| pRNAT-U6.1/Neo系统在E2F-3基因RNA干扰载体构建中的应用 |
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| 引用本文： | 胡海龙,吴长利,孙岩,张文岚,韩瑞发. pRNAT-U6.1/Neo系统在E2F-3基因RNA干扰载体构建中的应用[J]. 天津医药, 2009, 37(10) |
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| 作者姓名： | 胡海龙 吴长利 孙岩 张文岚 韩瑞发 |
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| 作者单位： | 1. 天津市泌尿外科研究所,天津医科大学第二医院,300211
2. 吉林大学第一医院移植中心 |
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| 基金项目： | 国家自然科学基金资助项目，天津市科技支撑计划重点项目，天津市高等学校科技发展基金计划项目，天津市卫生局科技基金资助项目 |
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| 摘 要： | 目的:构建针对E2F-3基因的siRNA表达载体.方法:化学合成3对编码短发夹RNA序列的、靶向E2F-3基因的寡核苷酸链,各64个碱基,退火、克隆到经BamHI、HindⅢ双酶切的pRNAT-U6.1/Neo载体上,重组构建RNAi质粒.通过双酶切、PCR鉴定及基因片段序列分析验证构建效果.结果:重组构建的pRNAT-U6.1/Neo载体经PCR分析及插入基因片段序列分析,结果表明64个碱基成功插入到预计位点,并且序列完全一致,插入序列无点突变位点.结论:载体的成功构建为进一步研究E2F-3基因在膀胱肿瘤中的作用奠定了基础,为进一步以其为靶点进行膀胱癌细胞系体外实验创造了条件.
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| 关 键 词： | RNA干扰 遗传载体 质粒 小分子干扰 |
Construction of pRNAT-U6.1/Neo siRNA System to Knockdown E2F-3 Activity |
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| HU Hailong,WU Changli,SUN Yan,ZHANG Wenlan,HAN Ruifa. Construction of pRNAT-U6.1/Neo siRNA System to Knockdown E2F-3 Activity[J]. Tianjin Medical Journal, 2009, 37(10) |
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| Authors: | HU Hailong WU Changli SUN Yan ZHANG Wenlan HAN Ruifa |
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| Abstract: | Objective: To construct siRNA plasmid expression vector in order to knockdown E2F-3 activity. Methods: Sixty-four base-pair oligos for hairpin RNA expression, which targeted E2F-3 gene, were chemically synthesized and annealed. The pRNAT-U6.1/Neo vector was linearized with Bam HI and HindⅢ. Finally, the annealed oligos were inserted into the lined pRNAT-U6.1/Neo to construct RNAi plasmid(pRNAT-U6.1-E2F-3/Neo). The reconstructed RNAi plasmids were i-dentified by electrophoresis after digestion with BamHI and Hind Ⅲ, and were confirmed by sequencing analysis. Results: The recombinant pRNAT-U6.1-E2F-3/Neo vector was identified by polymerase chain reaction, and confirmed by sequencing analysis. The results demonstrated that 64 bp had been inserted into the expected site. Furthermore, the insertion sequence was exactly correct and no mutation site was found. Conclusion: The pRNAT-U6.1-E2F-3/Neo RNAi system was constructed successfully. This will facilitate the study of E2F-3 in bladder cancer cell lines. |
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| Keywords: | RNA |
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