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Fusion promoter capable of realizing high-efficiency expression in pig muscular tissue

A muscle tissue and promoter technology, used in recombinant DNA technology, DNA preparation, DNA/RNA fragments, etc., can solve the problems of restricting the development of transgenic animals, protein overexpression, and few reports of muscle tissue-specific promoters.

Inactive Publication Date: 2012-05-16
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the number and types of promoters available for transgenics are very limited, mainly composed of cytomegalovirus (CMV), SV40, human immunodeficiency virus (HIV), human herpes simplex virus (HSV) and hepatitis B virus (HBV). They have no spatiotemporal or tissue specificity, and using them as promoters to guide protein expression in transgenic animals can easily lead to overexpression of certain proteins
The tissue-specific promoters reported in mammals are mainly concentrated in animal mammary gland tissues, such as β-lactoglobulin (BLG), casein gene and whey acid protein (WAP), etc. (Fan Baoliang et al., 2005; Cui Kuiqing et al., 2005; Hennighausen et al., 1992; Whitelaw et al., 2000), but there are few reports on muscle tissue-specific promoters, and there are relatively few muscle tissue-specific promoters that can be used for the preparation of transgenic pigs and have independent intellectual property rights, which greatly limits Therefore, obtaining a tissue-specific promoter with high expression efficiency has become an urgent problem to be solved

Method used

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  • Fusion promoter capable of realizing high-efficiency expression in pig muscular tissue
  • Fusion promoter capable of realizing high-efficiency expression in pig muscular tissue
  • Fusion promoter capable of realizing high-efficiency expression in pig muscular tissue

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Example 1: Acquisition of pig muscle tissue-specific expression promoter MSTNP fragments and different deletion fragments

[0061] 1. Main reagents:

[0062] Phenol (Sinopharm Chemical Reagent Co., Ltd.), chloroform (Sinopharm Chemical Reagent Co., Ltd.), isoamyl alcohol (Sinopharm Chemical Reagent Co., Ltd.), plasmid extraction kit (purchased from OMEGA), pGEM-Teasy vector (Pro Mag (Beijing) Biotechnology Co., Ltd. (i.e. Promega, USA), 2×GC Buffer (Bao Bioengineering Dalian Co., Ltd.), LA Taq polymerase (Bao Bioengineering Dalian Co., Ltd.), dNTP (purchased from Fermentas Company), DL-2000 Marker (purchased from Fermentas), AC Column Agarose DNA Recovery Kit (purchased from Bio Teke)

[0063] 2. Genomic DNA extraction from pig blood

[0064] The large white pig DNA sample used in the present invention is that the conventional phenol / chloroform extraction method extracts genomic DNA, and the specific operation steps are as follows:

[0065] (1) Prepare 0.5 mol / LEDTA ...

Embodiment 2

[0091] Example 2: Construction of the corresponding deletion fragment vector of the porcine MSTN promoter

[0092] 1. Main reagents

[0093] Kpn I and Nhe I endonucleases (purchased from Fermentas Company), T4 ligase (purchased from Bao Biological Engineering Dalian Co., Ltd.), plasmid extraction kit (purchased from OMEGA Company), pGL3-Basic vector (purchased from Promega ( Beijing) Biotechnology Co., Ltd.).

[0094] 2. Double digestion of vectors containing different deletion fragments and pGL3-Basic vector

[0095] Digest plasmids pGEM-T-MSTNp1, pGEM-T-MSTNp2, pGEM-T-MSTNp3, pGEM-T-MSTNp4, pGEM-T-MSTNp5, pGEM-T-MSTNp6 and pGL3 with Kpn I and Nhe I endonucleases -Basic carrier. The double enzyme digestion system is: 2 μl 10×Tango Buffer, 1 μl Kpn I, 1 μl Nhe I, 4 μl plasmid and 12 μl sterilized water, digest at 37°C for 6 hours. After enzyme digestion, use a purification kit to recover 6 promoter fragments of different sizes and the bands after enzyme digestion of the pG...

Embodiment 3

[0098] Embodiment 3: Determination of the activity of the promoter deletion fragment of the present invention

[0099] 1. Main reagents:

[0100] Fetal bovine serum (FBS, purchased from GBICO company), DMEM high glucose solution (purchased from GBICO company), 0.25% trypsin (purchased from GBICO company), Lipofectamin TM 2000 liposome (purchased from Invitrogen), Dual-LuciferaseReporter Assay System (purchased from Promega (Beijing) Biotechnology Co., Ltd.), PBS powder (purchased from DECENT BIOTECH, item number: p100p), pGL3-Control ( Purchased from Promega (Beijing) Biotechnology Co., Ltd.), PRL-TK (purchased from Promega (Beijing) Biotechnology Co., Ltd.)

[0101] 2. Main consumables:

[0102] 24-well cell culture plate (Costar), cell culture flask, disposable sterile pipette, disposable syringe, enzyme label plate (Costar), etc.

[0103] 3. Preparation of cell culture solution

[0104] PBS preparation: mix one packet of PBS powder with 1000ml ultrapure water, dissolve...

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Abstract

The invention belongs to the field of animal genetic engineering and is characterized in that: six different promoter deletion segments, which are MSTN-P1, MSTN-P2, MSTN-P3, PSTN-P4, MSTN-P5 and MSTN-P6 respectively, at the upstream of an MSTN gene are cloned from a large white pig; the segments are established in pGL3-Basic vectors to determine activities thereof; the MSTN-P2 has stronger activity and the nucleotide sequence thereof is shown as a sequence table SEQ ID NO:2. By adding SV40 enhancer segments into the MSTN-P2, the nucleotide sequence of the MSTN-P2 is shown as a sequence table SEQ ID NO:3. The promoter segments are found to be greatly enhanced in activities through transfected muscle-derived cells and non-muscle-derived cells. The invention discloses the establishment progress of the fusion promoters, thus providing a new genetic resource for genetically modified animals or induced foreign genes for establishing muscle specificity in high-efficiency expression in muscular tissues.

Description

technical field [0001] The invention belongs to the field of animal genetic engineering application and molecular biology technology, and specifically relates to the amplification of a porcine MSTN gene promoter region and the identification of its activity, and the sequence is modified so as to provide its transcriptional activity, but without destroy its muscle specificity. Background technique [0002] The growth and development of animals is the result of orderly expression and function of various functional genes in different time and space. The regulation of gene expression is regulated by internal and external factors, such as the influence of exogenous substances on growth and development, animal growth and development itself, etc. All these factors can regulate the gene expression of an individual. Gene regulation can be divided into pre-transcriptional regulation, transcriptional regulation, post-transcriptional regulation, translational regulation and protein re...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/10C12N15/85
Inventor 蒋思文邓兵
Owner HUAZHONG AGRI UNIV
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