[Construction and identification of RNA interference lentiviral expression vector of galectin-3 gene].
Nan Fang Yi Ke Da Xue Xue Bao
; 28(7): 1264-7, 2008 Jul.
Article
em Zh
| MEDLINE
| ID: mdl-18676279
ABSTRACT
OBJECTIVE:
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.METHODS:
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.RESULTS:
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.CONCLUSION:
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.
Buscar no Google
Base de dados:
MEDLINE
Assunto principal:
Lentivirus
/
Galectina 3
/
RNA Interferente Pequeno
/
Vetores Genéticos
Idioma:
Zh
Ano de publicação:
2008
Tipo de documento:
Article