Document Detail


Construction and detection of expression vectors of microRNA-9a in BmN cells.
MedLine Citation:
PMID:  21726059     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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:
Yong Huang; Quan Zou; Sheng-peng Wang; Shun-ming Tang; Guo-zheng Zhang; Xing-jia Shen
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Journal of Zhejiang University. Science. B     Volume:  12     ISSN:  1862-1783     ISO Abbreviation:  J Zhejiang Univ Sci B     Publication Date:  2011 Jul 
Date Detail:
Created Date:  2011-07-04     Completed Date:  2011-10-31     Revised Date:  2013-06-28    
Medline Journal Info:
Nlm Unique ID:  101236535     Medline TA:  J Zhejiang Univ Sci B     Country:  China    
Other Details:
Languages:  eng     Pagination:  527-33     Citation Subset:  IM    
Affiliation:
Key Laboratory of Silkworm and Mulberry Genetic Improvement, Ministry of Agriculture, Jiangsu University of Science and Technology, Zhenjiang 212018, China.
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MeSH Terms
Descriptor/Qualifier:
Animals
Blotting, Northern
Bombyx
Genetic Vectors*
Green Fluorescent Proteins / genetics
MicroRNAs / genetics*
Plasmids
Reverse Transcriptase Polymerase Chain Reaction
Chemical
Reg. No./Substance:
0/MicroRNAs; 0/enhanced green fluorescent protein; 147336-22-9/Green Fluorescent Proteins
Comments/Corrections

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