Pig intestinal endothelial cell line for stably expressing CaS9 protein

A technology of intestinal epithelial cells and stable expression, applied in the field of cell engineering

Inactive Publication Date: 2016-08-31
AGRO BIOLOGICAL GENE RES CENT GUANGDONG ACADEMY OF AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there is no commercial porcine intestinal epithelial cell line that can stably express Cas9 protein

Method used

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  • Pig intestinal endothelial cell line for stably expressing CaS9 protein
  • Pig intestinal endothelial cell line for stably expressing CaS9 protein
  • Pig intestinal endothelial cell line for stably expressing CaS9 protein

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Embodiment Construction

[0036] The present invention will be further described below in conjunction with embodiment.

[0037] The instruments, reagents, materials, etc. involved in the following examples, unless otherwise specified, are conventional instruments, reagents, materials, etc. in the prior art, and can be obtained through formal commercial channels. The experimental methods, detection methods, etc. involved in the following examples, unless otherwise specified, are conventional experimental methods, detection methods, etc. in the prior art.

[0038] (1) Construction of the lentiviral expression vector containing Cas9:

[0039] Amplify the SpCas9 gene from pX330 (Addgene #42230), construct the EFS:SpCas9:Flag:P2A:Blast fusion fragment expressing the Cas9 gene and the blasticidin (Blasticidin) resistance gene, and insert it into the lentiviral vector to obtain Cas9-containing The lentiviral expression vector LentiCas9-Blast (such as figure 1 shown); together with the purchased packaging pl...

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Abstract

The invention discloses a pig intestinal endothelial cell line for stably expressing a CaS9 protein. The pig intestinal endothelial cell line is characterized in that non-transformed intestinal endothelial cell line IPEC-J2 of a neonatal pig is taken as a host cell and is transfected with a lentiviral expression vector containing CaS9, and a transformant capable of stably expressing the CaS9 protein is obtained by screening of 20mu g/mL of Blasticidin. A construction method of the pig intestinal endothelial cell line comprises the following steps: firstly, constructing the lentiviral expression vector containing the CaS9; secondly, transforming an HEK293T cell; thirdly, transfecting an IPEC-J2 cell; fourthly, performing monoclonal screening. The pig intestinal endothelial cell line IPEC-J2-CaS9 disclosed by the invention can stably express the CaS9 protein, and has a growth curve identical with that of the non-transformed IPEC-J2 cell, and cell morphology identical with that of the IPEC-J2 cell. The pig intestinal endothelial cell line disclosed by the invention has important application value in aspects of researching the growth and differentiation of pig intestinal endothelial cells as well as dealing with the stimulation from exogenous substances.

Description

technical field [0001] The invention relates to constructing a porcine intestinal epithelial cell line stably expressing Cas9 protein, and belongs to the technical field of cell engineering. Background technique [0002] CRISPR-Cas9 is an adaptive immune system in bacteria against phage genomes or horizontally transferred plasmids. When foreign DNA invades bacteria, the CRISPR site is transcribed to produce two precursor small RNAs, namely crRNA and tracrRNA, and simultaneously synthesizes the protein Cas9 with endonuclease activity. Since Cas9 can recognize the PAM site in exogenous DNA and the 20 bases at the 5' end of crRNA can complement the bases in exogenous DNA, the nucleic acid-protein complex composed of crRNA, tracrRNA and Cas9 can specifically cut exogenous DNA. Bacterial-derived CRISPR-Cas9 or its slightly modified form sgRNA (crRNA and tracrRNA chimera)-Cas9 can be transplanted into eukaryotes and perform a programmable endonuclease function with double-strande...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/867
CPCC12N5/0679C12N15/86C12N2510/00C12N2740/15043C12N2800/107C12N5/10C12N15/867
Inventor 陈庄刘文华蒋宗勇陈中健朱翠张群洁朱庆锋吴秀菊张卫娜
Owner AGRO BIOLOGICAL GENE RES CENT GUANGDONG ACADEMY OF AGRI SCI
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