Construction method and application of axonal Charcot-Marie-Tooth drosophila model

A Charcot-Marie-Tooth disease and construction method technology, applied in the field of Drosophila model construction, to achieve the effects of simple drug feeding, rapid drug screening, and fast drug screening

Active Publication Date: 2020-07-17
FUZHOU UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no suitable simulated ATP1A1 mutant animal model that can be used for the study of the disease mechanism and the screening of related drugs.

Method used

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  • Construction method and application of axonal Charcot-Marie-Tooth drosophila model
  • Construction method and application of axonal Charcot-Marie-Tooth drosophila model
  • Construction method and application of axonal Charcot-Marie-Tooth drosophila model

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Gene editing and result identification of Drosophila ATPα mimicking human ATP1A1 amino acid mutations

[0026] Selection of Atpα mutation sites, such as figure 1 As shown, we simultaneously selected three mutations corresponding to human ATP1A1 for simulation, including p.A597T, p.P600T, and p.D601F, and the sites corresponding to the Drosophila Atpα gene were p.A576T, p.P579T, and p. D580F.

[0027] According to the genome sequence information where the site to be mutated is located, such as figure 1 As shown, a pair of CRISPR / Cas9 genome editing targets were designed, target 1: 5’-GCACGTGGGGGATCAATCA-3’ and target 2: 5’-GTCGGCACTTGGCAACGGCAT-3’. Such as figure 2 As shown, the two target sequences were respectively constructed on the pU6-BbsI-gRNA (Addgene: 45946) vector. Synthesize two target primers (forward: 5'-CTTC (target sequence)-3' and reverse: 3'-(target sequence)CAAA-5') according to the template for each target, and the The 5'-CTTC-3' and 3'-CAAA-5' ...

Embodiment 2

[0034] Pathological evaluation of the CMT2 model of Drosophila ATPα mimicking human ATP1A1 amino acid mutations

[0035] The established Drosophila model was subjected to immunostaining of the brain clock nervous system circuit and observation of circadian clock-related behaviors to evaluate pathological phenotypes. Immunostaining protocol: 3-5 day old Drosophila adults were collected and fixed in 4% paraformaldehyde fixative for 2 hours at room temperature. Dissect with pointed tweezers for dissection, remove the treated Drosophila brain from the worm body, put it into 150 μL PBST (PBS+0.5%Teiton X-100) for room temperature washing, 3 consecutive times, 15min each time . Then transfer to 150 μL PNT (PBST+10% goat serum for blocking) and incubate at room temperature for 2h for blocking. Afterwards, PNT was used to dilute the primary antibody (mouse anti-PDF 1:200, DSHB), and the blocked brain was transferred to the primary antibody at 4°C overnight. Wash 3 times with PBST a...

Embodiment 3

[0038] Example 3 Drosophila ATPα simulates the application of CMT2 model of human ATP1A1 amino acid mutation

[0039] Using the established Drosophila model, we evaluated the neurological drug oxcarbazepine. Specific scheme: firstly, the food containing the drug is prepared, and the neurological drug oxcarbazepine (O3764) from Sigma Company is dissolved in a solution containing 1‰ dimethyl sulfoxide to prepare a 120 mM stock solution, and then the final concentration is 120 mM The amount of μM is mixed with 0.75% soybean flour (g / ml), 4.5% corn flour (g / ml), 1.5% yeast (g / ml), 0.5% propionic acid (v / v), 0.1% paraben Methyl ester (g / ml), 0.02% corn syrup (v / v), 1.25% sucrose (g / ml), 1.25% glucose (g / ml), 0.5% agar (g / ml) food. The control food was prepared with normal food mixed with dimethyl sulfoxide at the corresponding concentration of drug food. Next, the control and pathological Drosophila models 2-3 days after eclosion were collected and put into the food containing th...

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Abstract

The invention relates to a construction method and application of a drosophila model for simulating human diseases, in particular to a construction method and application of a drosophila model for axonal Charcot-Marie-Tooth (CMT2). The model constructs the amino acid mutation of the endogenous corresponding human ATP1A1 gene of the Drosophila by a genome editing method, and evaluates the pathological phenotype by utilizing the changes of brain nervous system loop, biological clock related behavior, life span and exercise ability. The model and the pathological evaluation means established by the invention have the advantages of simple operation and strong repeatability, and the obtained CMT2 drosophila model can be used for researching the pathological process and mechanism of the disease,can also be used for screening and verifying medicines for delaying or treating the CMT2 disease on a large scale, and has a good application prospect.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a construction method and application of a drosophila model of axonal Charcot-Marie-Tooth disease (CMT2). Background technique [0002] Charcot-Marie-Tooth (CMT) was discovered as early as 1886, also known as hereditary motor sensory neuropathy (HMSN). CMT is an inherited peripheral nervous system disorder characterized by defects in the peripheral nervous system, including defects in sensory and motor neurons, often manifested by progressive loss of muscle tissue and the sense of touch in various parts of the body. The main clinical feature is progressive muscle weakness and atrophy of the distal extremities with sensory disturbance. The incidence of CMT is about 1 / 2500, and it is one of the most common hereditary peripheral neuropathies. According to clinical and electrophysiological features, CMT is divided into primary demyelinating neuropathy (CMT1, CMT3 and CMT4) ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/10C12N15/55A01K67/033A61K49/00
CPCC12N15/85C12N15/102C12N9/14A01K67/0339A61K49/0008C12Y306/03009C12N2800/105A01K2217/07A01K2227/706A01K2267/03
Inventor 陈文锋
Owner FUZHOU UNIV
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