Preparation and Application of Epilepsy Animal Models

Abstract

The present invention provides a method for preparing a refractory epilepsy animal model and its application. Specifically, the present invention provides a method for preparing a refractory epilepsy animal model of a non-human mammal, wherein the preparation method comprises the following steps: (1) providing a non-human mammal A and a non-human mammal B expressing 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 operably linked to the Cdkl5 gene for conditional knockout of the Cdkl5 gene, wherein, in the presence of the Cre recombinase, the conditional knockout element conditionally knocks out the Cdkl5 gene in the genome of neuron cells, thereby inactivating the Cdkl5 gene; (2) mating and breeding the animal A with the animal B to obtain a progeny non-human mammal C with neuron cell-specific knockout of the Cdkl5 gene; (3) culturing the progeny non-human mammal C to obtain the refractory epilepsy animal model. The present invention can also preliminarily evaluate the efficacy of anti-epileptic drugs according to the changes in the spontaneous epilepsy phenotype of the animals.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and more particularly to the preparation and application of an animal model of epilepsy. Background Art

[0002] Epilepsy is caused by synchronous abnormal discharge of neurons, which can lead to transient brain dysfunction.

[0003] In order to better understand the pathogenesis of epilepsy related to CDKL5 syndrome, researchers have established multiple transgenic mouse models, and these model mice exhibit many behavioral abnormalities, including phenotypes such as hindlimb clustering, changes in activity level, abnormal eye tracking, impaired learning and memory, and autistic-like social disorders. Unfortunately, however, none of these model mice have spontaneous epilepsy. And thus there is currently a lack of an animal model that mimics the refractory epilepsy of CDKL5 syndrome patients.

[0004] And currently there is no effective drug for treating epilepsy clinically, especially for the early-onset epilepsy symptoms in infants and young children of this disease, and the efficacy of traditional anti-epileptic drugs is poor. And in this field, due to the lack of a reliable animal model for in vivo drug screening, the efficiency of drug development for treating this disease has been greatly reduced.

[0005] Therefore, there is an urgent need in this field to develop a drug screening animal model that can greatly improve the efficiency and accuracy of drug research and development and reduce the risk of clinical research failure. Summary of the Invention

[0006] The object of the present invention is to provide a drug screening animal model that can greatly improve the efficiency and accuracy of drug research and development and reduce the risk of clinical research failure.

[0007] In a first aspect of the present invention, there is provided a method for preparing an animal model of refractory epilepsy in a non-human mammal, the method comprising the following steps:

[0008] (1) Provide a non-human mammal A and a non-human mammal B expressing neuron cell-specific Cre recombinase of the same species;

[0009] Wherein the genome of the non-human mammal A has: (E1) an endogenous Cdkl5 gene, and (E2) a conditional knockout element operably linked to the Cdkl5 gene and used for conditional knockout of the Cdkl5 gene, wherein, in the presence of the Cre recombinase, the conditional knockout element conditionally knocks out the Cdkl5 gene in the genome of neuron cells, thereby inactivating the Cdkl5 gene;

[0010] (2) Mate and breed the animal A with the animal B to obtain a filial non-human mammal C with neuron cell-specific knockout of the CDKL5 gene;

[0011] (3) Cultivate the offspring non-human mammal C as described above, thereby obtaining the refractory epilepsy animal model as described above.

[0012] In another preferred embodiment, the refractory epilepsy animal model has the following characteristics: characteristic refractory epilepsy brain waves.

[0013] In another preferred embodiment, the epilepsy brain waves described in the present invention are characterized by high amplitude and high frequency. Through EEG recording and analysis, they are easily distinguishable from the low-amplitude brain waves during the non-seizure period, and can also be distinguished from the single high-amplitude brain waves generated during accidental spasms in mice. This characteristic is more conducive to judging the seizure frequency of the epileptic mouse model.

[0014] In another preferred embodiment, the refractory epilepsy animal model has one or more of the following characteristics:

[0015] The duration of a single epileptic seizure is 30 - 90 seconds;

[0016] The number of seizures per day is 0 - 35 times, and as epilepsy progresses, the number of seizures gradually increases;

[0017] The characteristics of the animal model of the present invention compared with previous epilepsy models are spontaneous and high-frequency.

[0018] In another preferred embodiment, the non-human mammal A is female, and both X chromosomes have the endogenous CDKL5 gene and the conditional knockout element as described above, that is, the non-human mammal A is a homozygous female animal.

