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Myogenin (MyoG) gene enhancer

A generative and muscle cell technology, applied in the field of genetic engineering, can solve problems such as weak activity, and achieve the effect of diverse functions, obvious effects, and improved expression activity

Inactive Publication Date: 2013-06-19
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] In order to overcome the problem that the promoter of MyoG gene is very active when muscle differentiates to form myotubes, but its activity is very weak in myoblasts and adult muscle cells, the present invention provides an enhanced myogenin (MyoG) gene The enhancer of the myogenin (MyoG) gene is to enhance the promoter activity of the MyoG gene. Using a known experimental method, first clone the natural promoter of the bovine MyoG gene, and use the promoter deletion fragment activity analysis method , determine a core promoter fragment of appropriate size and activity, and detect the activity of the promoter deletion fragment by a dual-luciferase activity assay kit

Method used

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Examples

Experimental program
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Effect test

Embodiment 1

[0026] Example 1 Obtaining of pGL3-MyoGpro373

[0027] In the present invention, a nucleic acid sequence with a length of 2125 bp in the 5' end regulatory region of the MyoG gene is firstly obtained by PCR, and a method for analyzing the activity of the missing promoter fragment is used to obtain a nucleic acid sequence with a length of 373 bp and a relatively high muscle-specific promoter activity. The missing fragment of pGL3-MyoGpro373 (established through previous experiments, a vector that already exists in the laboratory).

[0028] The steps for extracting genomic DNA with Tissue DNA Kit are as follows:

[0029] 1. Discard the culture medium, wash the cells 3 times with 4ml PBS, then digest the cells with 500μl trypsin, transfer the cells to a centrifuge tube, centrifuge at 1000rpm for 5min at room temperature, and remove the supernatant thoroughly;

[0030] 2. Add 25ulOB protease, mix well, and treat in a water bath at 65°C for 5 minutes to completely lyse the cells; ...

Embodiment 2

[0064] Example 2 Artificial synthesis of enhancer fragment "SE" and construction of expression vector

[0065] According to the preliminary bioinformatics analysis of the MyoG natural promoter sequence and the sequence of its promoter regulatory elements, as well as the core sequences of some muscle-specific gene promoter regulatory elements found in bovine muscle cells, the present invention artificially designs and synthesizes A muscle-specific enhancer sequence, which is connected to the front of the 373 fragment of the pGL3-MyoGpro373 expression vector, to achieve the expression regulation of the MyoG gene in the early differentiation process of muscle cells by increasing the activity of the MyoG gene promoter (see figure 1 ). The gene expression regulatory elements contained in the enhancer sequence and their order are E-box (Seq ID No:2), SRE (Seq ID No:3), KLF3 (Seq ID No:4), E-box (Seq ID No: :5), Sp1 (Seq ID No:6), E-box (Seq ID No:7), Sp1 (Seq ID No:8), TEF-1 (Seq ...

Embodiment 3

[0079] Example 3 Analysis of MyoG promoter activity

[0080] The promoter activity analysis is carried out by using the host cell transfection experiment method, so as to finally obtain the high-efficiency and specific expression of regulating the transcription of the bovine MyoG gene. Through dual luciferase activity assay, it was found that the synthetic muscle enhancer increased the activity of MyoG promoter by about 2 times. Further studies have found that artificially synthesized muscle enhancers can increase the activity of the MyoG promoter in both the proliferation and differentiation stages of myoblasts ( image 3 and Figure 4 ).

[0081] Mouse C2C12 cells and bovine fibroblasts cultured. When the growth density of the cultured cells reaches about 80%-90%, discard the culture medium, rinse three times with PBS without calcium and magnesium ions, add 0.5ml of 0.25% trypsin for digestion, and place at 37°C for 1-2min. When 80% of the cells become round under the mi...

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Abstract

The invention relates to a myogenin (MyoG) gene enhancer, belonging to the technical field of genetic engineering. The MyoG gene enhancer consists of regulatory elements, namely E-box, SRE, KLF3, E-box, Sp1, E-box, Sp1 and TEF-1. The MyoG gene enhancer is characterized in that the sequence of the regulatory elements of the MyoG gene enhancer is E-box, SRE, KLF3, E-box, Sp1, E-box, Sp1 and TEF-1; and the SE nucleic acid sequence of the MyoG gene enhancer is shown in Seq ID No: 1 and has the length of 147 bp. Due to an artificially-synthesized enhancer sequence, the expression of a MyoG gene can be promoted at the proliferation and differentiation stages of myoblasts, and the expression activity of the MyoG gene can be remarkably improved.

Description

technical field [0001] The invention relates to an enhancer of myogenin (MyoG is an abbreviation of myogenin) gene, which belongs to the technical field of genetic engineering. Background technique [0002] Eukaryotic genomes contain transcriptional regulatory elements that regulate the expression of genes. Such as promoters, enhancers and silencers. Core promoters and enhancers, as well as transcription factors and promoter complexes play a very important role in the regulation of gene expression. In fact, in addition to the core promoter elements, the regulatory elements of other promoter regions also play a very important role in the process of cell-specific gene transcription. Therefore, the research on the regulatory elements of muscle-specific promoters has important research value. Among them, the enhancer can enhance the transcriptional activity of the promoter. It is mainly located upstream of the promoter and can be far away from the transcription start point (...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113
Inventor 佟慧丽李树峰严云勤金慧然王鑫李光鹏
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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