Method for detecting promoter activity using dual luciferase reporter gene

A dual-luciferase and reporter gene technology is applied in the field of determining the core promoter of Guanling cattle MSTN gene by using the dual-luciferase reporter gene, which can solve the problems of complex interaction between regulatory elements and trans-acting factors, and achieve the detection speed. Fast, high sensitivity, low cost results

Inactive Publication Date: 2015-09-30
GUIZHOU UNIV
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Problems solved by technology

However, the specificity of the promoter is controlled by multiple elements, and the regulatory elements that play key roles in different species and different developmental stages are not the same, and the interaction between regulatory elements and trans-acting factors is also very complicated

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  • Method for detecting promoter activity using dual luciferase reporter gene
  • Method for detecting promoter activity using dual luciferase reporter gene
  • Method for detecting promoter activity using dual luciferase reporter gene

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Embodiment Construction

[0021] The present invention can be better understood from the following examples. However, those skilled in the art can easily understand that the description of the embodiments is only for illustrating the present invention, and should not and will not limit the present invention described in the claims.

[0022] Embodiments of the present invention: a method for detecting promoter activity using a dual-luciferase reporter gene, specifically as follows:

[0023] 1. Preparation of strains, plasmids and reagents.

[0024] Strain: Escherichia coli competent cell DH5α was purchased from Dalian Takara Company;

[0025] Cell lines: mouse C2C12 cells and 3T3-L1 cells were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences;

[0026] Plasmids: Reporter vectors pGL3-Basic and pRL-TK were purchased from Promega.

[0027] Reagent source: 2×PCR Taq Mix was purchased from Tiangen Company;

[0028] T4 DNA Ligase and endotoxin-free plasmid mini-extraction kit were ...

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Abstract

The invention discloses a method for detecting promoter activity by utilizing double luciferase reporter genes. The method for detecting the promoter activity by utilizing the double luciferase reporter genes comprises step 1, building a pGL3-basic-MSTNpro recombinant which contains 7 sections of MSTN gene 5' control region fragments with different lengths; step 2, performing cultivation and planking on target cells, configuring a mixture of the pGL3-basic-MSTNpro of the step 1 and a lipidosome and enabling a renilla luciferase carrier pRL-TK to be served as an internal reference to perform cell co-transfection; step 3, performing detection on luciferase activity through the double luciferase reporter genes.

Description

technical field [0001] The invention relates to the field of molecular genetics, in particular to a method for determining the core promoter of Guanling cattle MSTN gene by using dual luciferase reporter genes. Background technique [0002] The timing and level of gene expression in eukaryotes follows a strict developmental sequence. The regulation of the expression of some specific genes is a key step in the initiation of transcription by transcription factors specifically binding to the relevant elements of the regulatory region, and finally leads to the temporal and spatial expression of the gene. Genes encoding tissue-specific proteins are expressed strictly according to tissue-specificity and developmental stages, providing a good research model for the regulation mechanism of gene expression. In transgenic animals with specific high expression, it is very important to obtain specific promoters that can be widely used. However, the specificity of promoters is controll...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12Q1/66
Inventor 许厚强刘敏陈伟陈祥赵佳福
Owner GUIZHOU UNIV
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