title: Construction and detection of expression vectors of microRNA-9a in BmN cells.
abstract: MicroRNAs (miRNAs) are small endogenous RNAs molecules, approximately 21-23 nucleotides in length, which regulate gene expression by base-pairing with 3' untranslated regions (UTRs) of target mRNAs. However, the functions of only a few miRNAs in organisms are known. Recently, the expression vector of artificial miRNA has become a promising tool for gene function studies. Here, a method for easy and rapid construction of eukaryotic miRNA expression vector was described. The cytoplasmic actin 3 (A3) promoter and flanked sequences of miRNA-9a (miR-9a) precursor were amplified from genomic DNA of the silkworm (Bombyx mori) and was inserted into pCDNA3.0 vector to construct a recombinant plasmid. The enhanced green fluorescent protein (EGFP) gene was used as reporter gene. The Bombyx mori N (BmN) cells were transfected with recombinant miR-9a expression plasmid and were harvested 48 h post transfection. Total RNAs of BmN cells transfected with recombinant vectors were extracted and the expression of miR-9a was evaluated by reverse transcriptase polymerase chain reaction (RT-PCR) and Northern blot. Tests showed that the recombinant miR-9a vector was successfully constructed and the expression of miR-9a with EGFP was detected.
authors: Key Laboratory of Silkworm and Mulberry Genetic Improvement, Ministry of Agriculture, Jiangsu University of Science and Technology, Zhenjiang 212018, China.
authors: Yong
authors: Y
authors: Huang
authors: Quan
authors: Q
authors: Zou
authors: Sheng-peng
authors: SP
authors: Wang
authors: Shun-ming
authors: SM
authors: Tang
authors: Guo-zheng
authors: GZ
authors: Zhang
authors: Xing-jia
authors: XJ
authors: Shen
mesh_terms: D000818:Animals; D015152:Blotting, Northern; D012831:Bombyx; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics; D035683:MicroRNAs / Q000235:genetics*; D010957:Plasmids; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D035683:MicroRNAs; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins