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Over-expression vector for muscle specific expression of pig IGF1 gene

An expression vector and overexpression technology, applied in the field of overexpression vector, can solve the problems of abnormal growth of the body, changing the characteristic phenotype of the host, disorder of the genome sequence, etc.

Inactive Publication Date: 2013-03-20
INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method is widely used in the study of gene expression and function, but many studies have exposed the shortcomings of this method: the introduction of heterologous gene sequences into the host often causes genome sequence disorder, abnormal expression of endogenous genes, and even changes A characteristic phenotype inherent to the host; also often sparks debate on the ethical issues of transgenics
In addition, the introduction of prokaryotic residual sequences such as viral promoter sequences or guiding plasmid DNA replication may easily lead to safety problems of transgenics, and long-term biosafety evaluations are required before they can be put into practical use, which is time-consuming and expensive.
At the same time, the general overexpression of foreign genes often causes abnormalities in body growth, etc.

Method used

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  • Over-expression vector for muscle specific expression of pig IGF1 gene
  • Over-expression vector for muscle specific expression of pig IGF1 gene
  • Over-expression vector for muscle specific expression of pig IGF1 gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment I

[0023] Embodiment 1, the construction of skeletal muscle specific expression pig IGF1 vector

[0024] 1. Obtaining the basic components of specific expression vectors

[0025] (1) Preparation of specific fragment of porcine sk-α-actin Promoter

[0026] Using Landrace pig genomic DNA as a template, the skeletal muscle-specific sk-α-actin promoter fragment was amplified according to the primers listed in Table 1 for amplifying the sk-α-actin Promoter. The reaction system was 50 μl, 5 μL 10×Buffer, 4 μL 2.5 mM dNTP, 1 μL 20 μM primer sk-α-actin Promoter F, 1 μL 20 μM primer sk-α-actin Promoter R (see Table 1 for the primer sequence), 0.5 μL 5 U / μL high-fiber True LA Tag polymerase, 2 μl Landrace pig genomic DNA template, add ultrapure water to 50 μL (high-fidelity enzyme purchased from TaKaRa Code: DR002A). PCR amplification program: 95°C for 10s, 60°C for 3min, 35 cycles. PCR amplification products were detected by 1% agarose gel electrophoresis (such as figure 1 shown).

...

Embodiment II

[0046] Embodiment II, the cell level expression test of GFP driven by the skeletal muscle-specific sk-α-actin promoter of the present invention

[0047] 1. Recovery and culture of cryopreserved cells

[0048] The pEGFP-N1 plasmid (purchased from Clontech with the product number PT3027-5) and the pT2 / α-actin-GFP plasmid were gel-recovered with the QIAGEN purification kit, and the final concentration of the product was adjusted to 500ng / ul for cell transfection.

[0049] Freeze-preserved pig fetal fibroblasts PEF (isolated and cultured by the Cell Engineering Laboratory of Beijing Institute of Animal Husbandry and Veterinary Medicine, Chinese Academy of Agricultural Sciences), mouse C2C12 cells and pig PK15 cells (purchased from the Cell Bank of the Chinese Academy of Sciences) were removed from liquid nitrogen Immediately put the small tube into warm water at 37-40°C and shake it quickly until the freezing solution is completely melted; complete rewarming within 1-2 minutes; tr...

Embodiment III

[0055] Embodiment III, verification of the transgenic mouse prepared by the expression vector containing pig IGF1

[0056] 1. Preparation of transgenic mice expressing porcine IGF1

[0057] The circular pT2 / α-actin-IGF1 plasmid and the transposase-expressing plasmid pCMV-SB11 were mixed at an equimolar concentration of 1:1, and transgenic mice were prepared by pronuclear microinjection.

[0058] 2. Integration detection of transgenic mice

[0059] Use detection primers for sk-α-actin Promoter (P 1), sk-α-actin 3'UTR (P2) and pIGF1 full-length (P3) respectively (see Table 2) to detect the corresponding carrier elements in the genomic DNA of transgenic mice integrate.

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Abstract

The invention provides a secure expression vector for specific over-expression of pig endogenous gene IGF1 (C1 : Ea) in pig skeletal muscle. The vector comprises an expression cassette composed of the pig endogenous sequence sk-alpha-actinPromoter / IGF1 (C1 : Ea) / sk-alpha-actin3'UTR / SV40 enhancer in series. The expression vector verifies on a cell scale that the skeletal muscle specific sk-alpha-actin promoter can be expressed efficiently in myoblasts, and expressed at lower level in PET cells having myogenic differentiation potential, but not expressed in other type cells. By using the constructed vector for preparing transgenic mice, the detected mRNA expression level and protein expression level of the IGF1 gene in the skeletal muscle of the transgenic mice via quantification PCR and WesterBlot are 15 times and 3.5 times that of the wild mice respectively; and the IGF1 gene can be only expressed in the skeletal muscle efficiently.

Description

technical field [0001] The invention relates to an overexpression vector for specifically expressing porcine insulin growth factor 1 (insulin-like growth factor 1, IGF1) gene in safe skeletal muscle. Background technique [0002] In the study of gene function and the production of transgenic animals, people often use eukaryotic expression vectors, such as pcDNA3.0 series vectors. Traditional methods often link target gene fragments to viral promoters (such as CMV promoters) or downstream of heterologous strong promoters by means of molecular biology to guide the expression of foreign genes. This method is widely used in the study of gene expression and function, but many studies have exposed the shortcomings of this method: the introduction of heterologous gene sequences into the host often causes genome sequence disorder, abnormal expression of endogenous genes, and even changes The characteristic phenotype inherent in the host; also often arouses debates on the ethical is...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12N15/63
Inventor 李奎宋成义吴晗鞠辉明白立景
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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