Construction and identification of lentiviral vector of siRNA specific for Beclin1 gene.
- Author:
Wenyu WANG
1
;
Guoqiang ZHAO
;
Yun ZHOU
;
Hongkun FAN
;
Gang WU
Author Information
1. Cancer Center, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430023, China.
- Publication Type:Journal Article
- MeSH:
Apoptosis Regulatory Proteins;
genetics;
Autophagy;
genetics;
Base Sequence;
Beclin-1;
Carcinoma, Non-Small-Cell Lung;
genetics;
pathology;
Cell Line, Tumor;
Genetic Vectors;
genetics;
Humans;
Lentivirus;
genetics;
Lung Neoplasms;
genetics;
pathology;
Membrane Proteins;
genetics;
Molecular Sequence Data;
RNA Interference;
RNA, Small Interfering;
genetics;
Transfection
- From:
Journal of Biomedical Engineering
2013;30(1):131-135
- CountryChina
- Language:Chinese
-
Abstract:
The lentiviral vector was used for construction of a recombinant mediating RNA interference (RNAi) against Beclin1 gene in this study. Recombinant vector plasmid was transfected into non small cell lung cancer (NSCLC) A549 cells by liposome. PCR results showed that three amplified positive fragments were inserted into pRNAT-U6. 2/Lenti vectors. DNA sequencing results showed that the three recombinant lentivirus plasmids, pRNAT-U6. 2/Lenti-si356, pRNAT-U6. 2/Lenti-si423 and pRNAT-U6. 2/ Lenti-si684 were constructed successfully. After transfection with liposome, RT-PCR and Western blot analysis confirmed that the expression of Beclin1 mRNA and protein was inhibited in the three recombinant lentivirus plasmids transfected groups, and gene silencing efficacy was 35.56%, 89.22% and 66.78%, respectively. The results demonstrated that the lentiviral vectors of RNAi targeting Beclin1 gene were successfully constructed, and NSCLC A549 stable cell line with Beclin1 gene knockdown was established. This study finally provided a new cell model to explore the biological behavior of the Beclin1 gene in NSCLC A549 cells.