[0019] In another preferred embodiment, when the non-human mammal A is female, the offspring non-human mammal C is a first-generation offspring animal, and the offspring animal is male.

[0020] In another preferred embodiment, the non-human mammal A is male, and one X chromosome has the endogenous CDKL5 gene and the conditional knockout element.

[0021] In another preferred embodiment, when the non-human mammal A is male, the offspring non-human mammal C is a second-generation offspring animal, and the second-generation offspring animal is male or female.

[0022] In another preferred embodiment, according to Mendel's genetic law, the parental animals A and B used to breed the offspring non-human mammal C can be any combination of male and female. Among the offspring produced (first-generation and above offspring), conditional knockout mice with a spontaneous epilepsy phenotype are selected through genotype identification.

[0023] In another preferred example, the intractable epilepsy includes spontaneous intractable epilepsy, early-onset epilepsy in infants, and CDKL5 syndrome epilepsy.

[0024] In another preferred example, the neuron cells include: excitatory neuron cells.

[0025] In another preferred example, the conditional knockout of the CDKL5 gene in the genome of the neuron cells includes partial or complete knockout of the CDKL5 gene.

[0026] In another preferred example, the conditional knockout element includes loxp sequences.

[0027] In another preferred example, loxp sequences are inserted on both sides of one or more exons in the CDKL5 gene of the genome of animal A.

[0028] In another preferred example, loxp sequences are inserted on both sides of exon 6 in the CDKL5 gene of animal A.

[0029] In another preferred example, a selection marker is also inserted between the loxp sequences.

[0030] In another preferred example, the selection marker is the neo gene.

[0031] In another preferred example, the genome of animal B contains an exogenous Cre expression cassette.

[0032] In another preferred example, the exogenous Cre expression cassette includes: (a) a neuron cell-specific promoter; and (b) the Cre gene located downstream of the neuron cell-specific promoter.

[0033] In another preferred example, the Cre gene includes the gene of Cre recombinase or improved Cre recombinase (iCre).

[0034] In another preferred example, the neuron cell-specific promoter includes the promoter of the Emx1 gene or the CamK2α gene.

[0035] In another preferred example, the CDKL5 gene of animal C is specifically knocked out in neuron cells starting from a specific time point (Emx1-Cre from embryonic day 12.5, CamK2α-iCre from postnatal).

[0036] In another preferred example, the knockout includes non-expression of the CDKL5 gene, or expression of an inactive CDKL5 protein, or expression of a pathogenic mutant CDKL5 gene.

[0037] In another preferred example, the specific knockout is achieved by knocking out exon 6 of the CDKL5 gene.

[0038] In another preferred example, the non-human mammal is a rodent or a primate, preferably including mice, rats, rabbits, and monkeys.

[0039] In another preferred example, compared with littermate controls or wild-type control animals, the refractory epilepsy animal model has one or more of the following characteristics:

[0040] (a) According to the Racine epilepsy grading standard, epilepsy develops from a lower level to a higher level.

[0041] (b) Granule cell axon fibers are distributed in a band-like pattern in the molecular layer of the dentate gyrus.

[0042] (c) The frequency of epileptic seizures increases.

[0043] (d) Epilepsy ultimately leads to the death of the animal.

[0044] (e) Characteristic epileptic brain waves are generated.

[0045] (f) Behaviors typical of grand mal epileptic seizures appear.

[0046] (g) Epileptiform discharges in the brain appear.

[0047] The second aspect of the present invention provides a use of a non-human mammal model prepared by the method of the first aspect of the present invention, and the model is used as an animal model for studying the pathogenesis of refractory epilepsy.

[0048] In another preferred example, the refractory epilepsy includes spontaneous refractory epilepsy, early-onset epilepsy in infants and young children, and CDKL5 syndrome epilepsy.

[0049] The third aspect of the present invention provides a use of a non-human mammal model prepared by the method of the first aspect of the present invention, and the model is used for screening or identifying substances (therapeutic agents) that can alleviate or treat refractory epilepsy.

[0050] The fourth aspect of the present invention provides a method for screening or identifying potential therapeutic agents for treating or alleviating refractory epilepsy, including the following steps:

[0051] (a) In the test group, in the presence of a test compound, the test compound is administered to a non-human mammal model prepared by the method of the first aspect of the present invention, and the phenotype of the animal model in the test group is analyzed; and in the control group where the test compound is not administered and other conditions are the same, the phenotype of the animal model in the control group is analyzed.

