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Construction method of SIRT1 gene knockout IPEC-J2 cell line

A technology of IPEC-J2 and construction method, applied in the field of gene knockout, can solve the problems such as unclear integrity of intestinal epithelium

Active Publication Date: 2019-08-30
ANIMAL SCI RES INST GUANGDONG ACADEMY OF AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, whether SIRT1 stimulates or inhibits intestinal stress remains controversial, and how intestinal epithelial SIRT1 mediates complex environment-host interactions to regulate intestinal epithelial integrity is unclear

Method used

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  • Construction method of SIRT1 gene knockout IPEC-J2 cell line
  • Construction method of SIRT1 gene knockout IPEC-J2 cell line
  • Construction method of SIRT1 gene knockout IPEC-J2 cell line

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Embodiment 1

[0041] A kind of embodiment of the construction method of the IPEC-J2 cell line of SIRT1 gene knockout of the present invention, comprises the steps:

[0042] 1. SIRT1 target site design and sgRNA sequence synthesis

[0043] According to the NCBI database, the ORF sequences of all transcripts of the porcine SIRT1 gene (Gene ID: NM_001145750.2) were obtained, and the first exon of the ORF region was located for target site design.

[0044] Use the online tool (http: / / tools.genome engineering.org) to design and screen the sgRNA guide sequence: 5'-CTCCGCGGTTTCTTGCGGAG-3', where the sgRNA action site is located in the first exon of the pig SIRT1 gene, at its 5' end Add CACCG to form positive oligo DNA.

[0045] Simultaneously synthesize the reverse complement of the sgRNA sequence:

[0046] 5'-CTCCGCAAGAAACCGCGGAG-3', and add AAAC at the 5' end and C at the 3' end to form a reverse OligoDNA. The base sequence of SIRT1-sgRNA is as follows:

[0047] 5'-CACCG CTCCGCGGTTTCTTGCGG...

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Abstract

The present invention discloses a construction method of a SIRT1 gene knockout IPEC-J2 cell line. The construction method comprises the following steps: (1) a gRNA probe is designed and synthesized for a first exon of a SIRT1 gene, a base sequence of gRNA is shown as SEQ ID NO.1, and a reverse complement sequence is shown as SEQ ID NO.2; (2) an inverted complementary double-stranded gRNA probe after annealing is cloned into a pSpCas9-2A-PuroV2.0 vector shown in figure 2, and a plasmid simultaneously expressing the sgRNA as shown in the SEQ ID NO.1 and Cas9 proteins is constructed; (3) the plasmid constructed in the step (2) is transfected into IPEC-J2 cells; and (4) a monoclonal mutant cell line is screened from the transfected IPEC-J2 cells in the step (3) to obtain the SIRT1 gene knockout IPEC-J2 cell line, and compared with a wild-type cell line, the SIRT1 gene knockout IPEC-J2 cell line lacks 5 bases in the first exon of the SIRT1 gene. The SIRT1 gene knockout IPEC-J2 cell line isfirstly constructed, the stable monoclonal SIRT1 gene knockout IPEC-J2 homozygous cell line is screened, and the SIRT1 gene knockout IPEC-J2 cell line can be used as a cell model for studying intestinal oxidative stress of piglets.

Description

technical field [0001] The invention relates to the technical field of gene knockout, in particular to a method for constructing a SIRT1 gene knockout IPEC-J2 cell line. Background technique [0002] IPEC-J2 cells are porcine intestinal columnar epithelial cells isolated from the jejunum of neonatal piglets. This cell line forms polarized monolayers with high transepithelial resistance when cultured on 0.4 μm pore size filters. Compared to common human colon-derived lines HT-29, T84 and Caco-2, IPEC-J2 cells are unique in that they are derived from porcine small intestine tissue and have not been transformed (compared to IPI-2I). Porcine intestinal epithelial cells more closely mimic the physiology of the human intestinal epithelium than rodent intestinal epithelial cell lines (such as IEC-6 or IEC-18), which is especially important in the study of zoonotic infections. In addition, IPEC-J2 cells are increasingly used in the study of intestinal oxidative stress, and they pr...

Claims

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Application Information

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IPC IPC(8): C12N15/90C12N15/85C12N9/22C12N5/10
CPCC12N15/907C12N15/85C12N9/22
Inventor 鲁慧杰马现永陈卫东邓盾容庭田志梅刘志昌
Owner ANIMAL SCI RES INST GUANGDONG ACADEMY OF AGRI SCI
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