| Arkadia表达载体转染肾小管上皮细胞条件的优化 |
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| 引用本文： | 廖芳芳,李娇,王惠川,张中文,吴国娟.Arkadia表达载体转染肾小管上皮细胞条件的优化[J].中国畜牧兽医,2012,39(8):44-47. |
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| 作者姓名： | 廖芳芳 李娇 王惠川 张中文 吴国娟 |
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| 作者单位： | 北京农学院动物科学技术学院,北京 102206 |
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| 基金项目： | 北京市自然科学基金项目，国家自然科学基金，北京市属高校人才强教计划资助项目 |
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| 摘 要： | 本试验旨在优化泛素化酶Arkadia质粒表达载体pGPU6/GFP/Neo转染小鼠肾小管上皮细胞(renal tubular epithelial cells,RTECs)的条件,为研究Arkadia表达载体在小鼠马兜铃酸肾病(aristolochic acid nephrohathy,AAN)中的作用奠定基础。以小鼠RTECs为转染对象、脂质体LipofectamineTM 2000为转染介质,采用Real-time PCR和CCK-8法检测不同的质粒与脂质体比例条件下Arkadia mRNA的表达差异及对细胞活性的影响;同时,用Real-time PCR检测不同的转染时间条件下表达载体对Arkadia mRNA沉默效果的差异。结果显示,Arkadia-pGPU6/GFP/Neo载体的转染效率表现出了一定的剂量和时间依赖性。当质粒DNA∶LipofectamineTM 2000为0.66 μg∶2 μL、转染时间为24 h时,对Arkadia mRNA表达的抑制最明显并且对细胞的毒性最小。最终确定了Arkadia-pGPU6/GFP/Neo载体转染小鼠RTECs的最佳条件。
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| 关 键 词： | Arkadia 表达载体 转染 肾小管上皮细胞 优化 |
| 收稿时间： | 2012-01-12 |
Transfection Optimization of Arkadia Expression Vector in Renal Tubular Epithelial Cells |
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| LIAO Fang-fang
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LI Jiao
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WANG Hui-chuan
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ZHANG Zhong-wen
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WU Guo-juan.Transfection Optimization of Arkadia Expression Vector in Renal Tubular Epithelial Cells[J].China Animal Husbandry & Veterinary Medicine,2012,39(8):44-47. |
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| Authors: | LIAO Fang-fang LI Jiao WANG Hui-chuan ZHANG Zhong-wen WU Guo-juan |
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| Institution: | College of Animal Science and Technology, Beijing University of Agriculture,Beijing 102206,China |
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| Abstract: | To lay the foundation for investigating the role of Arkadia expression vector in mouse aristolochic acid nephropathy(AAN),we optimized the transfection conditions of ubiquitination enzyme Arkadia plasmid expression vector pGPU6/GFP/Neo in mice renal tubular epithelial cells(RTECs).Mice RTECs were taken for transfection object,and liposome LipofectamineTM 2000 for transfection medium,we detected the Arkadia mRNA expressions differences and the affects of cell activity under the different ratios conditions of plasmid and liposome by Real-time PCR and CCK-8 assay;at the same time,the differences of Arkadia mRNA expressions silence under the different transfection time conditions by Real-time PCR.The results showed that transfection efficiency of Arkadia-pGPU6/GFP/Neo vector displayed dose and time dependents.When the ratio of plasmid DNA∶LipofectamineTM 2000 was 0.66 μg∶2 μL,and the transfection time was 24 h,Arkadia mRNA expression inhibition was very obvious and the cytotoxicity was the lowest.Lastly,we determined the best transfection condition of Arkadia-pGPU6/GFP/Neo vector. |
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| Keywords: | Arkadia expression vector transfection renal tubular epithelial cells optimization |
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