[Construction of FLT3 3'-UTR-luciferase reporter vector and evaluation of its activity].
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.
Gao
Xiao-Ning
XN
Department of Hematology, Chinese PLA General Hospital, Beijing 100853, China.
Lin
Ji
J
Li
Yong-Hui
YH
Wang
Li-Li
LL
Yu
Li
L
D020413:3' Untranslated Regions; D001483:Base Sequence; D002460:Cell Line; D005822:Genetic Vectors; D006801:Humans; D008156:Luciferases / Q000235:genetics*; D035683:MicroRNAs / Q000235:genetics*; D014162:Transfection; D051941:fms-Like Tyrosine Kinase 3 / Q000235:genetics*
D020413:3' Untranslated Regions; D035683:MicroRNAs; D008156:Luciferases; C497970:FLT3 protein, human; D051941:fms-Like Tyrosine Kinase 3