Clone and application of pig skeletal muscle specificity expression gene alpha-actin promoters

A skeletal muscle-specific gene expression technology, applied in the field of functional verification, can solve the problems of α-actin promoter function of skeletal muscle gene in pig muscle tissue, lack of targeted expression of regulatory genes, and low specificity.

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

The problem with these two high-efficiency promoters is obvious: they do not have the ability to regulate the targeted expression of genes in a specific tissue
[0007] From the analysis of the promoters obtained in plants and animals, it is found that the following problems generally exist: the specificity is not

Method used

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  • Clone and application of pig skeletal muscle specificity expression gene alpha-actin promoters
  • Clone and application of pig skeletal muscle specificity expression gene alpha-actin promoters
  • Clone and application of pig skeletal muscle specificity expression gene alpha-actin promoters

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1: Obtaining candidate promoter fragments and corresponding deletion fragments of porcine skeletal muscle-specific expression gene α-actin

[0036] The DNA sequence of the reported porcine α-actin gene (GenBank accession number: 100154254 ) is the probe sequence, and the 2006 bp upstream sequence before the candidate gene ATG was extracted from NCBI (http: / / www.ncbi.nlm.nih.gov / ) as a candidate promoter fragment. By designing specific primers (P1F: CCATCTGGAGGAAGCACG; P1R: CCACGATGGACGGGAACA), and using the blood genomic DNA of large white pigs as a template, the sequence was amplified by PCR. The amplification conditions were: 94°C for 5 min, 35* (94°C, 40sec, 57.8°C, 40sec, 72°C, 1min30sec); 72°C, 10min; 25°C, 2min. The amplified product was detected by 1.5% agarose electrophoresis, recovered and purified (operated by the UNIQ-10 column DNA gel recovery kit of Beijing Boda Tech Bio-Gene Technology Co., Ltd.), and TA cloning was performed. The ligation system ...

Embodiment 2

[0043] Example 2: Construction of the transfection vector of the promoter candidate fragment of the porcine skeletal muscle-specific expression gene α-actin and the corresponding deletion fragment.

[0044] (1) The enzyme-digested vector pGL3-basic (the vector was purchased from Promega (Beijing) Biotechnology Co., Ltd., that is, Promega Corporation in the United States. For information on the vector and multiple cloning sites, see figure 2 ) and the TA cloning plasmids TA-Q1, TA-Q2, TA-Q3, TA-Q4, TA-Q5, TA-Q6, TA-Q7, TA-Q8 with deletion fragments. Use KpnI, BglII double enzyme digestion pGL3-basic and TA-Q1, TA-Q2, TA-Q3, TA-Q4, TA-Q5, TA-Q6, TA-Q7, TA-Q8, the digestion system is:

[0045] 10×buffer2 2μL;

[0046] Plasmid DNA 5μL;

[0047] 100× bovine serum albumin (BSA) 0.2 μL;

[0048] Kpn I 0.5 μL;

[0049] BglII 0.5 μL;

[0050] Double distilled water 11.8μL

[0051]After digestion at 37°C for 3 hours, use 1.5% agarose gel electrophoresis to check the integrity of ...

Embodiment 3

[0059] Example 3: Liposome-mediated cell transfection

[0060] In the present invention, a 24-well cell culture dish is used for transfection. In order to eliminate experimental errors, three independent experiments were performed for each recombinant vector, and three replicate wells were performed for each experiment. According to Lipofectamine TM 2000 (invitrogen company) instructions, each well was transfected according to the ratio of the mass of the carrier: the volume of the liposome = 1 μg: 3 μL. The transfected receptors were cells from different sources, including mouse muscle cell line C2C12, porcine kidney cell PK and myotubes formed by the differentiation of C2C12 cells induced by 2% horse serum. 48h after the recombinant vector was transfected into the cells, the cell lysate was collected, and the activity of the missing promoter was analyzed by the dual luciferase detection system. The main steps of cell culture, induced differentiation and transfection of t...

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Abstract

The invention belongs to the technical field of animal gene engineering and particularly relates to isolated identification and functional verification of different-length promoter regions of pig skeletal muscle specificity expression genes alpha-actin. Eight upstream different-length promoters of the skeletal muscle specificity expression genes alpha-actin (the Gene Bank accession number is 100154254) are cloned from the pig genome, and the nucleotide sequences of the promoters are respectively shown as SEQ ID No: 2 to SEQ ID No: 9. The results show that the region with the length being 249bp has independent promoting activity and muscle tissue specificity, the nucleotide sequence is shown as the SEQ ID No: 9 in a sequence table. The invention also discloses a method for obtaining eight different deletion promoter segments, a method for preparing corresponding recombinant expression vectors and an application of a dual-luciferase enzymatic activity detection system to the promoter activity analysis.

Description

technical field [0001] The invention belongs to the technical field of animal genetic engineering, and particularly relates to the separation, identification and functional verification of promoter regions of different lengths of a skeletal muscle-specific expression gene α-actin in pigs. Background technique [0002] The expression of higher biological genes is finely regulated by the internal and external environment of cells, so it has strict temporal and spatial order. The regulation of gene expression is a complex and orderly process, which is completed by multi-stage regulation levels, which mainly include five levels: pre-transcription, transcription, post-transcription, translation and post-translation. The regulation at the transcriptional level is the most critical link. Promoter is an important regulatory element at the transcription level, a DNA sequence located in the upstream region of the 5' end of a structural gene, which can interact with numerous transcrip...

Claims

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Application Information

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IPC IPC(8): C12N15/113A01K67/027
Inventor 蒋思文刘京鸽
Owner HUAZHONG AGRI UNIV
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