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Construction of shRNA Eukaryotic Expression Vector Targeting DENN-SV Gene
Luo Zhenhua1, Li Jincheng1*, Zhang Xiumei2, Feng Zhuo1
1Department of Breast Surgery, First Affiliated Hospital of Liaoning Medical University, Jinzhou 121000, China;
2Department of Cardiology, First Affiliated Hospital of Liaoning Medical University, Jinzhou 121000, China
2Department of Cardiology, First Affiliated Hospital of Liaoning Medical University, Jinzhou 121000, China
Abstract: This research designed four target sequences (DS-1， DS-2， DS-3， DS-4) based on DENN-SV
sequence and shRNA design principles， and connected them to pRNAi-U6.1/Neo empty vector using recombinant
DNA technology after annealing， and transformed them into competent E.coli. Amplify strains， extract plasmids
and conduct digestion and DNA sequencing were obtained. After transfecting recombinant eukaryotic expression
vector into human breast cancer MCF-7 cells， we used RT-PCR and Western blot inhibition to restrain the efficiency
of DENN-SV mRNA expression， and got the growth curves of MCF-7 by MTT assay. Sequencing results conformed
that the designed sequence had been successfully transfected into human breast cancer MCF-7 cells. The expression
of GFP (green fluorescent protein) was visible but inhibited (P<0.01). The inhibitory effect of DS-1 group was the
greatest. Results of growth curves by MTT assay showed that after transfection， the cell prolife ratio of experimental
group reduced significantly (P<0.05). The study successfully， with the application of RNAi technology， built up small
interfering RNA recombinant， which laid the foundation for the further study of the breast cancer gene therapy.
sequence and shRNA design principles， and connected them to pRNAi-U6.1/Neo empty vector using recombinant
DNA technology after annealing， and transformed them into competent E.coli. Amplify strains， extract plasmids
and conduct digestion and DNA sequencing were obtained. After transfecting recombinant eukaryotic expression
vector into human breast cancer MCF-7 cells， we used RT-PCR and Western blot inhibition to restrain the efficiency
of DENN-SV mRNA expression， and got the growth curves of MCF-7 by MTT assay. Sequencing results conformed
that the designed sequence had been successfully transfected into human breast cancer MCF-7 cells. The expression
of GFP (green fluorescent protein) was visible but inhibited (P<0.01). The inhibitory effect of DS-1 group was the
greatest. Results of growth curves by MTT assay showed that after transfection， the cell prolife ratio of experimental
group reduced significantly (P<0.05). The study successfully， with the application of RNAi technology， built up small
interfering RNA recombinant， which laid the foundation for the further study of the breast cancer gene therapy.
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