[0052] (b) Compare the behaviors of the animal models in the test group and the control group. Among them, compared with the control group, if the phenotypes characterizing refractory epilepsy are improved in the animal models administered with the test compound, it indicates that the test compound can be used as a potential therapeutic agent for refractory epilepsy.

[0053] In another preferred embodiment, the phenotypes of refractory epilepsy are selected from the group consisting of: seizure frequency, the distribution of granule cell axon fibers in the molecular layer within the dentate gyrus, and typical epileptic brain waves.

[0054] In another preferred embodiment, the distribution of granule cell axon fibers in the molecular layer within the dentate gyrus includes that the granule cell axons project to the dendrites of adjacent granule cells, and the ends of granule cell axon fibers are distributed in a band-like manner in the molecular layer within the dentate gyrus.

[0055] In another preferred embodiment, the improvement of the phenotype includes: a decrease in seizure frequency, the granule cell axon fibers not projecting to the molecular layer within the dentate gyrus, and the disappearance of epileptic brain waves.

[0056] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0057] In another preferred embodiment, the method includes step (c) of administering the potential therapeutic agent screened or identified in step (b) to the non-human mammalian model prepared by the method of the first aspect of the present invention, so as to determine its effect on the phenotype of the animal model.

[0058] In another preferred embodiment, the improvement is a statistically significant improvement.

[0059] The fifth aspect of the present invention provides a non-human mammalian model prepared by the method of the first aspect of the present invention.

[0060] In another preferred embodiment, for CDKL5 gene knockout, the non-human mammalian model is heterozygous or homozygous.

[0061] In another preferred embodiment, the CDKL5 gene knockout is a specific knockout of the CDKL5 gene in neuronal cells.

[0062] The sixth aspect of the present invention provides a use of a cell in which the CDKL5 gene is conditionally knocked out for preparing a biological agent for constructing a refractory epilepsy animal model of a non-human mammal, and the cell is a neuronal cell.

[0063] In another preferred embodiment, the conditional knockout refers to specifically knocking out the CDKL5 gene in the neuronal cells using the Cre-LoxP recombinase system.

[0064] In another preferred example, the neuronal cells include excitatory neuronal cells.

[0065] In another preferred example, the biological agent is a liquid preparation.

[0066] It should be understood that within the scope of the present invention, the above-mentioned various technical features of the present invention and the various technical features specifically described hereinafter (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be elaborated one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Figure 1 (A) Seizures of different grades in epileptic mice. (B) Proportion of epileptic seizures in Cdkl5 conditional knockout mice and global knockout mice. (C) Seizure frequency of Cdkl5 conditional knockout mice.

[0068] Figure 2 It is a schematic diagram of an in vivo electrophysiological recording system.

[0069] Figure 3 It is the electroencephalogram of Emx1-cKO mice recorded, showing the changes in the electroencephalogram of the mice during epileptic seizures (lower figure), and the number of epileptic seizures of the mice can be easily seen through long-term monitoring (upper figure).

[0070] Figure 4 Using a new anti-epileptic drug can reduce the seizure frequency of epileptic mice, while there is no significant statistical difference in the seizure frequency of the control group of mice treated with the drug solvent before and after administration.

[0071] Figure 5 It is the result of Timm staining of mouse brain slices, showing that the axon fibers of granule cells in Emx1-cKO mice are distributed in a banded pattern in the molecular layer of the dentate gyrus (A-B, A'-B'), while there is no significant abnormality in VGAT-cKO mice (C-D, C'-D') and Knockout mice (E-F, E'-F') compared with the control. The statistical results show that there is a significant difference in the mossy fiber sprouting phenomenon between Emx1-cKO mice and control mice (G). DETAILED DESCRIPTION OF THE INVENTION

[0072] Through extensive and in-depth research, the present inventors have unexpectedly developed a spontaneous refractory epilepsy animal model for the first time. Specifically, the present invention provides a Cdkl5 neuronal cell (such as an excitatory neuronal cell) specific knockout animal, which can be used as a spontaneous refractory epilepsy animal model. The animal model of the present invention has an epilepsy phenotype, especially a refractory epilepsy phenotype, and can be used for the screening of anti-epileptic (especially refractory epilepsy) drugs. On this basis, the present inventors have completed the present invention.

