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A porcine ucoe regulatory element fragment that enhances exogenous gene expression

A kind of technology of regulating element, exogenous gene

Inactive Publication Date: 2017-03-15
HENAN AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to provide a pig UCOE regulatory element fragment, which can promote or enhance the stable and efficient expression of foreign genes in host cells, and propose new methods and solutions for solving the problem of gene silencing in transgenes

Method used

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  • A porcine ucoe regulatory element fragment that enhances exogenous gene expression
  • A porcine ucoe regulatory element fragment that enhances exogenous gene expression
  • A porcine ucoe regulatory element fragment that enhances exogenous gene expression

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] The base sequence of the porcine UCOE regulatory element fragment capable of enhancing the expression of exogenous genes provided by the present invention is shown in the sequence table. The following is a brief introduction to the screening process.

[0074] figure 1 It is a schematic diagram of the complete UCOE regulatory element of the pig in the present invention, which is obtained by alignment on the basis of human UCOE (hUCOE) sequence. The porcine UCOE regulatory element fragment claimed in the present invention is obtained by screening the complete UCOE regulatory element of pigs with different combinations of enzymes, and the specific screening process is as follows.

[0075] (1) Cloning of pig UCOE fragment and construction of recombinant plasmid

[0076] ① Extraction of pig genomic DNA

[0077] Genomic DNA from porcine liver tissue was extracted using an animal tissue genome extraction kit, and the quality of genomic DNA was detected by 0.8% agarose gel e...

Embodiment 2

[0223] In order to test the technical effect of the best pig UCOE fragment screened in Example 1, the inventors conducted further experimental verification, the brief introduction is as follows.

[0224] (1) Construction of pCpG-Mini-GFP-UCOE plasmid

[0225] ①Construction of pCpG-Mini-GFP plasmid

[0226] First construct the pCpG-GFP plasmid

[0227] The pDrive5-GFP-2 vector and pCpGfree-mcs were double-digested with restriction endonucleases NheI and NcoI respectively, and the digested products were electrophoresed on 1.0% agarose gel, gel-cut and purified, and the target fragment was recovered. The recovered two target fragments were ligated and transformed to construct pCpG-GFP plasmid. The recombinant plasmid was identified by single enzyme digestion using the restriction endonuclease PacI, and the identified correct plasmid was named pCpG-GFP.

[0228] The double enzyme digestion system of pDrive5-GFP-2 and pCpGfree-mcs is as follows:

[0229] NheI, 1 µL;

[0230] N...

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Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to a porcine UCOE regulatory element fragment for enhancing exogenous gene expression. The base sequence of the UCOE regulatory element fragment is shown in a sequence table. With respect to exogenous protein expression, the regulatory element fragment can enhance GFP expression by constructing recombinant plasmid expression vector pCpG-Mini-GFP-UCOE and transfecting HEK293T cells. According to the invention, with a human 1.5 kb UCOE fragment used as a control, a porcine UCOE fragment is cut into fragments with different lengths by an enzyme digestion method and an RT-PCR method; different expression vectors are constructed, and transfected into HEK293T cells; the relative expression level of gene GFP is reported by real-time fluorescence quantification PCR and Western blot technique detection, and the optimal UCOE fragment sequence for enhancing exogenous gene expression is screened, which is the gene sequence claimed in the invention.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a pig UCOE regulatory element fragment for enhancing exogenous gene expression. Background technique [0002] Chromosome opening element (ubiquitous chromatin opening element, UCOE) can change the chromosome structure to maintain transcriptional activity, this element mainly includes locus control regions (locus control regions, LCR) and UCOE two types. LCR can only participate in the regulation of transgene expression in several specific tissues. UCOE can regulate the expression of transgenes in a wide range of tissues. In terms of structural composition, those genomic regions containing bidirectional transcriptional promoters of housekeeping genes surrounded by abundant unmethylated CpG islands, which can give stable high-level expression of transgenes, are defined as UCOE. When regulating gene expression, UCOE has nothing to do with tissue specificity...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/85C12N15/67
Inventor 杨国宇高会贞郭豫杰王月影李和平朱河水钟凯鲁维飞韩立强查光明
Owner HENAN AGRICULTURAL UNIVERSITY
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