ErbB4基因miRNA干扰质粒的构建和有效性鉴定

Construction of miRNA RNAi expression vectors targeting ErbB4 and validation of their suppression effects

目的构建ErbB4基因特异性miRNA表达载体并检测其有效性。方法设计合成大鼠ErbB4基因特异性miRNA前体寡核苷酸,构建ErbB4基因特异性的重组miRNA干扰质粒。从大鼠脑分离提取总RNA,经RT-PCR构建靶序列质粒,并将其与miRNA干扰质粒共转染HEK293T细胞,经实时定量RT-PCR技术鉴定其干扰有效性。结果与结论针对ErbB4基因的miRNA干扰质粒构建成功,并能够有效抑制其靶基因的表达。该质粒为后续ErbB4基因特异性miRNA慢病毒表达载体的构建及ErbB4的相关功能研究奠定了基础。

Objective To construct specific miRNA RNAi expression vectors targeting ErbB4 gene and detect their sup- pression efficiency. Methods The specific pre-miRNA single stranded DNA oligos targeting rat ErbB4 were designed and synthesized. The recombined interfering plasmids containing miRNA targeting ErbB4 were constructed. Alter RNA was extracted from the rat brain, the ErbB4 targeting sequence expression vector was cloned by RT-PCR. HEK293T cell were cotransfected with ErbB4 targeting sequence expression vector and miRNA expression plasmids, and the suppression effects was identified by real time RT-PCR. Results and Conclusion The miRNA recombined plasmids targeting ErbB4 gene were successfully constructed and could effectively inhibit the expression of their targeting sequence, which can be used to construct lentiviral expression vector containing ErbB4 gene specific miRNA and facilitate ErbB4 function study.