Construction and identification of a lentiviral vector for RNA interference of human GLUT3 gene.
10.11817/j.issn.1672-7347.2016.05.002
- Author:
Chuanyi ZHENG
1
;
Zhenggang CHEN
1
;
Enqi BAI
1
;
Zhengzheng LI
1
;
Kun YANG
1
Author Information
1. Department of Neurosurgery, Affiliated Hospital of Hainan Medical College, Haikou 570102, China.
- Publication Type:Journal Article
- MeSH:
Genetic Vectors;
Glucose Transporter Type 3;
genetics;
HEK293 Cells;
HeLa Cells;
Humans;
Lentivirus;
Plasmids;
RNA Interference;
RNA, Messenger;
genetics;
RNA, Small Interfering;
genetics;
Transfection
- From:
Journal of Central South University(Medical Sciences)
2016;41(5):455-462
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVE:To construct an effective lentiviral vector for RNA interference (RNAi) with human glucose transporter 3 (GLUT3)gene.
METHODS:Four pairs of shRNA sequences against different parts of GLUT3-mRNA were separately cloned into the RNAi plasmid vector pLV-shRNA by recombinant DNA technology to construct shRNA expression vectors pLV-shRNA-GLUT3-1, pLV-shRNA-GLUT3-2, pLV-shRNA-GLUT3-3, and pLV-shRNA-GLUT3-4. The vectors were transfected into HeLa cells to detect the effectiveness of GLUT3 gene silencing. One of effective vectors was selected and co-transfected into 293T cells with lentivirus packaging plasmids to obtain packaged lentivirus particles LV-GLUT3. After viral titer determination, U251 glioblastoma cells were infected with LV-GLUT3 at a multiplicity of infection (MOI) of 10. Finally, the expression of GLUT3 protein was detected by Western blot.
RESULTS:DNA sequencing demonstrated that the shRNA sequences were successfully inserted into the pLV-shRNA vectors. In HeLa cells, the expression of GLUT3-mRNA was significantly down-regulated by the recombinant vectors compared with negative control. The recombinant lentivirus LV-GLUT3 harvested from 293T cells had a titer of 1.5×10(9) TU/mL. After infection with LV-GLUT3, the expression of GLUT3 protein in U251 glioblastoma cells was down-regulated.
CONCLUSION:An effective lentiviral shRNA expression vector targeting the GLUT3 gene is successfully constructed and can be used for further study on the functions of GLUT3 gene.
