Construction and identification of a lentiviral vector for RNA interference of human GLUT3 gene / 中南大学学报(医学版)
Journal of Central South University(Medical Sciences)
;
(12): 455-462, 2016.
Article
in Chinese
| WPRIM
| ID: wpr-815014
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.
Full text:
Available
Index:
WPRIM (Western Pacific)
Main subject:
Plasmids
/
RNA, Messenger
/
HeLa Cells
/
Transfection
/
Lentivirus
/
RNA, Small Interfering
/
RNA Interference
/
Glucose Transporter Type 3
/
HEK293 Cells
/
Genetic Vectors
Type of study:
Diagnostic study
Limits:
Humans
Language:
Chinese
Journal:
Journal of Central South University(Medical Sciences)
Year:
2016
Type:
Article
Similar
MEDLINE
...
LILACS
LIS