Method for constructing retinal neovascularization disease animal model by using gene manipulation technology, cultivation method and application

An animal model and new blood vessel technology, applied in the field of gene editing, can solve the problem of lack of animal models of retinal new blood vessel disease, and achieve the effect of ensuring sufficient supply

Active Publication Date: 2020-08-07
SICHUAN PROVINCIAL PEOPLES HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] Currently, there is a lack of animal models ...

Method used

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  • Method for constructing retinal neovascularization disease animal model by using gene manipulation technology, cultivation method and application
  • Method for constructing retinal neovascularization disease animal model by using gene manipulation technology, cultivation method and application
  • Method for constructing retinal neovascularization disease animal model by using gene manipulation technology, cultivation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] In this example, mouse is used as the target animal, and the Cre-loxp conditional knockout system is used to construct an animal model of retinal neovascular disease, and the method is as follows:

[0041] (1) mice carrying the loxp sequence (Ctnnd1 loxp / loxp , the loxp sequence is located on both sides of the 7th exon, the knockout target sequence is the 7th exon of the Ctnnd1 gene, named Ctnna1cKO mouse, purchased from Mutant MouseResource&Research Center at University of Californian at Davis, USA) and carrying Pdgfb- Cre tool mice (PDGFβ-cre transgenic mice, specifically expressing Cre in vascular endothelial cells) were mated to obtain mice carrying loxp sequences and Cre at the same time, expressed as Ctnnd1 loxp / + -Pdgfb-Cre, followed by Ctnnd1 loxp / loxp with Ctnnd1 loxp / + -Pdgfb-Cre mating to obtain Ctnnd1 loxp / loxp -Pdgfb-Cre mice (breeding ideas reference figure 1 Middle A and figure 2 ).

[0042] (2) Ctnnd1 loxp / loxp -Pdgfb-Cre mice were injected intra...

experiment example

[0048] The knockout mouse Ctnnd1 of embodiment 1 iECKO / iECKO Phenotypic identification of

[0049] 1 Western blotting was used to detect the expression of Ctnnd1 protein in the lung tissue of the target animal on day 25 after birth.

[0050] (1) Harvest the lung tissues of littermate wild-type and knockout-type target mice, put them in 1xPBS containing protease inhibitors, and ultrasonically lyse them on ice for 10 minutes; centrifuge at 4°C for 10 minutes at a speed of 16000g, and take the supernatant Transfer to another clean centrifuge tube, add protein loading buffer, mix well and heat at 95°C for 5min.

[0051] (2) After the samples were cooled, 20 μl of protein samples were taken respectively, and electrophoresis was performed at a constant voltage of 70v for 25 minutes and at 160v for 40 minutes with 10% separating gel.

[0052] (3) The membrane transfer conditions were as follows: transfer at a constant flow of 0.3A for 1 hour and 30 minutes, then wash the membrane o...

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Abstract

The invention discloses a method for constructing a retinal neovascularization disease animal model by using a gene manipulation technology, a cultivation method and application, and relates to the technical field of gene editing. According to the construction method disclosed by the invention, the Ctnnnd1 gene in the vascular endothelial cells of the target animal is not expressed or the expression of the Ctnnd1 gene is inhibited by utilizing a gene manipulation technology. By means of the method, the animal model of the retinal neovascularization disease can be obtained, the variety of the animal model of the retinal neovascularization disease is enriched, more diversified model selections are provided for related research of the retinal neovascularization disease, and scientific research of the retinal neovascularization disease, development of related treatment drugs and the like are promoted.

Description

technical field [0001] The present invention relates to the technical field of gene editing, in particular, to a method, breeding method and application for constructing an animal model of retinal neovascular disease using gene manipulation technology. Background technique [0002] Retinal neovascularization (RNV)-related eye diseases are a group of diseases characterized by pathological retinal neovascularization, mainly including proliferative diabetic retinopathy (PDR), retinopathy of prematurity (retinopathy) ofprematurity, ROP) and so on. This type of disease has a high incidence rate and poor treatment prognosis, so it has a high blinding rate and seriously endangers human vision health. At present, there is no more effective treatment for RNV clinically except laser photocoagulation in the early stage of the disease and intraocular injection of anti-vascular endothelial growth factor (VEGF) drugs. Moreover, studies have shown that anti-VEGF therapy is not effective ...

Claims

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Application Information

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IPC IPC(8): C12N15/90A01K67/027
CPCA01K67/0276A01K2217/075A01K2227/105A01K2267/03C12N15/907
Inventor 朱献军杨正林李姝锦杨牧
Owner SICHUAN PROVINCIAL PEOPLES HOSPITAL
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