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Fusion promoter efficiently expressed in pig muscular tissues

A muscle tissue and promoter technology, applied in the field of animal genetic engineering, can solve the problems of restricting the development of transgenic animals and the lack of reports on muscle tissue-specific promoters

Inactive Publication Date: 2013-12-04
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

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 efficiently expressed in pig muscular tissues
  • Fusion promoter efficiently expressed in pig muscular tissues
  • Fusion promoter efficiently expressed in pig muscular tissues

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Example 1: Acquisition of pig muscle tissue expression promoter MEF2CP fragment and different deletion fragments

[0059] Main reagents and sources:

[0060] Phenol (purchased from Sinopharm Chemical Reagent Co., Ltd.), chloroform (purchased from Sinopharm Chemical Reagent Co., Ltd.), isoamyl alcohol (purchased from Sinopharm Chemical Reagent Co., Ltd.), plasmid extraction kit (purchased from OMEGA Company), pGEM -Teasy vector (purchased from Promega, USA), 2×GC Buffer (purchased from Treasure Bioengineering Dalian Co., Ltd.), Ex Taq polymerase (purchased from Treasure Bioengineering Dalian Co., Ltd.), dNTP (purchased from Fermentas Co., Ltd.), DL-2000Marker , DL-10000Marker (purchased from Treasure Bioengineering Dalian Co., Ltd.), agarose DNA recovery kit (purchased from OMEGA Company)

[0061] (1) Genomic DNA extraction from pig blood

[0062] The DNA sample of the Meishan pig used in the present invention (the blood sample is collected from the high-quality pig fa...

Embodiment 2

[0076] Example 2: Construction of the corresponding deletion fragment vector of pig MEF2C promoter

[0077] Main reagents and sources

[0078] Kpn I and Bgl II endonucleases (purchased from Fermentas), T4 ligase and ligation reagents (purchased from NEB), plasmid extraction kits (purchased from OMEGA), pGL3-Basic vector (purchased from Promega, USA) .

[0079] (1) Double enzyme digestion of vectors containing different deletion fragments and pGL3-Basic vector

[0080] The cloning vector plasmids MEF2CP1, MEF2CP2, MEF2CP3 and MEF2CP4 and pGL3-Basic vector were digested with Kpn I and Bgl II endonucleases. The digestion system is: 2 μl 10×Fast Buffer, 1 μl Kpn I, 1 μl Bgl II, 4 μl plasmids (respectively MEF2CP1, MEF2CP2, MEF2CP3 and MEF2CP4) and 12 μl sterilized water, digest at 37°C for 30min-1h. After enzyme digestion, use a purification kit to recover 4 promoter fragments of different sizes and the band after enzyme digestion of the pGL3-Basic vector. The recovered produc...

Embodiment 3

[0083] Embodiment 3: Determination of the activity of the promoter deletion fragment of the present invention

[0084] Main reagents and sources:

[0085] Fetal bovine serum (ie 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.), phosphate buffer saline, namely PBS (purchased from Hyclone), pGL3-Control and pRL -TK vector (purchased from Promega, USA).

[0086] Main consumables:

[0087] 24-well cell culture plate (Costar), cell culture flask, disposable sterile pipette, disposable syringe, enzyme label plate (Costar), etc.

[0088] (1) Preparation of cell culture solution

[0089] Cell growth medium: Use a disposable syringe to absorb 9 parts of high-glucose medium (DMEM) and 1 part of fetal bovine serum (FBS), mix well and set asi...

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Abstract

The invention particularly relates to separation identification for a fusion promoter efficiently expressed in pig muscular tissues. According to the invention, cloning deletion segments of 4 different promoters on the upstream of an MEF2C gene of a Meishan pig, constructing the segments into a pGL3-Basic carrier and confirming the activity, wherein the activity of pGL3-MEF2CP3 is relatively stronger, and the nucleotide sequence of the promoter is as shown in SEQ ID NO:1. By replacing a luc segment of the pGL3-Basic carrier to be in sequence of PPAR gama CDS, the nucleotide sequence is as shown in SEQ ID NO:2 to construct a fusion promoter carrier pGL3-MEF2CP3-PPAR gama; the expression of the PPAR gama is improved through the verifications of both semiquantitative PCR and protein imprint.

Description

technical field [0001] The invention belongs to the technical field of animal genetic engineering, and specifically relates to the amplification of a pig MEF2C gene promoter region and the identification of its activity, and the vector constructed by the sequence is modified to maintain its transcriptional activity. Activity drives expression of downstream inserted exogenous genes. Background technique [0002] The expression of genes in higher organisms is finely regulated by the internal and external environment of cells, so it has strict temporal and spatial order. Gene expression regulation is a complex and orderly process, which is completed by multi-stage regulation levels, which mainly includes five levels: pre-transcription, transcription, post-transcription, translation, and post-translation. The regulation of transcription level is the most critical link. Transcriptional regulation is mediated by the coordination of various cis- and trans-acting elements. Promot...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/85
Inventor 蒋思文靳玮
Owner HUAZHONG AGRI UNIV
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