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Method for acquiring Nogo-B knockout mode mouse based on CRISPR/Cas9 technology and application thereof

A mouse and model technology, applied in the biological field, can solve the problems of limited success rate, time-consuming, high requirements, etc.

Pending Publication Date: 2018-10-12
GENERAL HOSPITAL OF PLA
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the traditional gene knockout method requires a series of steps such as complex targeting vector construction, embryonic stem cell screening, and chimera mouse breeding. The success rate is limited by many factors

Method used

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  • Method for acquiring Nogo-B knockout mode mouse based on CRISPR/Cas9 technology and application thereof
  • Method for acquiring Nogo-B knockout mode mouse based on CRISPR/Cas9 technology and application thereof
  • Method for acquiring Nogo-B knockout mode mouse based on CRISPR/Cas9 technology and application thereof

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Embodiment

[0034] The EGE-ZXH-007 gene is on the positive strand of chromosome 11, with a total length of about 51.1kb. NCBI ID: 68585 (such as figure 1 shown). The EGE-ZXH-007 gene has 10 transcripts, this design refers to transcript 005 (Rtn4-005), Transcript ID: ENSMUST00000102843 (such as figure 2 shown). The structural domain of the EGE-ZXH-007 gene is as follows image 3 shown. The regulatory region of the EGE-ZXH-007 gene is as follows Figure 4 shown. Targeting strategy: Analyze the structure of the EGE-ZXH-007 gene, which has three main transcripts A, B, and C. The design is to knock out the first exon of the two transcripts A and B, and transcript C The first exon of the EGE-ZXH-007 gene is located downstream, and the two sgRNAs can knock out the first exon of the two transcripts of EGE-ZXH-007 gene A and B about ~2kb. Model mice were prepared based on CRISPR / Cas9 technology. The schematic diagram of the targeting carrier is shown in Figure 5 shown.

[0035] The meth...

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Abstract

The invention relates to a method for acquiring a Nogo-B knockout mode mouse based on CRISPR / Cas9 technology and an application thereof, the method for acquiring the Nogo-B knockout mode mouse comprises the following steps of: designing a high-efficiency sgRNA for identifying EGE-ZXH-007 of a specific gene; connecting the sgRNA to a plasmid vector and then carrying out in-vitro transcription to obtain a Ca9 / sgRNA capable of being subject to microinjection; injecting the Ca9 / sgRNA capable of being subject to microinjection into a mouse fertilized egg, and then carrying out genotype detection and identification on the born mouse, and finally obtaining the Nogo-B knock-out mode mouse. According to the invention, the Nogo-B knockout mode mouse is obtained by using the CRISPR / Case9 technology,and compared with the traditional gene knockout technology, the operation is easy and the efficiency is higher, and the homozygous mutation is more easily obtained, thus laying a good foundation for further researching the function of Nogo-B.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method and application for obtaining Nogo-B knockout model mice based on CRISPR / Cas9 technology. Background technique [0002] Gene knockout is aimed at a sequence whose sequence is known but whose function is unknown, changing the genetic gene of the organism, making the specific gene function ineffective, so that part of the function is blocked, and can further affect the organism, and then it is speculated that the The biological functions of genes. [0003] Gene knockout technology was developed in the 1980s. It is a new molecular biology technology based on gene homologous recombination technology and embryonic stem cell technology. Gene homologous recombination technology refers to the homologous recombination of exogenous DNA and genes with the same or similar sequence in the genome of the recipient cell, thereby replacing the same / similar gene sequence in the genome of the...

Claims

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Application Information

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IPC IPC(8): C12N15/90A01K67/027
CPCA01K67/0276A01K2217/075A01K2227/105A01K2267/03C07K14/47C12N9/22C12N15/907
Inventor 高利利
Owner GENERAL HOSPITAL OF PLA
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