[0073] TERMINOLOGY

[0074] Cdkl5 gene

[0075] The Cyclin-dependent kinase-like 5 (CDKL5) gene is located at locus 22 on the short arm of the X chromosome and encodes a serine / threonine kinase expressed in the brains of humans and mice. This kinase consists of an N'-terminal kinase catalytic domain and a C'-terminal amino acid sequence. This gene was discovered by Montini et al. in 1998 during the study of disease-related genes. In 2003, Kalscheuer et al. found that in two female infants with X-linked infantile spasms and intellectual disabilities, the CDKL5 gene was disrupted due to the translocation of the X chromosome and autosomes. The exactly same symptoms of these two patients strongly suggest that the absence of CDKL5 protein can lead to neurological dysfunction. According to statistics, the pathogenic CDKL5 gene mutation sites are mainly concentrated in its N'-terminal kinase catalytic domain, and the symptoms of patients carrying mutations in the kinase domain are more severe than those of patients carrying mutations in other regions.

[0076] Epilepsy

[0077] Epilepsy is a chronic disease caused by sudden abnormal discharges of neurons, resulting in transient brain dysfunction. Epidemiological data show that the overall incidence of epilepsy in China is 7.0‰, and the prevalence of active epilepsy with seizures within 1 year is 4.6‰. Epilepsy has become the second most common disease in the department of neurology after headache in China. The causes of epilepsy are complex and diverse, including genetic factors, brain diseases, etc., and its pathogenesis is also very complex.

[0078] Gene inactivation

[0079] Many methods can be used to study genes with unknown functions. For example, inactivating the gene to be studied, analyzing the phenotypic changes of the resulting genetic modifications, and then obtaining the functional information of the gene. Another advantage of this research method is that it can associate gene functions with diseases, so that while obtaining gene functions, information about the diseases that the gene can treat as a potential drug or drug target and disease animal models can also be obtained. The method of gene inactivation can be achieved by gene knockout, gene disruption or gene insertion. Among them, gene knockout technology is a very powerful means to study the functions of human genes in the whole body.

[0080] As used herein, the terms "gene inactivation" and "gene knockout" are used interchangeably and refer to genetic manipulations such as interrupting and knocking out a certain target gene, so that the expression and / or activity of the target gene are significantly decreased or even completely lost.

[0081] The present invention utilizes the "tissue-specific Cre-LoxP system" to hybridize CDKL5 conditional knockout mice (Cdkl5 flox / flox ) with excitatory neuron cell-specific Cre expression mice (Emx1-Cre or CamK2α-iCre mice) to obtain Cdkl5-specific knockout mice in excitatory neuron cells (Emx1-Cre or CamK2α-iCre / Cdkl5 flox / flox mice). Among them, loxp markers are inserted on both sides of exon 6 of the Cdkl5 gene in the genome of Cdkl5 conditional knockout mice; the genome of excitatory neuron cell-specific Cre expression mice contains an exogenous Cre expression sequence, and the exogenous Cre expression sequence includes: (a) an excitatory neuron cell-specific promoter (the promoter of the Emx1 gene or the CamK2α gene), and (b) the Cre gene located downstream of the excitatory neuron cell-specific promoter. In the present invention, at the individual level, the above two types of mice are hybridized to obtain offspring mice that specifically express Cre recombinase in excitatory neuron cells, thereby excising the Cdkl5 gene between the LoxP sites in the genome of excitatory neuron cells and achieving the specific inactivation of the Cdkl5 gene in excitatory neuron cells.

[0082] Cre recombinase was discovered from phage P1 in 1981 and belongs to the λInt enzyme supergene family. The full length of the coding region sequence of the Cre recombinase gene is 1029 bp (EMBL database accession number X03453), encoding a 38 kDa protein. Cre recombinase is a monomeric protein composed of 343 amino acids. It not only has catalytic activity, but also, similar to restriction enzymes, can recognize specific DNA sequences, namely loxP sites, so that the gene sequence between loxP sites is deleted or recombined. Cre recombinase has a 70% recombination efficiency and can act on DNA substrates of various structures, such as linear, circular, and even supercoiled DNA, without the aid of any auxiliary factors. It is a site-specific recombinase that can mediate specific recombination between two LoxP sites (sequences), so that the gene sequence between LoxP sites is deleted or recombined.

