[Construction and identification of RNA interference lentiviral expression vector of galectin-3 gene].
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.


CONCLUSIONS
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.
Wang
Ming-dong
MD
Department of Neurosurgery, Affiliated Hospital of Hebei University, Baoding 071000, China. wangmd815@163.com
Shi
Yan-fang
YF
Wang
Hong
H
Ma
Wen-bin
WB
Wang
Ren-zhi
RZ
D001943:Breast Neoplasms / Q000235:genetics / Q000473:pathology; D002460:Cell Line; D045744:Cell Line, Tumor; D005260:Female; D037502:Galectin 3 / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D034622:RNA Interference; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
D037502:Galectin 3; D012333:RNA, Messenger; D034741:RNA, Small Interfering