| Construction and identification of a vector expressing TRAM siRNA in mammalian cells |
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| 作者姓名： | 陈力勇 朱佩芳 杨策 蒋建新 王正国 |
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| 作者单位： | StateKeyLaboratoryofTrauma,BurnsandCombinedInjury,Department4,InstituteofSurgeryResearch,DapingHospital,ThirdMilitaryMedicalUniversity,Chongqing400042,China |
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| 基金项目： | 国家重点基础研究发展计划(973计划) |
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| 摘 要： | Objective：To construct and identify a vector expressing TRAM siRNA in mammalian cells.Methods :It was constructed that the vector named R-pSUPER-EGFP used to transcribe functional TRAM siRNA. Two of pair 64 nt TRAM gene specific target sequences were inserted into the downstream of the H1 promoter. The recombinants were transformed into E. coli JM109, and finally their veracity was confirmed by double cutting with the enzymes and sequencing. R-pSUPER-EGFP was transfected into RAW264. 7 cell by using LipofectamineTM2000, and the expression of TRAM was detected by Western blotting. Results .. Two different recombinant plasmids containing corresponding TRAM gene specific target sequences were constructed, transfected into RAW264.7 cell line successively, which can specifically restrain expression of TRAM protein. Conclusion :The optimizing method in constructing the recombinant vector serves other plasmid-based RNA interference research. Therefore, the recombinant vectors establish the basis for research on the function of TRAM gene.
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| 关 键 词： | TRAM siRNA 哺乳动物 细胞学研究 鉴别诊断 基因表达 |
Construction and identification of a vector expressing TRAM siRNA in mammalian cells |
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| CHEN Li-yong,Zhu Pei-fang,YANG Ce,JIANG Jian-Xin,WANG Zheng-guo.Construction and identification of a vector expressing TRAM siRNA in mammalian cells[J].Journal of Medical Colleges of PLA(China),2005,20(3):135-140. |
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| Authors: | CHEN Li-yong Zhu Pei-fang YANG Ce JIANG Jian-Xin WANG Zheng-guo |
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| Abstract: | Objective:To construct and identify a vector expressing TRAM siRNA in mammalian cells.Methods:It was constructed that the vector named R-pSUPER-EGFP used to transcribe functional TRAM siRNA. Two of pair 64 nt TRAM gene specific target sequences were inserted into the downstream of the H1 promoter. The recombinants were transformed into E. coli JM109, and finally their veracity was confirmed by double cutting with the enzymes and sequencing. R-pSUPER-EGFP was transfected into RAW264. 7 cell by using LipofectamineTM2000, and the expression of TRAM was detected by Western blotting. Results:Two different recombinant plasmids containing corresponding TRAM gene specific target sequences were constructed, transfected into RAW264.7 cell line successively, which can specifically restrain expression of TRAM protein. Conclusion :The optimizing method in constructing the recombinant vector serves other plasmid-based RNA interference research. Therefore, the recombinant vectors establish the basis for research on the function of TRAM gene. |
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| Keywords: | TRAM siRNA pSUPER-EGFP vector mammalian cells |
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