Construction and Identification of Cyclin E siRNA Eukaryotic Expression Vectors
Construction and Identification of Cyclin E siRNA Eukaryotic Expression Vectors
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发表时间：
2007
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通讯作者：
H. Tao
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作者：
H. Tao
目的构建Cyclin E siRNA真核表达载体，并检测其对人乳腺癌MCF-7细胞cyclin E表达的抑制效果。方法根据Genebank中的cyclin E cDNA序列设计合成两对siRNA，分别插入pGENESIL-1质粒中。对重组质粒pGENESIL/Cyclin E-1和pGENESIL /Cyclin E-1进行测序和鉴定。转染MCF-7细胞，RT-PCR检测cyclin E的表达。结果成功构建了Cyclin E siRNA真核表达载体，双酶切鉴定结果表明，成功构建了Cyclin E siRNA真核表达载体。插入片段的序列分析显示，序列与合成的siRNA寡核苷酸的序列相同。结论Cyclin E siRNA真核表达载体的构建为Cyclin E靶向乳腺癌的基因治疗奠定了实验基础。
Objective To construct eukaryotic expression vectors of siRNA tageting Cyclin E, and measure its inhibition efficiency in cyclin E expression of the human breast cancer cell line MCF-7. Methods Two pairs of siRNA were designed and synthesized according to the cyclin E cDNA sequence in Genebank, and they were then inserted into pGENESIL-1 plasmids respectively. The recombinants (named pGENESIL/Cyclin E-1 and pGENESIL /Cyclin E-1) were sequenced and identified. The vector was transfected into MCF-7 cell line, and RT-PCR was performed to examine the expression of cyclin E. Results Cyclin E siRNA eukaryotic expression vectors were successfully constructed and identified by double endonuclease digestion. Sequence analysis of inserted fragments revealed that the sequences were identical to those of synthesized siRNA oligonucleotides. Conclusion Construction of cyclin E siRNA eukaryotic expression vectors lays an experimental foundation for gene therapy of breast cancer by tageting Cyclin E.