的可行且有效的方法。鼠LPB纤维肉瘤细胞的电转染，连续表达绿色荧光蛋白-GFP（LPBGFP），编码shRNA-GFP的质粒DNA，降低了GFP的表达，这在蛋白水平以及通过测量GFP荧光强度来确定。从治疗后第二天到第五天检测到GFP表达水平明显降低。此外，该方法易于实施并且显示出低的细胞破坏作用，这是用于进一步体内研究的最重要和优先因素。

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