[0083] LoxP (locus of X-over P1) sequence: Derived from phage P1, it is composed of two 13 bp inverted repeat sequences and an intervening 8 bp sequence. The 8 bp spacer sequence also determines the direction of LoxP. Cre covalently binds to DNA during the catalytic DNA strand exchange process, and the 13 bp inverted repeat sequence is the binding domain of Cre enzyme.

[0084] Animal model

[0085] An animal model of human disease refers to animals with manifestations simulating human diseases established in various medical scientific research. According to the cause of generation, it can be classified into spontaneous animal models and induced or experimental animal models.

[0086] Spontaneous Animal Models refer to diseases that occur in experimental animals without any conscious artificial treatment under natural conditions. It includes genetic diseases of mutant strains and tumor disease models of inbred strains. The greatest advantage of using such animal disease models to study human diseases is that the occurrence and development of the diseases are very similar to the corresponding human diseases, both occurring under natural conditions, with higher application value. However, the source of such models is relatively difficult.

[0087] In the present invention, a spontaneous epilepsy animal model of a non-human mammal is provided, and the animal model is as described in the fifth aspect of the present invention.

[0088] Specifically, the present invention uses mice (Cdkl5 flox / flox ) whose genome has: (E1) an endogenous CDKL5 gene, and (E2) a conditional knockout element (such as the Cre recombinase targeting sequence loxp sequence) operably linked to the CDKL5 gene and used for conditional knockout of the CDKL5 gene, and hybridizes them with excitatory neuron cell-specific Cre recombinase-expressing mice (Emx1-Cre or CamK2α-iCre mice) to construct a conditional knockout mouse of Cdkl5 in excitatory neuron cells, specifically knocking out the expression of Cdkl5 in excitatory neuron cells of the mice, enabling the inventors to study the function of Cdkl5 in excitatory neuron cells at the in vivo tissue-specific level.

[0089] The present invention takes an animal model with a Cdkl5 gene knockout having a refractory epilepsy phenotype as the research object, and judges the seizure frequency of mice by recording the electroencephalogram of animals (such as mice), studies the brain pathological morphology of mice, and preliminarily judges whether the drug has an effect against CDKL5-related epileptic seizures. Given the clear phenotypic advantages of the Cdkl5 conditional knockout animal model, the method of the present invention can quickly and efficiently conduct a preliminary evaluation of the effectiveness of the drug, thereby promoting the development and marketing of the drug.

[0090] The main advantages of the present invention include:

[0091] (a) The present invention discovers for the first time that conditional knockout of Cdkl5 in excitatory neuron cells can be used to prepare an animal model of refractory epilepsy.

[0092] (b) The refractory epilepsy animal model of the present invention can be used for screening anti-epileptic drugs.

[0093] (c) The present invention uses Cdkl5 knockout mice with spontaneous refractory epilepsy phenotype as research objects, determines the frequency of epileptic seizures in mice by recording the electroencephalogram of mice, studies the pathological morphology of mouse brain, and preliminarily determines whether the drug has an effect on anti-CDKL5 related epileptic seizures. In view of the clear phenotypic advantages of Cdkl5 conditional knockout mice, this method can quickly and efficiently conduct a preliminary evaluation of the effectiveness of the drug, thereby promoting the development and marketing of the drug.

[0094] (d) The present invention discovered for the first time that mice with conditional knockout of the Cdkl5 gene in excitatory neurons reproduced the refractory epilepsy phenotype of patients with CDKL5 deficiency. Therefore, using the mouse model constructed by this method to develop and screen disease therapeutic drugs will greatly improve the efficiency and accuracy of drug research and development and reduce the risk of clinical research failure.

[0095] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually carried out under conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight. The experimental materials and reagents involved in the present invention can be obtained from commercial channels unless otherwise specified.

[0096] General Methods

[0097] 1. Breeding Methods of Cdkl5 Gene-related Epilepsy Mice

[0098] By using Cre-LoxP technology, Cdkl5 flox / flox The conditional knockout mice of the present invention were obtained by mating Cdkl5 mice (gift from Joe Zhou's laboratory) with Emx1-Cre mice (purchased from Jackson Laboratory, USA) or CamK2α-iCre mice (gift from German Cancer Research Center). flox / flox Conditional knockout mice with Cdkl5 gene knockout in excitatory neurons were generated by mating Cdkl5 mice with male mice that specifically express Cre recombinase. When they become adults, they can be used to observe the occurrence of epilepsy and screen anti-epileptic drugs.

