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Clone and application of porcine skeletal muscle specific expression gene tnnc2 promoter

A promoter and skeletal muscle technology, applied in the field of functional verification, can solve problems such as the unseen research on the function of the promoter of the specific expression gene tnnc2 in porcine skeletal muscle

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 is no related report on the study of the promoter function of the porcine skeletal muscle-specific expression gene tnnc2

Method used

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  • Clone and application of porcine skeletal muscle specific expression gene tnnc2 promoter
  • Clone and application of porcine skeletal muscle specific expression gene tnnc2 promoter
  • Clone and application of porcine skeletal muscle specific expression gene tnnc2 promoter

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1: Acquisition of the promoter candidate fragment and the corresponding deletion fragment of the pig's skeletal muscle-specific expression gene tnnc2

[0035] Using the DNA sequence of the reported porcine tnnc2 gene (GenBank accession number: 414905) as the probe sequence, the 3000 to the upstream of the 5' end of the candidate gene mRNA was extracted at NCBI (http: / / www.ncbi.nlm.nih.gov / ). An intronic region serves as a regulatory region. Use MEME to predict the functional full-length promoter region, and predict the distribution of the core promoter region, cis-trans elements and CpG islands through bioinformatics software such as TFSEARCH and MethPrimer. According to the results of network prediction, primers for the full-length promoter and the 3′-terminal deletion fragment were designed (primer sequences are shown in Table 1; PCR reaction parameter settings are shown in Table 2). Among them, tnnc2-pro-F represents the common front primer of the 5 promoter...

Embodiment 2

[0040] Example 2: Construction of the promoter of the pig skeletal muscle-specific expression gene tnnc2 and the transfection vector of the corresponding deletion fragment.

[0041] (1) Restriction enzymes Mlu I and Nhe I cut the vector pGL3-basic (the vector was purchased from Promega (Beijing) Biotechnology Co., Ltd., which is the American Promega company, and the information on the vector and multiple cloning sites can be found in figure 2 ) and the recovery products tnnc2-pro, rnnc2-Q1, rnnc2-Q2, rnnc2-Q3, tnnc2-Q4 of each promoter fragment. The enzyme digestion system is:

[0042]

[0043] After digestion at 37°C for 6 hours, use 1.5% agarose gel electrophoresis to detect the digestion results and recover the target fragments: pGL3-basic recovers larger fragments, tnnc2-pro, tnnc2-Q1, tnnc2-Q2, tnnc2-Q3, tnnc2 -Q4 recycles fragments of corresponding size. Use the gel recovery kit of Omega Company to recover (operate according to the instructions of the kit), and sto...

Embodiment 3

[0048] Example 3: Cell Transfection of Promoter and Deletion Fragment Recombinant Plasmid

[0049] 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 carrier mass: volume of transfection reagent = 0.5 μg: 1.5 μL. The transfected recipients were 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 2% C2C12 cells (purchased from the Chinese Academy of Sciences Cell Bank) were induced to differentiate into myotubes with horse serum. The recombinant vectors (pGL3-tnnc2-pro, pGL3-tnnc2-Q1, pGL3-tnnc2-Q2,...

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Abstract

The invention belongs to the technical field of animal genetic engineering, and particularly relates to separation identification and functional verification of promoter areas with different lengths of porcine skeletal muscle specific expression gene tnnc2. Five promoters with different lengths in the upstream of a porcine skeletal muscle specific expression gene tnnc2 are cloned from a porcine genome, and the nucleotide sequences are shown as SEQ ID NO: 1 to SEQ ID NO: 5; the result shows that tnnc2-pro fragment of 2313 bp and tnnc2-Q2 fragment of 1048bp have independent promoter activity and muscular tissue specificity, and the nucleotide sequences are shown as SEQ ID NO: 1 and SEQ ID NO: 3. The invention discloses a preparation method of five different deletion promoter fragments, a dual-luciferase activity detecting system and application to promoter activity analysis by a quantitative PCR method.

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 different length promoter regions of pig skeletal muscle-specific expression gene tnnc2. 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 r...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/85
Inventor 蒋思文尚杨杨柴进张凤李倩倩马娟娟
Owner HUAZHONG AGRI UNIV
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