[Construction and identification of a lentiviral vector for RNA interference of human GLUT3 gene].
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.


CONCLUSIONS
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.
Department of Neurosurgery, Affiliated Hospital of Hainan Medical College, Haikou 570102, China.
Chuanyi
C
Zheng
Department of Neurosurgery, Affiliated Hospital of Hainan Medical College, Haikou 570102, China.
Zhenggang
Z
Chen
Department of Neurosurgery, Affiliated Hospital of Hainan Medical College, Haikou 570102, China.
Enqi
E
Bai
Department of Neurosurgery, Affiliated Hospital of Hainan Medical College, Haikou 570102, China.
Zhengzheng
Z
Li
Department of Neurosurgery, Affiliated Hospital of Hainan Medical College, Haikou 570102, China.
Kun
K
Yang
D005822:Genetic Vectors; D051274:Glucose Transporter Type 3 / Q000235:genetics*; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D016086:Lentivirus; D010957:Plasmids; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics*; D014162:Transfection
D051274:Glucose Transporter Type 3; D012333:RNA, Messenger; D034741:RNA, Small Interfering; C495687:SLC2A3 protein, human