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Cloning and application of swine liver specificity expression gene TTR promoter

A pig liver and promoter technology, applied in DNA/RNA fragments, introduction of foreign genetic material using vectors, recombinant DNA technology, etc., can solve the problems that have not been seen in the research on the function of TTR promoter of pig liver muscle-specific expression genes.

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

[0006] So far, there are no related reports on the study of the promoter function of the pig liver muscle-specific expression gene TTR

Method used

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  • Cloning and application of swine liver specificity expression gene TTR promoter
  • Cloning and application of swine liver specificity expression gene TTR promoter
  • Cloning and application of swine liver specificity expression gene TTR promoter

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1: Obtaining the promoter fragment of the pig's liver-specific expression gene TTR

[0035] NCBI database (http: / / www.ncbi.nlm.nih.gov / ) looks up the DNA sequence of the porcine TTR gene (GenBank accession number: 397419), and searches the region from 2000 bp upstream of the 5′ end of the gene mRNA to the first intron as a control area. Bioinformatics software such as TESS, TFSEARCH and MethPrimer were used to predict the distribution of cis-trans elements and CpG islands. The primers for the full-length promoter were designed according to the results predicted by the network (the primer sequences are shown in Table 1; the PCR reaction parameter settings are shown in Table 2). The restriction site Mlu I (ACGCGT) was added to the 5' end of the upstream primer, and the protective base CG was added; the Xho I (CTCGAG) restriction site was added to the 5' end of the downstream primer, with the protective base CCG. Using large white pig blood genomic DNA as a templ...

Embodiment 2

[0041] Example 2: Construction of a promoter transfection vector for pig liver-specific expression gene TTR.

[0042] (1) Restriction enzymes MluⅠ and XhoⅠ digested the vector pGL3-basic (the vector was purchased from Promega (Beijing) Biotechnology Co., Ltd., which is the American Promega company. For information on the vector and multiple cloning sites, see figure 2 ) and the recovered product of the TTR-pro promoter fragment. See Table 3 for enzyme digestion system

[0043] Table 3 enzyme digestion system

[0044]

[0045] After digestion at 37°C for 4 hours, use 1.5% agarose gel electrophoresis to detect the digestion results and recover the target fragments: pGL3-basic recovers larger fragments, and TTR-pro recovers corresponding 2205bp fragments. Use Omega's gel recovery kit to recover (operate according to the kit instructions), and store in a -20°C refrigerator.

[0046] (2) Ligate the recovered promoter fragment into the pGL3-basic vector (see image 3 ), the ...

Embodiment 3

[0050] Example 3: Cell Transfection of Recombinant Plasmid pGL3-TTR-pro

[0051] The present invention uses a 24-hole cell culture dish for transfection. In order to eliminate experimental errors, three independent experiments were performed for each recombinant vector, and each experiment was repeated three times. According to the instructions of Fugene HD (Roche Company), each well was transfected according to the ratio of the mass of the carrier: the volume of the transfection reagent = 0.5 μg: 1.5 μL. The transfected recipients are cells from different sources, mainly including mouse myogenic cell line C2C12 (purchased from the Cell Bank of the Chinese Academy of Sciences), mouse preadipocyte 3T3-L1 (purchased from the Cell Bank of the Chinese Academy of Sciences) and mouse liver cancer Cells Hepa1-6 (Wuhan Boster Bioengineering Co., Ltd.). After the recombinant vector pGL3-TTR-pro was transfected into the cells for 48 hours, the cell lysate was collected and the activit...

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Abstract

The invention belongs to the technical field of animal gene engineering, in particular to cloning and activity analysis of a swine liver specificity expression gene TTR promoter with the length of 2205 bp. A 2205 bp-long regulatory sequence at the upstream of a translation initiation codon ATG of the swine liver specificity expression gene TTR promoter is cloned from a swine genome, and the nucleotide sequence is shown as the SEQ ID NO: 3; the result shows that the TTR promoter with the length of 2205 bp has independent activity and liver tissue specificity with the nucleotide sequence shown as the SEQ ID NO: 3. The invention further discloses a preparation method of the promoter fragment, a dual-luciferase activity detection system and the application of the RT-PCR method for the activity analysis of the promoter.

Description

technical field [0001] The invention belongs to the technical field of animal genetic engineering, and in particular relates to the isolation, identification and functional verification of the TTR promoter region of pig skeletal muscle specific expression gene. 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. Promoter is an important regulatory element at the transcriptional level. It is a DNA sequence located in the upstream region of the 5' end of a structural gene, which can interact with many transcription factors to regulate gene exp...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/85
Inventor 蒋思文周鹏
Owner HUAZHONG AGRI UNIV
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