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Preparation and application of epilepsy animal model

A technology of epilepsy animals and animal models, applied in the direction of DNA / RNA fragments, recombinant DNA technology, hydrolytic enzymes, etc., can solve the problems of reduced efficiency, lack of refractory epilepsy in patients with simulated CDKL5 syndrome, and poor efficacy of antiepileptic drugs

Pending Publication Date: 2022-04-12
CENT FOR EXCELLENCE IN BRAIN SCI & INTELLIGENCE TECH CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Unfortunately, none of these model mice developed spontaneous epilepsy
And therefore there is currently a lack of animal models simulating refractory epilepsy in patients with CDKL5 syndrome
[0004] And currently there is no effective drug for the treatment of epilepsy, especially for infants with early-onset epilepsy symptoms, traditional antiepileptic drugs are less effective
And in this field, due to the lack of reliable animal models for in vivo drug screening, the efficiency of drug development for the treatment of this disease has been greatly reduced

Method used

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  • Preparation and application of epilepsy animal model
  • Preparation and application of epilepsy animal model
  • Preparation and application of epilepsy animal model

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0103] Example 1 Preparation of epileptic seizure mice

[0104] Age-appropriate female Cdkl5 flox / flox The mice were mated with male mice specifically expressing Cre recombinase, and the toes of the pups were numbered seven days after birth, and the tails of about 3 mm were taken for genotype identification. Submerge the mouse tail in 150ul of mouse tail lysis solution (containing 2ul 5% proteinase K solution), incubate overnight at 55°C; place the centrifuge tube containing the lysis solution on a metal bath at 95°C and heat for 15min; cool to After room temperature, centrifuge at 12000rpm / min for 5min; take the supernatant for PCR identification of genotype. Prepare the PCR reaction system, the formula is as follows:

[0105]

[0106] The PCR cycling conditions were:

[0107] 95°C for 15s, 60°C for 15s, 72°C for 45s, for 35 cycles.

[0108] The PCR product and 2x sample buffer were mixed and added to 1.5% agarose gel, electrophoresed in a constant voltage electric fie...

Embodiment 2

[0112] Example 2 Accurately determine the frequency of epileptic seizures in transgenic mice by means of EEG recording

[0113] The self-made electrode was installed on the mouse pia mater by surgery. During the operation, the mouse was anesthetized with 1.5% isoflurane and placed above the stereotaxic position so that the skull was in a horizontal position. The self-made electrode consists of two stainless steel screws (diameter 1mm), which are used as EEG recording electrodes, and the position of insertion into the skull is determined according to the mouse brain atlas (the coordinates of the bregma as the origin: front-to-back +1.0mm, mid-lateral +1.5mm and front-to-back- 3.2mm, mediolateral +1.5mm). Two insulated silver wires (Coonerwire, #AS633) were used as EMG recording electrodes, which were placed in the trapezius muscles on both sides. The electrodes are attached to miniature connectors and secured to the skull with dental cement. The mice's skin was then closed wi...

Embodiment 3

[0116] The method of embodiment 3 Timm staining determines that CDKL5-related epilepsy has occurred in mice

[0117] Mossy fiber sprouting in the dentate gyrus of the hippocampus is a characteristic morphological alteration in limbic epilepsy patients and mice Timm staining was performed on the hippocampal slices of CDKL5 conditional knockout mice with epilepsy, and it was found that the axon fibers of granule cells were distributed in bands in the molecular layer of the dentate gyrus ( Figure 5 , B-B'), while the control mice without epilepsy ( Figure 5 , A-A', C-C', E-E') and VGAT-Cre-mediated cKO mice ( Figure 5 , D-D'), CDKL5 whole body knockout mice ( Figure 5 , F-F') There was no phenomenon of granule cell axon fibers projecting to the inner molecular layer of the dentate gyrus. The statistical results also showed that the ectopic projection of granulosa cells caused by the conditional knockout of CDKL5 mediated by Emx1-Cre had a statistically significant differen...

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Abstract

The invention provides preparation and application of an intractable epilepsy animal model. Specifically, the invention provides a preparation method of an intractable epilepsy animal model of a non-human mammal, and the preparation method comprises the following steps: (1) providing a non-human mammal A and a non-human mammal B expressed by neuron cell specific Cre recombinase of the same species; wherein the genome of the non-human mammal A has (E1) an endogenous Cdkl5 gene and (E2) a conditional knockout element which is operatively connected with the Cdkl5 gene and is used for conditional knockout of the Cdkl5 gene, and the conditional knockout element is used for conditional knockout of the Cdkl5 gene of the genome of neuronal cells in the presence of the Cre recombinase, so that the Cdkl5 gene is inactivated; (2) carrying out mating breeding on the animal A and an animal B to obtain a filial generation non-human mammal C of which the Cdkl5 gene is specifically knocked out in neuronal cells; and (3) culturing the filial generation non-human mammal C to obtain the intractable epilepsy animal model. According to the invention, the curative effect of the anti-epilepsy drug can be preliminarily evaluated according to the change of spontaneous epilepsy phenotype of animals.

Description

technical field [0001] The invention relates to the field of biotechnology, more specifically to the preparation and application of epilepsy animal models. Background technique [0002] Seizures are caused by abnormal firing of neurons in sync, leading to transient brain dysfunction. [0003] In order to better understand the pathogenesis of epilepsy associated with CDKL5 syndrome, the researchers established multiple transgenic mouse models, which showed many behavioral abnormalities, including hindlimb huddling, changes in activity level, abnormal eye tracking, learning and memory phenotypes such as impaired and autistic-like social impairments. Unfortunately, none of these model mice developed spontaneous epilepsy. And thus there is currently a lack of animal models simulating treatment-resistant epilepsy in patients with CDKL5 syndrome. [0004] And currently there is no effective drug for the treatment of epilepsy clinically, especially for infants with early-onset e...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01K67/033C12N9/22C12N15/11
Inventor 熊志奇王红涛程学文朱姊艾
Owner CENT FOR EXCELLENCE IN BRAIN SCI & INTELLIGENCE TECH CHINESE ACAD OF SCI
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