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Application of Myf6 promoter in driving expression of MyoD gene in muscular tissue

A muscle tissue and promoter technology, which is used in applications, genetic engineering, plant genetic improvement, etc.

Inactive Publication Date: 2015-11-11
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] So far, there have been no published studies on the expression of the pig muscle tissue-specific gene MyoD driven by the promoter of the pig muscle tissue-specific gene Myf6 (herein referred to as the Myf6 promoter) in model animals.

Method used

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  • Application of Myf6 promoter in driving expression of MyoD gene in muscular tissue
  • Application of Myf6 promoter in driving expression of MyoD gene in muscular tissue
  • Application of Myf6 promoter in driving expression of MyoD gene in muscular tissue

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] 1.1 Construction and identification of intermediate vector pMyoDEGFP-N1

[0058] 1.1.1 Amplification of MyoD gene CDS sequence

[0059] Obtain the CDS sequence of MyoD from the GenBank database, design specific primers, add HindIII restriction site and CCC protection base at the 5' end of the upstream primer, remove the stop codon sequence of CDS in the downstream primer, and add A KpnI restriction site and a CGG protection base were added to the end. The primer sequences are shown in Table 1-1.

[0060] Table 1-1 MyoD gene CDS sequence PCR amplification primer design

[0061]

[0062] Using pig muscle tissue cDNA as a template for PCR amplification, a band of 960 bp is expected to be amplified. The PCR reaction system is shown in Table 1-2.

[0063] Table 1-2 PCR amplification parameter list

[0064]

[0065] The PCR reaction conditions were: pre-denaturation at 94°C for 4 min; denaturation at 94°C for 40 s; annealing at 70°C for 40 s; extension at 72°C for 1...

Embodiment 2

[0134] Example 2: Identification of Offspring Positive Transgenic Mice

[0135] 2.1 Extraction of genomic DNA from transgenic mice

[0136] The tail tip (about 1 cm) was taken from the transgenic mice 2 weeks after birth, and the total genomic DNA was extracted by phenol-chloroform extraction.

[0137]Specific steps are as follows:

[0138] (1) Cut the tail of the transgenic mouse about 1 cm and place it in a 2ml EP tube, cut up the tissue block with sterilized scissors, add 1ml of tissue lysate (see Table 2-1 for the preparation method of tissue lyse), and turn it upside down repeatedly Several times, add proteinase K10ul (0.2mg / ml), and mix thoroughly.

[0139] (2) Place in a 55°C water bath and digest overnight (shake every 30 minutes at the beginning).

[0140] (3) After fully digested by the lysate, take out the EP tube, add an equal volume of Tris-saturated phenol, slowly invert for 10 min, centrifuge at 12000g at 4°C for 10 min, and transfer the supernatant to anothe...

Embodiment 3

[0160] Example 3 Identification of exogenous gene copy number in transgenic mouse genome

[0161] 3.1 Construction of single-copy gene Pthlh recombinant plasmid

[0162] Know the single-copy gene Pthlh (Umemorietal2013) of mouse from literature, design has the primer of restriction site (primer sequence sees Table 2-2), the underline part of primer sequence is restriction site; PCR amplification parameter and The amplification program is shown in Table 2-3. The mouse single-copy gene Pthlh partial fragment (see SEQIDNO: 6) was connected to the pGL3-Basic vector to construct the pGL3-Pthlh-Basic recombinant plasmid. The specific plasmid construction steps refer to the plasmid construction methods in 1.1 and 1.2, pGL3-Pthlh -Basic recombinant plasmid construction process such as figure 2 .

[0163] 3.2 Identification of copy number in the genome of transgenic mice

[0164] (1) Preparation of standard products

[0165] 1) Selection of standard products: The constructed reco...

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Abstract

The invention belongs to the field of animal genetic engineering, and in particularly relates to an application of a promoter region of a specific expression gene Myf6 of skeletal muscle of pigs in level verification of a model animal transgenic mouse living body. According to the invention, a constructed eukaryotic expression vector pSV40-Myf6-MyoDEGFP-N1 is subjected to linear treatment, and a transgenic mouse is prepared through micro-injection; MyoD gene expression conditions in different tissues of the positive transgenic mouse are verified, a tissue expression profile is established, and the horizontal expression conditions of MyoD in skeletal muscle of the transgenic mouse and wild type mouse in mRNA and protein are verified, the result shows that Myf6 gene promoter can specifically up-regulate the overexpression of the MyoD gene in the skeletal muscle; and the growth condition of muscle fiber is observed through a muscular tissue slice, the result shows that the area and the diameter of the muscle fiber in the muscular tissue slice of the transgenic mouse are significantly higher than those of the wild type mouse, and the density of the muscle fiber in unit area is obviously lower than that of the wild type mouse.

Description

technical field [0001] The invention belongs to the technical field of animal genetic engineering, and in particular relates to the expression application of Myf6 promoter driving muscle tissue MyoD gene. Background technique [0002] Promoter is an important cis-element for gene expression regulation, and also an important part of genetic engineering expression vector. RNA polymerase and transcription factors can recognize and bind specific DNA sequences on the promoter to initiate transcription (Landoline et al2010). With the development of genetic engineering, it is often necessary to construct an expression vector capable of expressing heterologous proteins at a high level. The promoter has a great influence on the expression level of the foreign gene, and is an important element of the expression vector of genetic engineering (Li Shanshan et al., 2005). [0003] Transgenic technology began in the 1970s, and researchers introduced foreign genes of interest into host ce...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/12
Inventor 蒋思文马娟娟彭健柴进李凤娥
Owner HUAZHONG AGRI UNIV
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