[0099] 2. Methods for accurately recording epileptic seizure frequency using EEG

[0100] The self-made electrode was surgically installed above the pia mater of the mouse. After the mouse recovered for one week, it was connected to a long-term electroencephalogram recording system, and the electroencephalogram activity of the mouse was monitored for 24 hours. The frequency of epilepsy in the mouse was judged according to the electroencephalogram, and the frequency of seizures before and after drug administration was compared to judge the therapeutic effect of the drug on refractory epilepsy.

[0101] 3. Method for judging epilepsy occurrence in mice by Timm staining

[0102] The axon fibers of granule cells in CDKL5 conditional knockout mice with epilepsy occurred in a banded distribution in the molecular layer of the dentate gyrus. This method was used to judge whether drug treatment could prevent the occurrence of epilepsy.

[0103] Example 1 Preparation of epileptic seizure mice

[0104] Age-appropriate female Cdkl5 flox / flox mice were mated with male mice specifically expressing Cre recombinase. After the pups were born for seven days, their toes were clipped for numbering, and about 3 mm of their tails was taken for genotype identification. The mouse tails were immersed in 150 μl of mouse tail lysis buffer (containing 2 μl of 5% proteinase K solution) and incubated overnight at 55 °C; the centrifuge tube containing the lysis buffer was heated on a 95 °C metal bath for 15 min; after cooling to room temperature, it was centrifuged at 12,000 rpm for 5 min; the supernatant was taken for PCR genotype identification. The PCR reaction system was prepared with the following formula:

[0105]

[0106] The PCR cycling conditions were:

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

[0108] The PCR product was mixed with 2x loading buffer and added to 1.5% agarose gel, and electrophoresed in a 120 mV constant voltage electric field for 35 min. The results were observed and photographed in a gel imager. Conditional knockout mice with the Cdkl5 gene knocked out in excitatory neurons were identified according to the gel electrophoresis results. After they reached adulthood, they could be used to observe the occurrence of epilepsy and the screening of antiepileptic drugs.

[0109] Emx1-Cre mediates the recombination of LoxP in forebrain excitatory neuron cells at embryonic day 12.5 in mice, while CamK2α-iCre specifically knocks out the target gene in excitatory neuron cells starting from the birth of mice.

[0110] By long-term video recording, it was observed that Emx1-Cre-driven CDKL5 conditional knockout mice successively developed spontaneous epilepsy symptoms starting from 2 months old. According to the Racine epilepsy grading standard, it was clearly visible that the epilepsy in mice developed from low grade to high grade ( Figure 1 A). Further statistics showed that 80% of the Emx1-Cre-mediated conditional knockout mice (n = 15) exhibited symptoms of grand mal epilepsy ( Figure 1 B), and the seizure frequency was 1 - 23 times per day ( Figure 1 C). Among the CamK2α-iCre-driven CDKL5 conditional knockout mice (n = 15), 66.7% had grand mal epilepsy ( Figure 1 B), and the seizure frequency was 0 - 3 times per day ( Figure 1 C), which even directly led to the death of the mice. This indicates that the method of knocking out the CDKL5 gene in mice through excitatory neurons can produce mice with refractory epileptic seizures.

[0111] However, no epilepsy was observed in VGAT-Cre-mediated inhibitory neuron-specific Cdkl5 gene knockout mice and Cdkl5 global knockout mice before 6 months old ( Figure 1 B), indicating that there is likely a compensatory mechanism for CDKL5 in the early stage of embryonic development.

[0112] Example 2 Accurately judging the seizure frequency of transgenic mice by EEG recording

[0113] Through surgery, a self-made electrode was installed on the pia mater of the mice. During the surgery, the mice were anesthetized with 1.5% isoflurane and placed above the stereotaxic apparatus to keep their skulls in a horizontal position. The self-made electrode consisted of two stainless steel screws (diameter 1 mm) as electroencephalogram recording electrodes. The insertion positions into the skull were determined according to the mouse brain atlas (coordinates with the bregma as the origin: anteroposterior +1.0 mm, mediolateral +1.5 mm and anteroposterior -3.2 mm, mediolateral +1.5 mm). Two insulated silver wires (Coonerwire, #AS633) were used as electromyogram recording electrodes and were placed in the trapezius muscles on both sides respectively. The electrodes were connected to a mini connector and fixed to the skull with dental cement. Then the skin of the mice was sutured with surgical sutures to reduce direct contact with the outside world, and then the mice were placed in a warm environment until they resumed normal activities.

