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

A technology of promoter and skeletal muscle, applied in the field of functional verification, can solve the problems such as the promoter function of myoz1 gene specifically expressed in porcine skeletal muscle that has not been studied.

Inactive Publication Date: 2014-12-31
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 relevant report on the study of the promoter function of the porcine skeletal muscle-specific expression gene myoz1

Method used

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1: Acquisition of the promoter candidate fragment and the corresponding deletion fragment of the porcine skeletal muscle-specific expression gene myoz1

[0036] NCBI database (http: / / www.ncbi.nlm.nih.gov / ) searches for the DNA sequence of the pig's myoz1 gene (GenBank accession number: 574060), and searches the region from 3000 bp upstream of the 5' end of the gene mRNA to the first intron as a control area. The approximate region of the full-length promoter was predicted by MEME, and the distribution of cis-trans elements and CpG islands were predicted by bioinformatics software such as TFSEARCH and MethPrimer. The primers for the full-length promoter and the 5' and 3' end deletion fragments were designed according to the results predicted by the network (primer sequences are shown in Table 1; PCR reaction parameter settings are shown in Table 2). Wherein myoz1-pro-R represents the common back primer of 6 promoter fragments, adds the enzyme cutting site NheI (...

Embodiment 2

[0041] Example 2: Construction of a transfection vector for the promoter of the pig skeletal muscle-specific expression gene myoz1 and the corresponding deletion fragment.

[0042] (1) Restriction endonucleases Nhe I and Hind III digested the vector pGL3-basic (the vector was purchased from Promega (Beijing) Biotechnology Co., Ltd., namely the American Promega Company, see figure 2 ) and the recovery products of each promoter fragment myoz1-pro, myoz1-Q1, myoz1-Q2, myoz1-Q3, myoz1-Q4, myoz1-Q5. The enzyme digestion system is:

[0043]

[0044] 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, myoz1-pro, myoz1-Q1, myoz1-Q2, myoz1-Q3, myoz1 -Q4, myoz1-Q5 recovered fragments of corresponding size. Recover with the gel recovery kit of Omega Company (operate according to the instructions of the kit), and store in a -20°C refrigerator.

[0045] (2) L...

Embodiment 3

[0049] Example 3: Cell Transfection of Promoter and Deletion Fragment Recombinant Plasmid

[0050] 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) differentiated myotubes induced by horse serum. The recombinant vectors pGL3-myoz1-pro, pGL3-myoz1-Q1, pGL3-myoz1-Q3, pGL3-myoz1-Q4,...

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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 a porcine skeletal muscle specific expression gene myozl. Six promoters of different lengths in the upstream of 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: 6; the result shows that myozl-Q4 fragment of 648bp has independent promoter activity and muscular tissue specificity, and the nucleotide sequence is shown as SEQ ID NO: 5. The invention discloses a preparation method of six 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 the pig skeletal muscle-specific expression gene myoz1. 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 ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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