Promoter for specifically expressing gene in poultry skeletal muscle and application

A promoter and skeletal muscle technology, applied in the direction of using vectors to introduce foreign genetic material, peptides, DNA/RNA fragments, etc., can solve the problem of not having the ability to target gene expression regulation, limiting the application of tissue-specific promoters, and specific initiation Problems such as the small number of children

Inactive Publication Date: 2019-03-22
AFFILIATED HOSPITAL OF GUANGDONG MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The problem with these two highly efficient promoters is obvious, and they do not have the ability to target and regulate gene expression
[0004] However, from the analysis of tissue-specific promoters obtained in animals and plants, the following problems generally exist: the specificity is not very high; the number of specific promoters isolated and applied is small; the activity is low, which is a great Limitation of the application of tissue-specific promoters in genetic engineering
However, there are currently no reports of promoters that can efficiently and specifically promote gene expression in avian skeletal muscle, which greatly hinders the application of specific expression of exogenous genes in avian skeletal muscle

Method used

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  • Promoter for specifically expressing gene in poultry skeletal muscle and application
  • Promoter for specifically expressing gene in poultry skeletal muscle and application
  • Promoter for specifically expressing gene in poultry skeletal muscle and application

Examples

Experimental program
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Effect test

Embodiment 1

[0044] In the embodiment of the present invention, a fragment from 1000 bp upstream of the transcription initiation point of CARP to 203 bp downstream of the transcription initiation point is amplified by PCR method (i.e. -1000 to +203 bp, (-): indicates upstream of transcription initiation, (+): indicates Downstream of transcription initiation), Gene ID of CARP gene: 378926. This fragment was cloned into the luciferase reporter vector pGL3-basic (Promega). Using a series of primers with restriction sites, a series of vectors with 5' deletion structures were constructed by PCR. P-386(-1000~+203bp), P-386(-386~+203bp), P-355(-355~+203bp), P-337(-337~+203bp), P-319( -319~+203bp), P-286(-286~+203bp), P-269(-269~+203bp), P257(-257~+203bp), P227(-227~+203bp), P-189 (-189~+203bp), (-): indicates upstream of transcription initiation, (+): indicates downstream of transcription initiation, for example: P-337 (-337~+203bp) is upstream of the transcription initiation point linked to CA...

Embodiment 2

[0047] The vector of Example 1 was transfected into CFM, chicken fibroblasts and DT40 cells, and CFM, chicken fibroblasts and DT40 cells were cultured in DMEM containing 10% fetal bovine serum and 1% double antibody, and placed in Containing 5% carbon dioxide, 37°C incubator. Transfection experiments were performed with lip2000 (Invitrogen, USA) when the cell density reached 70%.

Embodiment 3

[0049] Carry out the luciferase reporter assay on the CFM, chicken fibroblasts and DT 40 cells that have been transfected in Example 2, the steps are as follows: inoculate CFM, chicken fibroblasts and DT 40 cells in a 12-well plate, transfect with Lip2000 After 24 h, the luciferase activity was detected with a luciferase reporter system (Promega, USA). All transfections were performed in triplicate, and firefly luciferase activity was normalized to kidney luciferase activity for each sample, and data are presented as mean ± standard deviation and included at least three separate experiments. Two independent samples were compared using t test. All analyzes were conducted with two-tailed test, and the difference was significant (Pfigure 1 as shown, figure 1 It can be seen that in CFM cells, the deletion of -1 kb to -373 base pairs (bp) has no significant effect on the activity of the CARP promoter. However, deletion of this region from -337bp to -227bp resulted in a significan...

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Abstract

The invention belongs to the technical field of biology and in particular relates to a promoter for specifically expressing a gene in poultry skeletal muscle and application. The promoter for specifically expressing the gene in the poultry skeletal muscle, provided by the invention, comprises at least one E-box; the nucleotide sequence of the E-box CANNTG, wherein N is selected from A, T, C or G.The invention further provides application of the gene promoter capable of being specifically expressed in the poultry skeletal muscle. The promoter for specifically expressing the gene in the poultryskeletal muscle, provided by the invention, can be used for constructing various specific transgenic expression vectors of the poultry skeletal muscle; and the specific transgenic expression vectorsare used for high-abundance expression of the promoter gene in poultry skeletal muscle tissue or skeletal muscle derived cells or the poultry meat quality and muscle-related disease therapy are improved through a gene engineering technology.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a promoter for gene specific expression in poultry skeletal muscle and its application. Background technique [0002] Whether exogenous genes can be efficiently and stably expressed in the body is the key to genetic engineering research. At the same time, the problem to be solved is that the expression of exogenous genes should be turned on as needed, that is, have strict temporal and spatial specificity. It is a common technology to construct various eukaryotic expression systems in eukaryotic cells by using genetic engineering technology to express exogenous genes. The most critical is the regulation of transcription level. A promoter is a DNA sequence that can correctly and effectively initiate RNA transcription, and can interact with transcription factors to bind RNA polymerase. Promoter is an important part of gene, which controls the expression of gene in time, sp...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/85
CPCC07K14/465C12N15/85
Inventor 马国达汪亚君梁春梅李友
Owner AFFILIATED HOSPITAL OF GUANGDONG MEDICAL UNIV
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