sgRNA targeting knockout of TNFα gene and porcine embryonic fibroblast cell line with knockout of TNFα gene and its application

A fibroblast and α gene technology, applied in the field of genetic engineering, can solve problems such as incomplete silencing, inability to explain gene functions well, safety risks, etc., achieve great application research value, complete knockout effect, improve efficiency and specific effect

Active Publication Date: 2022-05-27
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, most of the research on TNFα gene is to use inhibitors to explore its role in humans and mice. There are few reports on the function of porcine TNFα, and it is difficult to guarantee the specificity and effectiveness of drug blocking. A good explanation of gene function also has certain safety risks
[0004] At present, methods such as inhibitors, silencing, knockdown, and interference in the prior art are not specific, incomplete or unable to silence TNFα gene expression, so it is necessary to develop a pig TNFα that can achieve complete silencing and long-term stable in vitro culture The gene-deleted cell line is expected to prepare transgenic pigs through the TNFα gene-deficient fibroblast cell line, which will provide a disease model for the study of immune and inflammatory responses, and provide a basis for further research on the regulatory role of TNFα gene in pig muscle development

Method used

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  • sgRNA targeting knockout of TNFα gene and porcine embryonic fibroblast cell line with knockout of TNFα gene and its application
  • sgRNA targeting knockout of TNFα gene and porcine embryonic fibroblast cell line with knockout of TNFα gene and its application
  • sgRNA targeting knockout of TNFα gene and porcine embryonic fibroblast cell line with knockout of TNFα gene and its application

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Experimental program
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Embodiment 1

[0035] Example 1 Construction and detection of CRISPR-Cas9 targeting vector targeting TNFα gene

[0036] 1. Sequence design of TNFα gene sgRNA

[0037] Download the porcine TNFα gene sequence (accession number NC_010449.5) from the NCBI website, and use the website http: / / crispr.mit.edu / to design knockout target sites on the first exon of the gene, and a total of 10 were designed Target, according to the PCR sequencing peak map, the sgRNA with the highest set of peaks is screened to be the sgRNA with high editing efficiency, and the final selected sgRNA target sequence is: GCGCTCGCCAAGAAGGCCG (as shown in SEQ ID NO: 1), according to the BbsI restriction Dicer designs restriction sites at both ends of the sgRNA, and adds CACCG to the 5' end of the sgRNA to form Oligo1, the sequence is shown in SEQ ID NO: 2; the 5' end of the reverse complementary sequence is added with AAAC, and the 3' end Add C to form Oligo2, and the sequence is shown in SEQ ID NO: 3. Finally, the designed ...

Embodiment 2

[0056] Example 2 Construction and genomic identification of TNFα gene knockout pig embryonic fibroblast cell line

[0057] 1. Screening of positive monoclonal cells

[0058] (1) When the porcine embryonic fibroblasts grow to a confluence of 70% to 90%, prepare a mixture containing 150 μL of electroporation solution, 6 μg of expression vector pX330-TNFα and 3 μg of G418 resistance plasmid, and use the A024 program for electroporation.

[0059] (2) After 6 hours of transfection, it was changed to growth medium containing 10% fetal bovine serum, and after 24 hours, the cells were digested and divided into plates, and the cells in the 6-well plate were passaged into 10 to 20 10 cm dishes, and 600 μg / mL of G418 was added for For screening, the medium was changed every 4 to 5 days, and the monoclonal cell mass could be obtained by changing about 2 times.

[0060] (3) Select monoclonal cells with regular morphology and good condition, and transfer them to 48-well plates with a cloni...

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Abstract

The present invention relates to the technical field of genetic engineering, in particular to sgRNA targeting knockout of TNFα gene and porcine embryonic fibroblast cell line of knockout TNFα gene and its application; The dot sequence is located on the first exon, and the target sequence is shown in SEQ ID NO:1. After constructing the sgRNA into the vector pX330, the expression vector pX330-TNFα was used as the targeting vector of the TNFα gene to transfect porcine embryonic fibroblasts to obtain a monoclonal cell line of porcine embryonic fibroblasts with knockout of the TNFα gene. A variety of mutants with different editing types can cause frameshift mutations, and the editing efficiency is high. The pig embryonic fibroblast cell line prepared by the invention can be used as a donor of somatic cell nuclear transplantation for the preparation of transgenic pigs, and has great application research value.

Description

technical field [0001] The present invention relates to the technical field of genetic engineering, in particular to a sgRNA targeting TNFα gene knockout and a pig embryonic fibroblast cell line knocking out TNFα gene and its application. Background technique [0002] The CRISPR-Cas system is an adaptive immune system that exists in most archaea and most bacteria to resist foreign virus or DNA invasion. Type II RISPR-Cas9 system is currently the most successful and widely used. It is mainly through a short RNA sequence and a DNA target sequence to form a double-strand according to the principle of base complementarity, combined with Cas9 protein to induce formation at the DNA target site. Double-strand break damage, and special gene mutations can be introduced at the target site of the genome during damage repair. The CRISPR-Cas9 system has the advantages of simple design, high targeting efficiency and the ability to form a variety of mutations at the target site. It has be...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/85C12N5/10C12N15/11C12Q1/6888C12Q1/02A01K67/027C12R1/91
CPCC12N15/113C12N15/8509C12N5/0656C07K14/525A01K67/0276G01N33/5005C12Q1/6888C12N2310/20C12N2510/00A01K2217/075A01K2227/108A01K2267/03
Inventor 张浩付玉张盼张博凌遥
Owner CHINA AGRI UNIV
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