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CXCR4基因shRNA表达质粒的构建及筛选鉴定

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摘要 目的构建编码CXCR4 mRNA的短发卡样RNA(shRNA)表达质粒用以筛选出基因沉默效果最明显的shRNA质粒表达载体。方法根据GenBank中登录的CXCR4基因的核苷酸序列,应用shRNA设计软件设计并合成4条短发卡shRNA的oligo DNA,构建shRNA重组质粒。采用酶切电泳和测序方法进行鉴定。将鉴定后的CXCR4基因shRNA重组质粒经脂质体介导转染胶质瘤U251细胞,24 h后经RT-PCR筛选有效的shRNA重组质粒。结果构建的质粒表达载体PCR鉴定均可扩增出目的条带,DNA测序结果与理论序列完全一致。RT-PCR检测显示,转染pGPU6/GFP/Rac1-421的U251细胞CXCR4基因mRNA的转录水平下降。结论成功构建CXCR4基因shRNA表达质粒,并筛选出效果最为明显的shRNA质粒表达载体为pGPU6/GFP/Rac1-421。
作者 胡志洪 王晓婓 国春龙 宋新灵
机构地区 白城中心医院 吉林大学第二医院 吉林省申邦细胞工程研究所
出处 《中国老年学杂志》 CAS CSCD 北大核心 2013年第17期4192-4194,共3页 Chinese Journal of Gerontology
基金 吉林省自然科学基金(No.201015175)
关键词 CXC趋化因子受体4 RNA干扰 U251细胞
分类号 R739.4 [医药卫生—肿瘤]
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参考文献12

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中国老年学杂志
2013年 第17期

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