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A method for preparing ps1 gene conditional knockout flox rats

A gene and female mouse technology, applied in biochemical equipment and methods, genetic engineering, preparations for in vivo experiments, etc., can solve the problems of unknown PS1 gene function, no rat model, etc., and achieve simple preparation process and easy operation , high efficiency effect

Active Publication Date: 2020-05-19
BIOCYTOGEN PHARMACEUTICALS (BEIJING) CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] In summary, the function of the PS1 gene is currently unknown, and there is no relevant rat model for research. Therefore, there is an urgent need in this field to study the PS1 gene, and then use it for the development of related drugs. Suitable animal models

Method used

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  • A method for preparing ps1 gene conditional knockout flox rats
  • A method for preparing ps1 gene conditional knockout flox rats
  • A method for preparing ps1 gene conditional knockout flox rats

Examples

Experimental program
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Effect test

Embodiment 1

[0042] Example 1 Construction of PS1 Gene Homologous Recombination Vector

[0043] The design of the vector is to knock out the fourth exon of PS1 gene, so as to realize the inactivation of the whole gene. Firstly, three homologous recombination fragments (A, B, and C) were amplified from the genomic DNA of wild-type SD rat tissue, and connected to the LScKO vector by enzyme digestion to construct the PS1 conditional gene knockout vector plasmid. LScKO vector map see figure 2 . The source of the backbone (the vector backbone is pUC19, from Takara, product number 3219) was designed and modified by the inventor, and LoxP sites and multiple cloning sites were introduced into the vector, see figure 2 . The specific transformation method is as follows: a fragment DNA (such as SEQ ID NO.10) is synthesized by a plasmid synthesis company, the pUC19 vector is digested with EcoRI and HindIII, the synthesized fragment is connected to the pUC19 vector, and the sequence verification b...

Embodiment 2

[0068] Example 2 Construction of CRISPR / Cas9 Targeting Plasmid

[0069] 1. Design, synthesis and construction of sgRNA fragments:

[0070] Table 3 sgRNA fragment sequence

[0071]

[0072]

[0073] 1. The specific design is as follows:

[0074] (a) Find information about the PS1 gene on NCBI; the gene ID number is 29192, located on chromosome 6, about 47.98kb,

[0075] (b) Design sgRNA fragments using Crispr software (http: / / crispr.mit.edu / )

[0076] (c) The selected targets are analyzed on the NCBI database to determine knockout regions. Select the sgRNA site with fewer off-target sites, and finally locate the target site on exon 4.

[0077] 2. The 8 groups of sgRNA designed for the 5' end target site are numbered sgRNA1-sgRNA8, and the 8 groups of sgRNA designed for the 3' end target site are numbered sgRNA9-sgRNA16. The sequences are shown in Table 3. Use the UCA activity detection method independently developed by our company to detect sgRNA activity, and the 5'...

Embodiment 3

[0086] Example 3 Production of flox rats using CRISPR-Cas9 system mRNA targeting PS1 gene

[0087] 1. Microinjection and fertilized egg transfer

[0088] Get the pronuclear fertilized eggs of SD rats, use the microinjector to inject the mixture of the transcripts of the two groups of sgRNAs obtained in the premixed embodiment 2, Cas9mRNA and the recombinant vector prepared in the embodiment 1, into the rats for fertilization In the cytoplasm of the eggs, the injected fertilized eggs were transferred to the culture medium for short-term culture, and then transplanted into the oviducts of recipient female mice to develop, and 177 embryos were transferred to obtain 32 first-established mice (namely founder mice).

[0089] The specific method for preparing transgenic rats by microinjection is as follows:

[0090] 1) Choose well-developed SD female mice aged 4-5 weeks, intraperitoneally inject 20 IU of pregnant horse serum gonadotropin (PMSG), and inject 20 IU of human chorionic g...

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Abstract

The invention provides a method for preparing flox rats for PS1 gene conditional knockout. According to the method, a conditional gene knockout carrier is built by adding Cre recombinant enzyme targeting sequence loxP loci at the two ends of a PS1 gene key exon, then a micro-injection method is conducted, and the flox rats capable of being used for PS1 gene conditional knockout are prepared. After the flox rats and Cre rats are mated, PS1 gene knockout rats with tissue specificity or inductive tissue specificity can be obtained. The conditional gene knockout method based on the CRISPR / Cas9 technology can reduce harm to other cells, and specific gene knockout can be achieved only with the need of Cre tool rats. The PS1 gene conditional knockout rats can also serve as anima models for researching the Alzheimer's disease and other diseases, and the method has important and high application value in the aspect of research of functions of the PS1 gene, particularly research of functions of the PS1 gene in nervous tissue.

Description

technical field [0001] The invention belongs to the field of animal genetic engineering and genetic modification, and in particular relates to a method for constructing a flox rat model of PS1 gene conditional knockout based on CRISPR / Cas9 technology. Background technique [0002] In the field of experimental animals, although mice are currently the more popular animal model, for many researchers, rats are still the first choice. The reason is that compared with mice, rats are larger in size and more suitable for in vivo imaging, electrophysiology, surgery, etc., and are closer to humans in terms of physiology, behavior, and metabolism. In some disease models, genetically engineered rats make up The limitations of genetically engineered mice, such as many cardiovascular diseases can be achieved in rats but not in mice, studies have shown that rats have more accurate inflammatory diseases and diseases including Parkinson's, Huntington's disease, Alzheimer's Nervous system di...

Claims

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Application Information

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IPC IPC(8): C12N15/85A01K67/027C12N15/113A61K49/00
CPCA01K67/0276A01K2217/075A01K2227/105A01K2267/0312A61K49/0008C07K14/4711C12N15/113C12N15/8509C12N2310/10C12N2800/107C12N2800/30
Inventor 沈月雷
Owner BIOCYTOGEN PHARMACEUTICALS (BEIJING) CO LTD
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