sgRNA for targeted knockout of TNF[alpha] gene, porcine embryo fibroblast cell line for knocking out TNF[alpha] gene and applications of fibroblast cell line

Abstract

The invention relates to the technical field of genetic engineering, and especially relates to a sgRNA for targeted knockout of a TNF[alpha] gene, a porcine embryo fibroblast cell line for knocking out the TNF[alpha] gene and applications of the fibroblast cell line. The target sequence of the provided sgRNA on the TNF[alpha] gene is located on the first exon and shown as SEQ ID NO: 1. After the sgRNA is constructed into the vector px330, an expression vector pX330-TNF[alpha] is taken as the targeting vector of the TNF[alpha] gene; and porcine embryo fibroblast cells are transfected so as to obtain the TNF[alpha] gene-knocked-out monoclonal cell line of porcine embryo fibroblast cells, and the obtained mutant having many different editing types can cause frameshift mutation, so that high editing efficiency can be achieved. The prepared cell line can be used as the donor of somatic cell nuclear transfer for the preparation of transgenic swine, so that the cell line has large applicationand research values.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a sgRNA targeted to knock out the TNFα gene and a porcine embryonic fibroblast cell line for knocking out the TNFα gene and applications thereof. Background technique [0002] The CRISPR-Cas system is an acquired 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 transformation and the most widely used, mainly through a short RNA sequence and the DNA target sequence to form a double strand according to the principle of base complementarity, then combined with the Cas9 protein to induce the formation of a Cas9 protein at the DNA target site Double-strand break damage can introduce specific gene mutations at genomic target sites during damage repair. The CRISPR-Cas9 system has the advantages of simple design, high targeting efficiency, and the ability to form mu...

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

Images