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Construction method of congenital cataract disease animal model and application

A congenital cataract, animal model technology, applied in the field of biological gene editing, can solve problems such as complex genetic patterns, lack of animal models of lens degenerative diseases, and increased difficulty in the study of pathogenic molecular mechanisms.

Active Publication Date: 2021-04-06
中国人民解放军陆军特色医学中心
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The reasons for this problem mainly include the following aspects: 1) Due to the scope of medical ethics and the randomness of the disease, human congenital cataract embryos cannot be obtained
2) Strong heterogeneity of clinical manifestations
3) Many pathogenic genes and complex genetic patterns
4) The pathogenic molecular mechanism is complex
It has been found that the pathogenic genes of congenital cataract participate in the lens and related cell biochemical processes are very complex, such as cytoskeleton, ion channel, RNA splicing, endoplasmic reticulum pressure, etc. come many obstacles
At present, there is still a lack of animal models of lens degenerative diseases, which brings inconvenience to further in-depth and systematic research on the diseases

Method used

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  • Construction method of congenital cataract disease animal model and application
  • Construction method of congenital cataract disease animal model and application
  • Construction method of congenital cataract disease animal model and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Figure 6 A schematic diagram of the design of the CRISPR targeting sequence provided for the embodiments of the present invention, such as Figure 6As shown, in this example, CRISPR-Cas9 technology is used to point-mutate the GJA8(c.134G>T) gene to construct an animal model of congenital cataract disease. The specific steps are as follows:

[0034] 1. Design the gRNA sequence targeting the point mutation GJA8(c.134G>T) gene, and obtain the F0 generation GJA8(c.134G>T) point mutation mice

[0035] 1) Design the gRNA target site sequence for the second exon region of the mouse GJA8(C.134G>T) gene, as follows:

[0036] 5'-CCTGTATCACATGCGGCTCTCGG-3'(SEQ ID NO.1); RNA synthesized in vitro, microinjected into mouse fertilized eggs together with Cas9 endonuclease, DTA and Neo cassette (SDA-Neo-SDA) is the plasmid backbone Self-contained, just bring the homology arm and cKO region into the skeleton, such as Figure 7 shown; the primers used to connect the transformed fragme...

Embodiment 2

[0048] Using the genomic DNA of the mouse, the fragment near the CRISPR target site was sequenced, and it was determined that the first builder mouse had a frameshift mutation that affected the reading frame.

[0049] details as follows:

[0050] (1) Extract samples for amplification

[0051] ①Cut a little tissue sample from the tail tip of the founder mouse obtained in step 1, and place it in a clean 1.5ml centrifuge tube;

[0052] ② Add 100 μl lysate solution (40mM NaOH, 0.2mM EDTA solution) to the centrifuge tube, and heat in a metal bath at 100°C for 1 hour;

[0053] ③ Take out the centrifuge tube, cool to room temperature, add 100 μl of neutralizing solution (40 mM Tris-HCl, pH 5.5), centrifuge at 10,000 g for 2 min, and take the supernatant for identification of mouse genotype.

[0054] (2) Mouse genotype identification

[0055] ①PCR amplification: Configure the PCR reaction system according to the following system.

[0056] ②Purification:

[0057] The amplified PCR...

Embodiment 3

[0078] (1) Obtain F1 generation GJA8 (C.134G>T) gene knockout mice

[0079] Mate the mutant founder mice with wild-type mice to obtain the F1 generation of mutant heterozygous mice (WT / mut), and perform DNA sequencing to confirm that the target gene has occurred image 3 The target protein is inactivated by the frameshift mutation, and the GJA8(C.134G>T) gene knockout is realized.

[0080] (2) Obtain F2 generation GJA8 (C.134G>T) gene mutant mice

[0081] The mutant heterozygous mice (WT / mut) with the same genotype were mated with each other to obtain the F2 generation of GJA8 (C.134G>T) gene mutant homozygous mice (mut / mut).

[0082] (3) Obtain F1' generation GJA8 gene knockout mice

[0083] The above F2 generation GJA8 (C.134G>T) mut / mut mice were crossed with Cre tool mice to obtain F1' generation GJA8 (C.134G>T) mut / mut; WT / KO heterozygous mice, GJA8(C.134G>T)mut / mut; WT / KO heterozygous mice were mated to obtain F2' generation GJA8 KO / KO Homozygous non-mutant mice.

...

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Abstract

The invention relates to a construction method of a congenital cataract disease animal model, which comprises the steps of firstly, designing a gRNA target site sequence for a second exon region of a mouse GJA8 gene, amplifying a homologous arm and a cKO region, and respectively connecting the homologous arm and the cKO region to a vector; injecting positive clones into blastula by electroporation ES cells of the constructed plasmids, transferring the blastula into a mouse body, and obtaining a first builder mouse after a birth mouse is mature; mating with a wild type mouse to obtain a heterozygote mouse F1 generation with gene mutation; finally, mating the F1 generation of heterozygote mice with the same genotype to obtain F2 generation of gene mutation homozygote mice. According to the construction method of the congenital cataract disease animal model provided by the invention, typical characteristics of congenital cataract, such as crystalline lens degeneration opacity and crystalline lens epithelial apoptosis, can be obtained, the animal model can be used for researching congenital cataract diseases and screening medicines for treating the congenital cataract diseases, and application prospect is promising.

Description

technical field [0001] The invention belongs to the technical field of biological gene editing, and relates to a method for constructing an animal model of congenital cataract disease and its application. Background technique [0002] Congenital cataract refers to the crystal opacity that occurs at birth or shortly after birth. Its incidence rate is 0.6-6 / 10000 population. It is an important cause of blindness and amblyopia in children, accounting for the second blinding eye disease in children. Based on the huge population of our country, the number of congenital cataract patients can reach one million, which brings a heavy burden to the family and society. Although congenital cataract surgery techniques continue to improve, and the success rate continues to increase. However, children's ocular anatomy, physiological characteristics, and postoperative inflammatory response are more complex than those of adults, and postoperative complications such as amblyopia and hindranc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85A01K67/027A61K49/00
CPCC12N15/8509A01K67/0276C07K14/47A61K49/0008C12N2800/107C12N2800/30C12N2999/007A01K2217/075A01K2227/105A01K2267/03
Inventor 林森刘闻一李雪罗琳林叶剑剑
Owner 中国人民解放军陆军特色医学中心
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