Skeletal muscle specific promoter and its application

A skeletal muscle-specific promoter technology, which is applied in genetic engineering applications and biological fields, can solve the problem of no relevant reports on the role of miRNA, and achieve high specificity, reduced difficulty, and high activity.

Active Publication Date: 2021-04-09
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although miR-208 has been deeply studied in myocardial diseases, there is no relevant report on the role of miRNA in the 5'UTR region.

Method used

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  • Skeletal muscle specific promoter and its application
  • Skeletal muscle specific promoter and its application
  • Skeletal muscle specific promoter and its application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Construction of pGL3-2eMCK-pMCK-T208-mCherry-EGFP vector:

[0022] 1) Construction of pGL3-mCherry-EGFP vector

[0023] A. Design primers to amplify EGFP, SV40poly(A) and mCherry sequences according to the pCMV-C-EGFP, pGL3-Control and pBABE-puro mCherry-EGFP-LC3B vector sequences, respectively. See Table 1 for primers.

[0024] Table 1 EGFP, SV40poly(A) and mCherry primers

[0025]

[0026] Note in Table 1: The underlined part is the added restriction site, and the bold part is the protected base.

[0027] Table 2 PCR system

[0028]

[0029] PCR reaction procedure: pre-denaturation at 98°C for 3min, denaturation at 98°C for 10s, annealing at 60°C for 10s, extension at 72°C for 2s, denaturation to extension for 35 cycles, and extension at 72°C for 3min.

[0030] Purification and recovery of PCR products: The amplified PCR products were detected by 1% agarose gel electrophoresis, after 20 minutes of electrophoresis, the target band was cut off under ultraviole...

Embodiment 3

[0096] Identification of promoter specificity

[0097] intraperitoneal injection of mice

[0098](1) Dilute 120 μg of plasmid (pGL3-2eMCK-pMCK-mCherry-EGFP or pGL3-2eMCK-pMCK-T2 08-mCherry-EGFP) in 300 μl of 5% sterile glucose solution, shake and mix.

[0099] (2) Dilute, 18 μl HiGene in 300 μl 5% glucose, shake and mix.

[0100] (3) Add the diluted HiGene to the plasmid solution, shake and mix immediately, and centrifuge briefly at low speed to shake the liquid to the bottom of the tube.

[0101] (4) Stand at room temperature for 20 minutes.

[0102] (5) intraperitoneal injection into mice.

[0103] (6) After 48 hours, the myocardial and leg muscles of the mice were collected and stored in liquid nitrogen.

[0104] Tissue-like RNA extraction

[0105] RNA was extracted from heart and muscle tissue by Trizol method, and cDNA was synthesized by reverse transcription kit from TAKARA company

[0106] RNA level detection of promoter specificity.

[0107] According to the mCh...

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Abstract

This application belongs to the field of genetic engineering application and biotechnology. The present invention provides a modified skeletal muscle-specific promoter 2eMCK-pMCK-T208, the nucleotide sequence of which is shown in SEQ ID NO: 1 in the sequence table. After connecting it with the target gene, it was found that the expression level of the target protein activated by 2eMCK‑pMCK‑T208 in the myocardium was lower than that of 2eMCK‑pMCK through intraperitoneal injection, showing a significant difference, while in skeletal muscle There was no significant difference in the expression level of the target protein initiated by 2eMCK-pMCK. The promoter disclosed in the present invention can enable high-efficiency and specific expression of foreign genes in skeletal muscle, which can not only improve pig muscle quality, but also reduce potential harm caused by low specificity of the promoter.

Description

technical field [0001] This research belongs to the field of genetic engineering application and biotechnology, involving skeletal muscle-specific promoter and miR-208 and its application. The modified promoter reduces the expression level of the target gene in myocardium, thereby improving the tissue specificity of the promoter. Background technique [0002] Now that gene editing technology is becoming more and more mature, enabling the specific expression of foreign genes in vivo has become an urgent problem to be solved in the application of gene editing technology. We have also encountered similar problems when using transgenic technology to improve pig muscle quality. [0003] At present, there are many promoters for muscle-specific regulation of exogenous genes. Muscle-specific UNC45B contributes to the folding of sarcomere myosin The zebrafish muscle-specific gene unc45b minimal promoter 503unc can drive gene expression in zebrafish muscle tissue (Berger and Currie ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/85
CPCC07K14/435C12N15/85
Inventor 任竹青左秀
Owner HUAZHONG AGRI UNIV
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