LncRNA XIST gene knock-in animal model construction method based on RNA targeted binding site

Abstract

The invention discloses a construction method of an LncRNA XIST gene knock-in animal model based on an RNA targeted binding site. Comprising the following steps: bioinformatics analysis of a seventh exon of an XIST gene on a targeted binding site of the XIST gene and mmu-miR-140-5p, in-vitro experiments prove that the targeted binding site of the XIST gene and mmu-miR-140-5p in bioinformatics analysis, sgRNA vector construction of the seventh exon containing the targeted binding site of a targeted mouse XIST gene, in-vitro transcription of sgRNA, and acquisition and identification of an F0 generation mouse. The XIST gene is knocked into an animal model in the acquisition process of F1-generation hybrid mice and the acquisition process of homozygous offspring, so that the problem of high off-target rate of the gene caused by overlong fragment in the knock-in process of the CRISPR / Cas9 for the XIST gene with the full length reaching 17.9 kb and containing seven exons is solved.

Description

【Technical field】 [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a method for constructing an LncRNAXIST gene knock-in animal model based on RNA targeting binding sites. 【Background technique】 [0002] The CRISPR / Cas system is a technology derived from the acquired immunity of bacteria for targeted modification of target genes by RNA-mediated Cas proteins. After successfully knocking out mammalian cells in 2013, the Type II CRISPR / Cas9 system modified by researchers has now been applied to gene knockout in various model organisms. The CRISPR / Cas9 system vector construction is simple, fast, easy to operate, saves time and effort, and is applicable to almost all species. The roles of CRISPR / Cas9 and TALEN (Transcription Activator-like Effector Nucleases) are to achieve double-strand breaks at specific sites on the chromosome, and then trigger autonomous damage repair, which will lead to insertion or deletion, resulting in p...

AI Technical Summary

Problems solved by technology

[0004] Aiming at the problem that the traditional CRISPR / Cas9 technology in the prior art has a full length of 17.9kb and contains seven exons, the LncRNA XIST gene knock-in process has a high gene off-target rate caused by too long fragments, the present invention provides a The construction method of LncRNA XIST gene knock-in animal model based on RNA targeting binding site is a CRISPR / Cas9 technology LncRNA XIST gene knock-in animal model construction based on LncRNAXIST and microRNA mmu-miR-140-5p targeting binding site Method, by knocking in the exon of the LncRNA containing the targeting binding site of LncRNA and microRNA, the purpose of studying the effect of LncRNA targeting and binding to microRNA can be achieved, which can reduce the gene knockdown caused by the excessive length of LncRNA and containing multiple exons. The difficulty of entry, realize the research on the direction of lncRNA targeting and binding microRNA

Images