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Methods and applications for constructing disease models based on gene manipulation strategies

A technology of disease model and genetic manipulation, applied in other methods of inserting foreign genetic materials, animal husbandry, stably introducing foreign DNA into chromosomes, etc., can solve problems such as research, clinical typing difficulties, and diagnosis difficulties

Active Publication Date: 2021-07-16
浙江鼎益生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Moreover, its clinical involvement is wide, and there is a large overlap of clinical phenotypes among various types, making clinical typing very difficult. Therefore, the final diagnosis of cerebellar ataxia must rely on genetic testing
However, due to the large number of types and the complexity of the pathogenic genes, it brings great difficulties to the diagnosis
At present, there is still a lack of specific and effective treatments for cerebellar ataxia, the main reason being the lack of detailed pathogenesis studies

Method used

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  • Methods and applications for constructing disease models based on gene manipulation strategies
  • Methods and applications for constructing disease models based on gene manipulation strategies
  • Methods and applications for constructing disease models based on gene manipulation strategies

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0078] Mutation of mouse Emc3 gene by CRISPR / Cas9 technology

[0079] 1 Design the gRNA sequence targeting knockout of the Emc3 gene, and obtain the F0 generation of Emc3 gene conditional knockout mice. The gRNA sequence was designed for the second exon region of the mouse Emc3 gene: 5'-CCTTTGTGAAAACCTGTGTGGCA-3'. Synthesize RNA in vitro, microinject into mouse fertilized eggs together with Cas9 protein and repair DNA template, and obtain the first founder mouse (founder) with gene mutation; the schematic diagram of CRISPR design is as follows figure 1 shown. Repair DNA template sequence: the DNA repair template sequence is as follows (SEQ ID NO.1):

[0080]CcttcctcagaaacctttatttgttcagagcccagtcactctggaagactgccttggatccATAACTTCGTATAATGTATGCTATACGAAGTTATggctgtgaaaacctgtgtggcaagttgaatatgtttcattgatttttagggtttttggaaagtttccacacccagcttttgccaaccaggtcctcccttatgtctgagtgggccttctactttacgcttgctttctttgcgtttcacattttaacatctctttgtcttgtgtttttcagTCAGGTCCTAATTCGAAGCAGAGTCCTCAGGGAAAATGGAAAATACATT...

Embodiment 2

[0082] Knockout of mouse Emc3 gene by Cre-loxp gene knockout technology

[0083] 1. Using mice as the target animals, the Cre-loxp conditional knockout system was used to construct a mouse model of retinal neovascular disease.

[0084] (1) mice carrying the loxp sequence (Emc3 loxp / loxp ) is mated with a tool mouse carrying Pdgfb-Cre specifically expressed in vascular endothelial cells to obtain a mouse carrying loxp sequence and Cre at the same time, expressed as Emc3 loxp / + -Pdgfb-Cre, followed by Emc3 loxp / loxp with Emc3 loxp / + -Pdgfb-Cre mice were mated to obtain Emc3 loxp / loxp -Pdgfb-Cre mice ( figure 2 A, image 3 ).

[0085] The mouse Emc3 genome sequence is shown in SEQ ID NO.2. Among them, the 501-825th position is the first exon, the 9346-9403rd position is the second exon, the 9746-9839th position is the third exon, and the 11572-11676th position is the fourth exon, The 12157-12238 position is the fifth exon, the 14192-14271 position is the sixth exon, the ...

Embodiment 3

[0109] Staining of frozen sections of eyeballs and separation of hyaloids.

[0110] (1) Eyeball frozen section preparation: After the mice were killed, the eyeballs were put into 4% PFA solution and fixed at room temperature for 20 minutes, then a hole was made in the center of the cornea with a 30G needle, and the entire cornea was cut off with ophthalmic scissors. The fixed eyeballs were rinsed once with 1×PBS, placed in 30% 1×PBS sucrose solution, and dehydrated at 4°C for 2 hours. Then the lens of the eyeball was taken out, the water was blotted, and the eyeball was placed in a frozen section embedding box equipped with OCT in the same direction, and after being solidified at -80°C, it was stored at -20°C. All sections were 10 μm thick.

[0111](2) Vitreous vessel separation: Take mouse eyeballs, place them in a 24-well plate filled with 4% PFA and fix them at room temperature for 15 minutes, then perforate the cornea and completely remove the corneas, and continue to fix...

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Abstract

The invention discloses a method and application for constructing a disease model based on a gene manipulation strategy, and relates to the field of biotechnology. In the method, the Emc3 gene is not expressed in the vascular endothelial cells or the cerebellar Purkinje cells of the target animal through a gene manipulation technique. Using this method, a disease animal model showing typical characteristics of retinal neovascular disease or cerebellar ataxia disease can be obtained. This method provides a new idea or strategy for constructing a retinal neovascular disease model or a cerebellar ataxia disease model. In addition, the disease model obtained by this method also provides a model basis for researchers to study retinal neovascular diseases and screen drugs for the treatment of retinal neovascular diseases or cerebellar ataxia diseases.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method and application for constructing a disease model based on a gene manipulation strategy. Background technique [0002] Retinal neovascular disease is one of the serious blinding eye diseases in my country and even in the world. It mainly refers to the blinding vitreoretinal disease caused by the growth of new blood vessels accompanied by pathological changes such as hemorrhage, exudation, and proliferation. Including diabetic retinopathy, retinal vascular occlusive disease, retinal periphlebitis, retinopathy of prematurity, etc., the threat to the visual health of the Chinese population has become increasingly prominent. For a long time, there are different opinions on the mechanism of retinal neovascularization. Therefore, it is of great clinical significance to carry out research on the immunological mechanism related to retinal neovascular diseases in order to find target...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/90
CPCA01K67/02
Inventor 朱献军朱雄杨牧李姝锦杨业明刘文静孙宽祥
Owner 浙江鼎益生物科技有限公司
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