CN114304068A - Preparation and application of epilepsy animal model - Google Patents

Abstract

The invention provides preparation and application of an intractable epilepsy animal model. Specifically, the invention provides a preparation method of a non-human mammal animal model of refractory epilepsy, wherein the preparation method comprises the following steps: (1) providing a non-human mammal A and a non-human mammal B expressed by a neuron cell-specific Cre recombinase of the same species; wherein the genome of said 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 said conditional knockout element conditionally knocks out the Cdkl5 gene of the genome of the neuronal cell in the presence of said Cre recombinase, thereby inactivating the Cdkl5 gene; (2) mating and breeding the animal A and the animal B to obtain a progeny non-human mammal C with a Cdkl5 gene specifically knocked out in neuron cells; (3) the offspring non-human mammal C is cultured to obtain the intractable epilepsy animal model, and the curative effect of the antiepileptic drug can be preliminarily evaluated according to the change of the spontaneous epilepsy phenotype of the animal.

Description

Preparation and application of epilepsy animal model

Technical Field

The invention relates to the technical field of biology, in particular to preparation and application of an epilepsia animal model.

Background

Epilepsy is caused by abnormal firing of neurons in synchrony, resulting in transient cerebral dysfunction.

To better understand the pathogenesis of epilepsy associated with the CDKL5 syndrome, researchers have established transgenic mouse models that exhibit a number of behavioral abnormalities, including phenotypes such as hind limb clasping, altered activity, abnormal eye tracking, impaired learning and memory, and autistic-like social disturbance. Unfortunately, none of these model mice had spontaneous epileptic development. And therefore there is currently a lack of animal models that mimic the refractory epilepsy of patients with CDKL5 syndrome.

At present, no effective medicine for treating epilepsy exists clinically, especially for early onset epilepsy of infants with the epilepsy, and the traditional antiepileptic medicine has poor curative effect. And because reliable animal models are lacked for in vivo drug screening in the field, the efficiency of drug development for treating the disease is greatly reduced.

Therefore, there is an urgent need in the art to develop a drug screening animal model that can greatly improve the efficiency and accuracy of drug development and reduce the risk of clinical study failure.

Disclosure of Invention

The invention aims to provide a drug screening animal model which can greatly improve the efficiency and accuracy of drug research and development and reduce the risk of clinical research failure.

In a first aspect of the present invention, there is provided a method of preparing a non-human mammal animal model of refractory epilepsy, the method comprising the steps of:

(1) providing a non-human mammal A and a non-human mammal B expressed by a neuron cell-specific Cre recombinase of the same species;

wherein the genome of said 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 said conditional knockout element conditionally knocks out the Cdkl5 gene of the genome of the neuronal cell in the presence of said Cre recombinase, thereby inactivating the Cdkl5 gene;

(2) mating and breeding the animal A and the animal B to obtain a progeny non-human mammal C with the CDKL5 gene specifically knocked out in neuron cells;

(3) culturing said progeny in a non-human mammal C, thereby obtaining said animal model of refractory epilepsy.

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

In another preferred embodiment, the epileptic brain waves have the characteristics of high amplitude and high frequency, and can be easily distinguished from low-amplitude brain waves in the non-seizure period and single high-amplitude brain waves generated when a mouse is accidentally spastic through EEG recording analysis, and the characteristics are more favorable for judging the frequency of epileptic seizure of an epileptic mouse model.

In another preferred embodiment, the animal model of refractory epilepsy 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, and gradually increases with the progress of epilepsy;

the animal model of the invention is spontaneous and high frequency compared to previous models of epilepsy.

In another preferred example, said non-human mammal a is female and both X chromosomes have said endogenous CDKL5 gene and said conditional knock-out element, i.e. said non-human mammal a is a homozygous female.

In another preferred embodiment, when the non-human mammal a is female, the progeny non-human mammal C is a first generation progeny, and the progeny is male.

In another preferred embodiment, said non-human mammal a is male and has said endogenous CDKL5 gene and said conditional knockout element on one X chromosome.

In another preferred embodiment, when the non-human mammal a is male, the progeny non-human mammal C is a second generation progeny animal, and the second generation progeny animal is male or female.

In another preferred embodiment, both parent animal A and animal B used to breed offspring non-human mammal C can be any combination of female and male, and among the resulting offspring (first and beyond), conditional knock-out mice with spontaneous epileptic phenotype are selected by genotyping according to Mendelian's law of inheritance.

In another preferred example, the refractory epilepsy comprises spontaneous refractory epilepsy, infantile early onset epilepsy, CDKL5 syndrome epilepsy.

In another preferred embodiment, the neuronal cells comprise: an excitatory neuronal cell.

In another preferred example, the CDKL5 gene of the genome of the conditional knockout neuron cell comprises a partial or complete knockout CDKL5 gene.

In another preferred embodiment, the conditional knockout element comprises a loxp sequence.

In another preferred example, the loxp sequence is inserted on both sides of one or more exons in the CDKL5 gene of the animal a genome.

