Dual fluorescent reporter gene vector for identifying miRNA targets, preparation method and application thereof

A reporter gene and dual fluorescence technology, applied in the field of dual fluorescence reporter gene vector and its preparation, can solve the problem of not being able to detect miRNA targets, and achieve the effects of short experimental period, high efficiency and simple operation

Inactive Publication Date: 2013-08-28
HUNAN NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the existing reporter gene carriers for detecting miRNA target genes (targets), the multiple cloning sites are all located outside the coding region of the reporter gene carrier, which can only be used to predict the miRNA target of the 3′UTR of the target gene, and cannot be used to detect miRNA targets located in coding regions

Method used

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  • Dual fluorescent reporter gene vector for identifying miRNA targets, preparation method and application thereof
  • Dual fluorescent reporter gene vector for identifying miRNA targets, preparation method and application thereof
  • Dual fluorescent reporter gene vector for identifying miRNA targets, preparation method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0026] Example 1 Construction of dual fluorescent reporter gene vector pmirCDS

[0027] 1. Linearized carrier: the dual fluorescent reporter gene carrier pmirGLO (purchased from Promega) was digested with restriction enzymes PmeI and XhoI (purchased from Fermentas), and the digested product was electrophoresed on 1% agarose gel, and excised under ultraviolet light The target DNA band (molecular weight is 7340bp). The target DNA band was recovered and purified with a gel recovery kit (purchased from Changsha Ambio Company).

[0028] 2. Obtain the insert: synthesize an oligonucleotide chain and its complementary chain by conventional chemical methods, and its forward chain sequence is:

[0029] 5′-AAGAAGGGCGGCAAGATCGCCGTGTTTAAACGAGCTCAGCTAGCCTCGAGTTAGATCTTCGACCTGCAGGCATGCAAGCTGATCC-3′,

[0030] Its complementary strand sequence is

[0031] 5'-GGATCAGCTTGCATGCCTGCAGGTCGAAGATCTAACTCGAGGCTAGCTGAGCTCGTTTAAACACGGCGATCTTGCCGCCCTTCTT-3'.

[0032] The two oligonucleotide chains synt...

Embodiment 2

[0042] Example 2 Application of the dual fluorescent reporter gene carrier pmirCDS in the identification of miRNA targets located in the gene coding region

[0043] 1. Insert the predicted candidate target sequence into the dual fluorescent reporter vector pmirCDS

[0044] Based on the prediction of bioinformatics software, the predicted miRNA target sequence and the mutated miRNA target sequence located in the coding region were respectively inserted into the dual fluorescent reporter gene vector pmirCDS. The following takes the known miR-148b target sequence located in the coding region of the human DNMT3B gene (Duursma AM et al. RNA, 2008, 14:872–877) as an example to illustrate the role of the dual fluorescent reporter gene carrier pmirCDS in identifying the gene coding region Applications within miRNA targets.

[0045] ①Synthesis of miRNA target sequence: The miRNA target sequence is relatively short, 18-30 nucleotides in length. Therefore, artificial synthesis is genera...

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Abstract

The invention discloses a dual fluorescent reporter gene vector for identifying miRNA targets, a preparation method thereof, and an application thereof in identifying the miRNA targets in coding regions and detecting functions of the miRNA on the coding regions. The carrier has a renilla luciferase gene and a firefly luciferase gene, and multiple cloning sites (MCS) are located between a terminal amino acid codon and a stop codon of a firefly luciferase reporter gene, thereby facilitating insertion of the miRNA targets into the coding regions of the firefly luciferase reporter gene, so as to identify the miRNA targets in coding regions and detect the functions of the miRNA on the coding regions. The preparation method of the carrier provided by the invention does not require a special treatment for terminals of the carrier or target fragments, is free of connection reaction and PCR amplification, and is simple to operate, short in experiment period and high in efficiency.

Description

technical field [0001] The invention belongs to the technical field of miRNA function detection, and in particular relates to a dual fluorescent reporter gene carrier for identifying miRNA targets, a preparation method and application thereof. Background technique [0002] miRNA (microRNA, microRNA) is a kind of small molecule RNA with a length of about 22 nucleotides that widely exists in eukaryotes. miRNA specifically binds to the mRNA of the target gene, causing mRNA degradation or repressing the translation of the mRNA, thereby inhibiting the expression of the target gene at the post-transcriptional or translational level. It is speculated that about one-third of the genes in the human body are regulated by miRNAs. More and more studies have shown that miRNAs are involved in the regulation of various life processes including cell proliferation and apoptosis, tissue differentiation, organ formation, and physiological metabolism. [0003] miRNA research has become a hot ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C12N15/65C12Q1/68
Inventor 周建林徐枣旭刘雨婷
Owner HUNAN NORMAL UNIVERSITY
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