Method for increasing activity of myogenin (MyoG) gene promoter

A promoter and generative technology, applied in the field of genetic engineering, can solve problems such as inconvenient research on transgenic animals, achieve good muscle quality improvement effects, improve muscle quality and growth speed, and increase expression activity

Inactive Publication Date: 2013-07-10
NORTHEAST AGRICULTURAL UNIVERSITY
View PDF1 Cites 2 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At the same time, longer promoters are not convenient for transgenic animal research

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Method for increasing activity of myogenin (MyoG) gene promoter
  • Method for increasing activity of myogenin (MyoG) gene promoter
  • Method for increasing activity of myogenin (MyoG) gene promoter

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 Construction of Japanese Wagyu MyoG gene promoter deletion sequence pGL3-MyoGpro expression vector

[0031] Sufficient Japanese Wagyu fibroblasts were collected, genomic DNA was extracted using Tissue DNA Kit, and stored at -70°C in aliquots. According to the MyoG gene sequence in NCBI, primers were designed using the software Primer Premier 5. The sequence of the 5' end regulatory region of Japanese Wagyu MyoG gene with a length of 2125bp was obtained by PCR reaction.

[0032]

[0033] Use the transcription factor prediction software: MatInspector (http: / / www.genomatix.de / en / index.html) to predict the transcription factor binding sites of the cloned fragments. A series of promoter deletion fragments of different lengths and positions were obtained through bioinformatics analysis, and the corresponding MyoG gene promoter deletion sequence pGL3-MyoGpro expression vectors were constructed respectively. By transfecting mouse C2C12 cells and bovine fibroblast...

Embodiment 2

[0077] Example 2 Construction of Japanese Wagyu pGL3-MyoGpro373-double expression vector

[0078] As shown in the figure below, use the PCR method to amplify the MyoG promoter regulatory element between the red arrows, and connect it to the front of the 373bp deletion fragment of pGL3-MyoGpro373, which is the promoter nucleic acid sequence of the MyoG gene with "double" characteristics. The Nhel and BglII introduced by restriction sites were introduced during PCR amplification, and the promoter nucleic acid sequence of the MyoG gene with "double" characteristics was connected to the upstream of the MyoG gene through double restriction and ligation reactions, thereby constructing pGL3-MyoGpro373 -double.

[0079]

[0080]

[0081] Preparation of the system used for PCR amplification of target fragments

[0082]

[0083] Using pGL3-MyoGpro as a template and using the primers in Table 2-17, perform PCR amplification with LA Taq polymerase. The PCR reaction program was:...

Embodiment 3

[0085] Example 3 Cell Transfection and Detection of pGL3-MyoGpro373-double Expression Vector

[0086] Transfect mouse C2C12 cells and bovine fibroblasts with pGL3-MyoG-CMV (MyoG gene expression vector with CMV (cytomegalovirus) promoter), pGL3-MyoGpro373, pGL3-MyoGpro373-double and pGL3-Basic expression vectors, respectively cells, and a dual luciferase activity assay was performed. The specific implementation method is the same as in Example 1. The test results showed that the activity of the modified pGL3-MyoGpro373-double promoter was 3 times higher than that of pGL3-MyoGpro373. And it maintains good muscle specificity, and can be well applied in the research of transgenic animals.

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention relates to a method for increasing the activity of a myogenin (MyoG) gene promoter, and belongs to the technical field of genetic engineering. The method for increasing the activity of the myogenin (MyoG) gene promoter is characterized in that a deletion fragment which is 373 bp in length and has higher muscle specific promoter activity, namely pGL3-MyoGpro373, is obtained by adopting a method for analyzing the activity of a promoter deletion fragment by means of cloning a nucleotide sequence, which is at the 5' end control region of the MyoG gene and is 2125 bp in length; a promoter element therein having the SP1 positive regulation effect is cloned; the two fragments are connected in series, so that an expression carrier containing two copying numbers of promoter regulatory elements is constructed; the expression carrier is called as pGL3-MyoGpro373-double, namely a promoter sequence having double characteristics; and the base composition of the expression carrier is represented by Seq ID No:2.

Description

technical field [0001] The invention relates to a method for improving the activity of a myogenin (MyoG is an abbreviation of myogenin) gene promoter, which belongs to the technical field of genetic engineering. Background technique [0002] The regulation of gene expression in eukaryotes is a complex and rigorous process. In the study of transgenic animals, it is of great significance to obtain highly efficient and specific promoters that can be widely used for the expression of foreign genes. The mammalian core promoter includes a TATA box as well as promoter proximal elements located approximately 200 to 300 bp upstream of the transcription start site. A generalized promoter includes all cis-regulatory elements such as promoter proximal elements, enhancers and silencers, and generally has a length of at least 2 to 3 kb or more. In fact, the core promoter is the main site where RNA polymerase recognizes and binds, and makes gene transcription. The core promoters of gene...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/10C12N15/85
Inventor 李树峰佟慧丽严云勤金慧然王鑫李光鹏
Owner NORTHEAST AGRICULTURAL UNIVERSITY
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap