Construction of FLT3 3'-UTR-luciferase reporter vector and evaluation of its activity / 中国实验血液学杂志
Journal of Experimental Hematology
;
(6): 694-697, 2010.
Artigo
em Chinês
| WPRIM
| ID: wpr-243283
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.
Texto completo:
DisponíveL
Índice:
WPRIM (Pacífico Ocidental)
Assunto principal:
Sequência de Bases
/
Transfecção
/
Linhagem Celular
/
Regiões 3' não Traduzidas
/
MicroRNAs
/
Tirosina Quinase 3 Semelhante a fms
/
Vetores Genéticos
/
Genética
/
Luciferases
Limite:
Humanos
Idioma:
Chinês
Revista:
Journal of Experimental Hematology
Ano de publicação:
2010
Tipo de documento:
Artigo
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