[0114] One week after the mice recovered, they were connected to a long-term electroencephalogram recording system ( Figure 2) First, let the mice adapt continuously for two days, and then monitor the electroencephalogram (EEG) activity of the mice for 24 hours. When synchronously recording the EEG and electromyogram (EMG) of freely moving mice, the activities of the mice are videotaped. This long-term EEG recording system includes: a two-channel amplifier (A-MSYSTEMS, Model 1800), a digital-to-analog converter (CED Ltd., Micro1401mkⅡ), and Spike2 software (CED Ltd.).

[0115] Judge the frequency of epilepsy in mice based on the EEG, compare the seizure frequencies of mice before and after drug administration, and judge the therapeutic effect of the drug on refractory epilepsy. Figure 3 The EEG shows the seizure Figure 4 The figure shows the changes in the epilepsy frequency of mice before and after administration of a certain drug.

[0116] Example 3 The method of Timm staining was used to determine that the mice had CDKL5-related epilepsy

[0117] The mossy fiber sprouting in the dentate gyrus of the hippocampus is a characteristic morphological change in patients with limbic system epilepsy and mice. Timm staining was performed on hippocampal slices of CDKL5 conditional knockout mice with epilepsy, and it was found that the axon fibers of granule cells were distributed in a band-like pattern in the molecular layer within the dentate gyrus ( Figure 5 , B-B’), while in control mice without epilepsy ( Figure 5 , A-A’, C-C’, E-E’) and VGAT-Cre-mediated cKO mice ( Figure 5 , D-D’), CDKL5 global knockout mice ( Figure 5 , F-F’), there was no phenomenon that the axon fibers of granule cells projected into the molecular layer within the dentate gyrus. The statistical results also showed that the phenomenon of ectopic projection of granule cells caused by Emx1-Cre-mediated CDKL5 conditional knockout was significantly different statistically compared with the control group ( Figure 5 G). Therefore, this method can be used to judge whether drug treatment can prevent the occurrence of epilepsy in mice with CDKL5 knockout in excitatory neuron cells.

[0118] First, anesthetize the mice by intraperitoneal injection with 8% chloral hydrate, and then sequentially perfuse the mice through the heart with 1xPBS buffer, sulfide solution (1.2% (wt / vol) Na2S·9H2O, 1% (wt / vol) NaH2PO4 in distilled water), and 4% paraformaldehyde. Then, remove the mouse brains and fix them overnight in 4% PFA. After dehydration in 15% and 30% sucrose gradients, perform cryosectioning. Horizontally section the mouse brains with a slice thickness of 30 μm. Subsequently, attach the brain slices to glass slides and let them air-dry. After rehydration with gradient ethanol, treat the brain slices with a mixed solution of gum arabic (50%, wt / vol), hydroquinone (5.67%, wt / vol), citrate-sodium citrate buffer (pH = 3.6), and silver nitrate (17%, wt / vol) in a volume ratio of 12:6:2:1, and place them in the dark at room temperature for 45 minutes. After the brain slices are significantly stained, dehydrate them with gradient ethanol, clear them with xylene, and mount them with neutral gum and let them air-dry. Take bright-field images using an Olympus VS120 microscope, then quantify the staining in Image J software, and evaluate the degree of mossy fiber sprouting using the Timm index (total area of Timm granules divided by the length of the dentate gyrus). For each animal, the Timm index is the average of the calculated values of at least three adjacent brain slices.

[0119] Comparative example

[0120] Using the method of the example of the present invention, except that male mice expressing Cre recombinase in germ cells and female Cdkl5 flox / flox mice were mated to produce Cdkl5 global knockout mice.

[0121] The results showed that the epilepsy symptoms of these mice were not obvious. Before six months, there was no typical grand mal behavior, and epileptiform discharges in the brain were not detected.

[0122] Discussion

[0123] According to existing research reports and recent research results of our laboratory, the global knockout CDKL5 model will show obvious gene compensation, making it impossible for Cdkl5 knockout mice to exhibit obvious epilepsy symptoms. Although it has been reported that mouse spasms can be recorded in old female heterozygous mice, this model mouse lacks high-amplitude and high-frequency epileptiform discharges, and the seizure time is relatively late, which is not suitable for screening anti-epileptic drugs. After specifically knocking out CDKL5 in excitatory neuron cells in the present invention, mice can produce typical epileptiform behaviors and epileptiform brain waves starting from 3 months. Therefore, it is more suitable for screening drugs for refractory epilepsy, which is an important application direction of the present invention.

