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目的构建WT1(Wilms’tumor susceptibility gene,WT1)基因siRNA(small interference RNA,siRNA)载体, 为进一步探讨WT1的生物学功能奠定基础。方法根据针对WT1 mRNA的有效的siRNA序列,设计合成两条shRNA(small hair RNA,shRNA)的DNA模板单链,同时模板链两端分别设计不同的两个限制酶切位点。退火形成siRNA载体插入片断。用限制性内切酶将siRNA空载体线性化,T4连接酶将插入片断插入siRNA空质粒中。结果经酶切、PCR及测序鉴定确定构建成功。结论成功构建了WT1的干扰质粒,可对其生物学行为作进一步研究。
Objective To construct a small interfering RNA (siRNA) vector for WT1 gene and lay a foundation for further study on the biological function of WT1. Methods According to the effective siRNA sequence targeting WT1 mRNA, a single strand of DNA template for the synthesis of two small hair RNA (shRNA) shRNAs was designed. Two different restriction sites were designed at both ends of the template. Annealed to form siRNA vector inserts. The siRNA empty vector was linearized with a restriction enzyme and the T4 ligase inserted into the siRNA empty plasmid. Results confirmed by restriction enzyme digestion, PCR and sequencing. Conclusion The interference plasmid of WT1 was successfully constructed and its biological behavior could be further studied.