Construction and identification of RNA interference lentiviral expression vector of galectin-3 gene / 南方医科大学学报
Journal of Southern Medical University
; (12): 1264-1267, 2008.
Article
in Chinese
| WPRIM (Western Pacific)
| ID: wpr-270159
Responsible library:
WPRO
ABSTRACT
<p><b>OBJECTIVE</b>To construct a recombinant lentiviral U6 plasmids for RNA interference (RNAi) of galectin-3 gene and select the optimal target sequence of galectin-3 gene for RNAi.</p><p><b>METHODS</b>Double-stranded oligo DNAs were designed and synthesized according to the sequence of galectin-3 gene, and ligated into linearized pGCL-GFP/U6 plasmid followed by transformation into competent DH5alpha cells. After PCR and sequence analysis for verification of the positive clones, the plasmid pGCL-GFP/U6 Gal-3shRNA-1 was extracted and transfected into CaCl2-treated 293T cells to obtain the viral vectors containing the RNAi sequence. MCF-7 cells were infected with pGCL-GFP/U6 Gal-3shDNA-1, and at the infection rate over 50%, the cells were harvested to extract the RNA. Real time-PCR was performed to determine the expression level of galectin-3 mRNA in the infected cells.</p><p><b>RESULTS</b>The recombinant vector was successfully constructed as confirmed by sequence analysis. High titer of the virus was obtained, and after infection of MCF-7 cells, RNAi targeting the 1# and 3# sequences in galectin-3 gene resulted in suppression of galectin-3 mRNA expression by 95% and 85%, respectively.</p><p><b>CONCLUSION</b>The recombinant lentiviral U6 plasmid for RNAi of Galectin-3 gene has been successfully constructed, which provides the basis for further study of the role of galectin-3 gene in tumor cells.</p>
Full text:
Available
Database:
WPRIM (Western Pacific)
Main subject:
Pathology
/
Breast Neoplasms
/
RNA, Messenger
/
Transfection
/
Cell Line
/
Lentivirus
/
Reverse Transcriptase Polymerase Chain Reaction
/
Galectin 3
/
RNA, Small Interfering
/
RNA Interference
Type of study:
Diagnostic study
Limits:
Female
/
Humans
Language:
Chinese
Journal:
Journal of Southern Medical University
Year:
2008
Document type:
Article