Construction method, construction kit and application of optic atrophy animal model

A technology of optic atrophy and animal models, applied in biochemical equipment and methods, animal/human peptides, chemical instruments and methods, etc., can solve the problems of lack of animal models of optic atrophy, achieve rich categories, short modeling time, and promote The effect of research and development

Active Publication Date: 2018-12-18
EAST CHINA NORMAL UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] Although there are dozens of pathogenic genes that cause hereditary optic atrophy due to mutations, the existing animal models are only OPA1 (Q285STOP) mice and OPA3 (L122P) mice induce

Method used

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  • Construction method, construction kit and application of optic atrophy animal model
  • Construction method, construction kit and application of optic atrophy animal model
  • Construction method, construction kit and application of optic atrophy animal model

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Experimental program
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Embodiment

[0045] In this embodiment, mice are taken as an example to illustrate the method for constructing the mouse model of optic atrophy in this embodiment. The specific operation is as follows:

[0046] (1) Using the CRISPR design tool (online design tool http: / / tools.genome-engineering.org), select the CRISPR / Cas system knockout target, that is, the 18bp DNA ending with NGG as the target sequence: CATCTTCATTGTGGG CGG (5'-3'); the underline is the position of the PAM motif.

[0047] (2) For the above-mentioned target, an oligonucleotide chain with a T7 promoter sequence and a 60bp target sequence (ie, the sgRNA precursor sequence) synthesized in vitro, the sequence of the sgRNA precursor sequence is as follows:

[0048] GATCACTAATACGACTCACTATAGG CATCTTCATTGTGGGCGG GTTTTAGAGCTAGAAAT; target sequence is underlined.

[0049] (a) Using this as a template, perform PCR reaction with high-fidelity KOD enzyme to obtain a DNA template that can transcribe sgRNA.

[0050] PCR reaction s...

experiment example

[0074] Phenotypic Characterization of a Mouse Animal Model of Optic Atrophy

[0075] (1) Measurement of visual evoked potential (VEP)

[0076] Take the mice under the natural light adaptation state, respectively connect the recording electrode to the cheek, the reference electrode to the cranium, and the ground electrode to the tail, and use the visual electrophysiological recorder (VEP) to measure the visual evoked potential, and detect the waveform (N1 wave and P1 wave ) is abnormal.

[0077] The result is as figure 2 As shown, heterozygous OPA3 G93S / + and homozygous OPA3 G93S / G93S Both animal models showed abnormal visual evoked potential (VEP), and the amplitude difference between N1 wave and P1 wave decreased.

[0078] (2) Ex vivo tissue detection: After the mice were sacrificed, the eyeball and optic nerve were bluntly separated, the scissors went deep into the deep orbit, cut off the optic nerve, and the eyeball and optic nerve were taken out.

[0079] (a) Retinal...

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Abstract

The invention discloses a construction method, a construction kit and an application of an optic atrophy animal model, and relates to the biotechnical field. The construction method is characterized in that a base at the 277th locus of an exon 2 of an animal OPA3 gene mutates from G to A to cause missense mutation, an amino acid residue mutating from G to S, of the 93rd position of an encoded OPA3protein, and an animal containing the mutation exhibits typical optic atrophy disease characteristics and can be used as the optic atrophy animal model. The animal model fills a gap in human autosomal dominant optic atrophy animal models, enriches the type of existing optic atrophy animal models, and facilitates the development of researches on ophthalmic diseases.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a construction method, kit and application of an animal model of optic atrophy. Background technique [0002] The human OPA3 gene is located at 19q13.32, and the OPA3 protein encoded by it is one of the mitochondrial fusion proteins. This gene has multiple mutation sites, such as IVS1-1G-C, 18-BP DEL, and 313C-G(Q105E). The first two mutations lead to clinical disease type III 3-methylglutaconic aciduria, manifested as symptoms of central nervous system diseases such as rigidity, ataxia, and cognitive impairment, and are autosomal recessive genetic diseases; the third This mutation causes optic atrophy with or without cataracts and mild neurological symptoms, and is an autosomal dominant genetic disease. [0003] Although there are dozens of pathogenic genes that cause hereditary optic atrophy due to mutations, the existing animal models are only OPA1 (Q285STOP) mice and OPA3 (L122...

Claims

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Application Information

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IPC IPC(8): C12N15/85A01K67/027
CPCA01K67/0275A01K2267/03C07K14/47C12N2800/80C12N2810/10
Inventor 田颖李大力刘明耀邵艳姣尹树明席在喜
Owner EAST CHINA NORMAL UNIV
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