| 贠喆1 马云雷1 张涛1 蔡承魁1 韩康1 杨彤涛1 庞炜2 周勇1.hERG shRNA 表达载体构建及其稳定转染骨肉瘤细胞系MG-63、
SOSP-9607 的建立[J].,2012,12(11):2031-2034 |
| hERG shRNA 表达载体构建及其稳定转染骨肉瘤细胞系MG-63、
SOSP-9607 的建立 |
| Construction of hERG shRNA Expression Vectors and Establishment of itsStably Transfected Osteosarcoma Cell lines MG-63 and SOSP-9607 |
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| DOI： |
| 中文关键词: hERG 钾离子通道 骨肉瘤 shRNA 载体构建 |
| 英文关键词: hERG Potassium channel Osteosarcoma shRNA Vector construction |
| 基金项目: |
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| 摘要点击次数: 958 |
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| 中文摘要: |
| 目的：构建hERG 钾离子通道蛋白（human ether-a-go-go-related gene potassium channel）shRNA 表达载体质粒，获得稳定转染
干扰质粒的人骨肉瘤细胞系MG-63、SOSP-9607。方法：将4 对合成的寡核苷酸链退火形成双链，连接入pGPU6/GFP/Neo 表达载
体，并测序鉴定。使用脂质体法将重组的质粒转染至MG-63、SOSP-9607，通过G418 筛选建立稳定转染的两种细胞系，采用免疫
印迹（Western blot）技术检测hERG 蛋白的表达。结果：测序结果证实shRNA 与载体连接正确，免疫印迹实验证实hERG 蛋白表
达显著降低。结论：成功构建了hERG shRNA 真核表达载体，获得了稳定表达hERG shRNA 的人骨肉瘤细胞系MG-63 和
SOSP-9607。 |
| 英文摘要: |
| Objective: To construct and screen the human ether-a-go-go-related gene potassium channel (hERG) shRNA expression
vectors and to obtain the stably transfected osteosarcoma cell lines MG-63 and SOSP-9607. Methods: Four target gene segments
were synthesized and cloned respectively into pGPU6/GFP/Neo expression vector to construct four recombinant eukaryotic vectors which
were identified by DNA sequencing. The recombinant plasmids were transfected into MG-63 and SOSP-9607 cells by lipofectanite. The
resistant MG-63 and SOSP-9607 cells were selected by G418. The expression of hERG was detected by western blot. Results: The
shRNAs and vectors were linked corrected. The expression of hERG protein in both cell lines decreased significantly. Conclusions: The
hERG shRNA recombinant plasmids vectors were constructed successfully and the recombinant plasmids significantly knocked down
hERG protein in MG-63 and SOSP-9607 cells. |
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