[0124] All documents mentioned in this invention are cited herein by reference as if each individual document was cited by reference. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims

1. A method for preparing a refractory epilepsy animal model of a non-human mammal, characterized in that, The method includes the following steps: (1) Provide a non-human mammal A and a non-human mammal B that expresses Cre recombinase specifically in excitatory neuron cells 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 operably linked to the Cdkl5 gene for conditional knockout of the Cdkl5 gene. Among them, in the presence of the Cre recombinase, the conditional knockout element conditionally knocks out the Cdkl5 gene in the genome of excitatory neuron cells, thereby inactivating the Cdkl5 gene; (2) Mate and breed the non-human mammal A with the non-human mammal B to obtain a progeny non-human mammal C with specific knockout of the CDKL5 gene in excitatory neuron cells; (3) Cultivate the progeny non-human mammal C to obtain the refractory epilepsy animal model.

2. The preparation method according to claim 1, wherein The refractory epilepsy animal model has the following characteristics: characteristic refractory epilepsy brain waves.

3. The preparation method according to claim 1, characterized in that The refractory epilepsy animal model has one or more of the following characteristics: The duration of a single epileptic seizure is 30 - 90 seconds; The number of seizures per day is 0 - 35 times, and as epilepsy progresses, the number of seizures gradually increases; The characteristics of the refractory epilepsy animal model are spontaneous.

4. The preparation method according to claim 1, characterized in that, The refractory epilepsy includes spontaneous refractory epilepsy, early-onset epilepsy in infants and young children, and CDKL5 syndrome epilepsy.

5. The preparation method according to claim 1, characterized in that, The conditional knockout of the CDKL5 gene in the genome of neuron cells includes partial or complete knockout of the CDKL5 gene.

6. The preparation method according to claim 1, characterized in that, Loxp sequences are inserted on both sides of one or more exons in the CDKL5 gene of the genome of the non-human mammal A.

7. The preparation method according to claim 1, characterized in that, The genome of the non-human mammal B contains an exogenous Cre expression cassette.

8. The preparation method according to claim 7, characterized in that, The exogenous Cre expression cassette includes: (a) a neuron cell-specific promoter; and (b) a Cre gene located downstream of the neuron cell-specific promoter.

9. The preparation method according to claim 8, wherein, The Cre gene includes the gene of Cre recombinase or improved Cre (iCre).

10. The preparation method according to claim 8, characterized in that, The neuron cell-specific promoter includes the promoter of the Emx1 gene or the CamK2α gene.

11. The preparation method according to claim 1, wherein, Compared with littermate controls or wild-type control animals, the refractory epilepsy animal model has one or more of the following characteristics: (a) According to the Racine epilepsy grading standard, epilepsy develops from a lower level to a higher level; (b) Granule cell axon fibers are distributed in a banded pattern in the molecular layer of the dentate gyrus; (c) The frequency of epileptic seizures increases; (d) Epilepsy ultimately leads to the death of the animal; (e) Characteristic epilepsy brain waves are generated; (f) Behaviors typical of grand mal seizures appear; (g) Epileptiform discharges in the brain appear.

12. Use of a refractory epilepsy animal model prepared by the preparation method according to claim 1, characterized in that, The refractory epilepsy animal model is used to study the pathogenesis of refractory epilepsy.

13. Use of a refractory epilepsy animal model prepared by the preparation method according to claim 1, characterized in that, The refractory epilepsy animal model is used to screen or identify therapeutic agents that can alleviate or treat refractory epilepsy.

14. A method for screening or identifying potential therapeutic agents for treating or alleviating refractory epilepsy, characterized in that, It includes the following steps: (a) In the test group, in the presence of the test compound, the test compound is administered to a refractory epilepsy animal model prepared by the method according to claim 1, and the phenotype of the refractory epilepsy animal model in the test group is analyzed; and in a control group where the test compound is not administered and other conditions are the same, the phenotype of the refractory epilepsy animal model in the control group is analyzed; (b) The behaviors of the refractory epilepsy animal models in the test group and the control group are compared. Among them, compared with the control group, if the phenotype characterizing refractory epilepsy in the refractory epilepsy animal model administered with the test compound is improved, it indicates that the test compound can be used as a potential therapeutic agent for refractory epilepsy.

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