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Construction method of IPEC-J2 cell with APN gene knockout

A technology of IPEC-J2 and construction method, which is applied in the field of construction of porcine intestinal epithelial cell line (IPEC_J2) model, and can solve problems such as not yet obtained

Inactive Publication Date: 2018-06-29
YANGZHOU UNIV
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Problems solved by technology

[0004] The APN gene can encode a functional receptor protein for host cells infected by a variety of diarrheal pathogens. There have been related studies on sgRNA and modified vectors for editing the pig APN gene (patent application number: 201710424887.0), but researchers have not yet obtained APN gene knockout. intestinal epithelium

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  • Construction method of IPEC-J2 cell with APN gene knockout
  • Construction method of IPEC-J2 cell with APN gene knockout
  • Construction method of IPEC-J2 cell with APN gene knockout

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Embodiment Construction

[0028] Target site design and sgRNA sequence synthesis:

[0029] The CDs sequences of all transcripts of the porcine APN gene (Gene ID: 397520) were obtained according to the NCBI database, and the first exon of the CDs region was found for target site design. Use the online tool CRISPR Design (http: / / crispr.mit.edu / ) to design the sgRNA guide sequence:

[0030] 5'-TCTGTCTGTGGTGTATGCCC-3', in which the sgRNA action site is located in the first exon of the porcine APN gene, and CACCG is added to its 5' end to form a positive strand Oligo DNA.

[0031] At the same time, the reverse complementary sequence of the sgRNA sequence was synthesized: 5'-GGGCATACCACCAGACAGA-3', and AAAC was added at the 5' end to form a negative strand Oligo DNA.

[0032] The result is as follows:

[0033] Positive strand Oligo DNA: 5'-CACCGTCTGTCTGTGGTGTATGCCC-3';

[0034] Negative strand Oligo DNA: 5'-AAACGGGCATACCACCAGACAGA-3'.

[0035] . Construction of targeting vector:

[0036] (1) Anneal the...

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Abstract

The invention discloses a construction method of an IPEC-J2 cell with APN gene knockout and belongs to the technical field of gene engineering. According to the construction method, a CRISPR / Cas9 technology is adopted to knock out an APN gene sequence in an IPEC-J2 cell line genome, and an established intestinal epithelial cell with APN gene knockout can provide more direct and effective study model to deeply reveal a diarrhea pathogen pathogenic mechanism and to cultivation and application of a diarrheal disease resistance transgenic pig.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, specifically relates to the application technology of CRISPR / Cas9, and also relates to the construction technology of the pig small intestinal epithelial cell line (IPEC_J2) model. Background technique [0002] CRISPR / Cas9 technology is an adaptive immune system mediated by sgRNA (small guide RNA) derived from bacteria and archaea. Among them, sgRNA is used to recognize and bind target DNA. When the Cas9 protein activity is activated, it can bind to the DNA downstream of the region and cut it, generating double-strand DNA breaks and inducing frameshift mutations caused by non-homologous end-joining repair mechanisms. , so as to realize the editing of the target gene. Due to its simplicity and high efficiency, CRISPR / Cas9 technology has been widely used in the field of gene function research in humans, animals and plants in recent years as an important genetic method for site-specific...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/90C12N9/22C12N15/66
CPCC12N15/907C12N9/22C12N15/66
Inventor 包文斌戴开宇王诗琴王海飞吴圣龙
Owner YANGZHOU UNIV
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