Construction and characterization of a eukaryotic expression vector for small interfering RNA targeting the NEDD9 gene

Chang,Jing-Xia; Wang,Hua-Qi; Zhao,Guo-Qiang; Chu,He-Ying; Zhang,Guo-Jun

doi:10.3892/ijmm.2012.1137

December 2012 Volume 30 Issue 6

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December 2012 Volume 30 Issue 6

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Article

Construction and characterization of a eukaryotic expression vector for small interfering RNA targeting the NEDD9 gene

Authors:
- Jing-Xia Chang
- Hua-Qi Wang
- Guo-Qiang Zhao
- He-Ying Chu
- Guo-Jun Zhang
View Affiliations / Copyright

Affiliations: Department of Respiratory Medicine, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, P.R. China, Department of Microorganisms and Immunization, Preclinical Medicine, Zhengzhou University, Zhengzhou, Henan, P.R. China
Pages: 1343-1348
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Published online on: September 20, 2012

https://doi.org/10.3892/ijmm.2012.1137
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Abstract

The aim of this study was to construct a eukaryotic expression vector for a small interfering RNA (siRNA) targeting the neural precursor cell expressed, developmentally downregulated 9 (NEDD9) gene, and to investigate the effects of RNA interference (RNAi) on NEDD9 expression in human lung adenocarcinoma A549 cells. We used the siRNA design and analysis software to determine the target oligonucleotides according to the sequence of NEDD9 mRNA available in GenBank. Four siRNA sequences were obtained, and the corresponding cDNAs were synthesized and inserted into the pRNAT-CMV3.2 plasmid to construct the recombinant plasmids. These were transformed into the E. coli strain DH5α. The plasmids, after identification by PCR and DNA sequencing, were transfected into the A549 cell line via the liposome method. NEDD9 mRNA and protein in the cells were determined by fluorescence quantitative RT-PCR (FQ-PCR) and western blotting, respectively. The pRNAT-CMV3.2-transfected plasmid was used as a control. Four recombinant plasmids were identified by PCR and sequence analysis, which contained the correct insertion of the designed sequences in the plasmids. FQ-PCR and western blotting showed substantially decreased mRNA and protein expression of the NEDD9 gene in the transfected cells, compared with the control group. In conclusion, the recombinant plasmids expressing the siRNA targeting the NEDD9 gene were successfully constructed, and the siRNA expression vectors inhibited the expression of NEDD9 in A549 cells.

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Journals

International Journal of Molecular Medicine

International Journal of Oncology

Molecular Medicine Reports

Oncology Reports

Experimental and Therapeutic Medicine

Oncology Letters

Biomedical Reports

Molecular and Clinical Oncology

World Academy of Sciences Journal

International Journal of Functional Nutrition

International Journal of Epigenetics

Medicine International

Construction and characterization of a eukaryotic expression vector for small interfering RNA targeting the NEDD9 gene

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