Construction of FLT3 3'-UTR-luciferase reporter vector and evaluation of its activity.
- Author:
Xiao-Ning GAO
1
;
Ji LIN
;
Yong-Hui LI
;
Li-Li WANG
;
Li YU
Author Information
1. Department of Hematology, Chinese PLA General Hospital, Beijing 100853, China.
- Publication Type:Journal Article
- MeSH:
3' Untranslated Regions;
Base Sequence;
Cell Line;
Genetic Vectors;
Humans;
Luciferases;
genetics;
MicroRNAs;
genetics;
Transfection;
fms-Like Tyrosine Kinase 3;
genetics
- From:
Journal of Experimental Hematology
2010;18(3):694-697
- CountryChina
- Language:Chinese
-
Abstract:
To analyze the possible microRNA (miRNA) target sites in the 3' untranslated region (3'-UTR) of FMS-like tyrosine kinase 3 (FLT3) gene, a FLT3 3'-UTR-luciferase reporter vector was constructed and the effect of miRNA on its activity was evaluated in 293T cell line. The 3'-UTR fragment of FLT3 gene was amplified by PCR from genomic DNA of HepG2 cells. PCR products were cloned into PstI/EcoRI-modified pGL3-control reporter vector (pGL3-control-m). The miRNA targeting FLT3 3'-UTR was predicted by Target Scan 5.1 software. The luciferase reporter vector and miRNA eukaryotic expression vector were transferred into 293T cells using FuGENE HD transfection reagent. The Dual-Luciferase Reporter Assay System was used to quantitate the reporter activity. The results showed that a 804-bp 3'-UTR fragment of FLT3 gene was successfully cloned into the pGL3-control-m reporter vector, which authenticated by PstI/EcoRI digestion and DNA sequencing. The predicted miRNA targeting FLT3 3'-UTR included miRNA-15a, miRNA-15b, miRNA-16, miRNA-195, miRNA-424 and miRNA-497. The luciferase activity of reporter construct treated with miRNA-15a, miRNA-15b or miRNA-195 was decreased respectively about 20% compared with the control group. It is concluded that the FLT3 3'-UTR-luciferase reporter vector has been successfully constructed. The luciferase activity of the reporter can be suppressed by miRNA-15a, miRNA-15b or miRNA-195.