In another preferred example, the loxp sequence is inserted on both sides of exon 6 in the CDKL5 gene of the animal a.

In another preferred embodiment, a screening marker is inserted between the loxp sequences.

In another preferred embodiment, the screening marker is a neo gene.

In another preferred embodiment, the genome of animal B comprises an exogenous Cre expression cassette.

In another preferred embodiment, the exogenous Cre expression cassette comprises: (a) a neuronal cell specific promoter; and (b) a Cre gene located downstream of the neuronal cell specific promoter.

In another preferred embodiment, the Cre gene comprises a Cre recombinase or a modified Cre recombinase (imimproved Cre, icar) gene.

In another preferred embodiment, the neuron cell specific promoter comprises Emx1 gene or CamK2 alpha gene promoter.

In another preferred example, the CDKL5 gene of animal C is specifically knocked out in neuronal cells starting from a specific time point (Emx1-Cre from embryonic day 12.5, CamK2 alpha-iCre from postnatal).

In another preferred example, the knockout comprises no expression of the CDKL5 gene, or expression of an inactive CDKL5 protein, or expression of a pathogenicity mutated CDKL5 gene.

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

In another preferred embodiment, the non-human mammal is a rodent or primate, preferably including a mouse, rat, rabbit, monkey.

In another preferred embodiment, the animal model of refractory epilepsy has one or more of the following characteristics compared to littermate or wild-type control animals:

(a) epilepsy progresses from low to high according to the Racine epilepsy rating scale;

(b) granular cell axon fibers are distributed in a band shape in a dentate gyrus inner molecular layer;

(c) an increased frequency of epileptic seizures;

(d) epilepsy eventually leads to death of the animal;

(e) characteristic epileptic brain waves are generated;

(f) the behavior of typical grand mal seizures;

(g) epileptiform discharges of the brain occur.

In a second aspect, the invention provides the use of a non-human mammalian animal model prepared by the method of the first aspect of the invention for studying an animal model of the pathogenesis of refractory epilepsy.

In another preferred example, the refractory epilepsy comprises spontaneous refractory epilepsy, infantile early onset epilepsy, CDKL5 syndrome epilepsy.

In a third aspect, the invention provides the use of a non-human mammalian model prepared by a method according to the first aspect of the invention to screen or identify substances (therapeutic agents) that reduce or treat refractory epilepsy.

In a fourth aspect, the present invention provides a method of screening for or identifying a potential therapeutic agent for treating or ameliorating refractory epilepsy, comprising the steps of:

(a) administering a test compound to a non-human mammalian model prepared by a method according to the first aspect of the invention in the presence of the test compound in a test panel, and analysing the phenotype of said animal model in the test panel; and analyzing the phenotype of said animal model in a control group not administered said test compound and otherwise identical;

(b) comparing the behavior of the test and control animal models, wherein an improvement in the phenotype characterizing refractory epilepsy in the animal model to which the test compound is administered as compared to the control group indicates that the test compound is a potential therapeutic agent for refractory epilepsy.

In another preferred embodiment, the phenotype of refractory epilepsy is selected from the group consisting of: seizure frequency, distribution of granular cell axon fibers in the inner molecular layer of the dentate gyrus, typical epileptic brain waves.

In another preferred embodiment, the distribution of the granular cell axon fibers in the dentate gyrus inner molecular layer comprises the projection of granular cell axons to dendrites adjacent to granular cells, and the distribution of the granular cell axon fiber ends in a band shape in the dentate gyrus inner molecular layer.

In another preferred embodiment, said phenotype is improved comprising: the epileptic seizure frequency is reduced, granular cell axon fibers are not projected to the dentate gyrus inner molecular layer, and epileptic brain waves disappear.

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

In another preferred embodiment, the method comprises the step of (c) administering the potential therapeutic agent screened or identified in step (b) to a non-human mammalian model prepared by the method of the first aspect of the invention, thereby determining its effect on the phenotype of said animal model.

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

In a fifth aspect, the invention provides a non-human mammalian model prepared by a method according to the first aspect of the invention.

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

In another preferred example, the CDKL5 gene knockout is a CDKL5 gene knockout specifically in neuronal cells.

The sixth aspect of the invention provides a use of a cell, wherein the CDKL5 gene of the cell is subjected to conditional knockout, the cell is used for preparing a biological agent for constructing a non-human mammal animal model with refractory epilepsy, and the cell is a neuron cell.

In another preferred embodiment, the conditional knock-out refers to a specific knock-out of the CDKL5 gene in the neuronal cell using the Cre-LoxP recombinase system.

In another preferred embodiment, the neuronal cells comprise excitatory neuronal cells.

In another preferred embodiment, the biological agent is a liquid agent.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIG. 1(A) seizures of epileptic mice at different levels. (B) Ratio of seizures in Cdkl5 conditional and systemic knockout mice. (C) Frequency of attacks in Cdkl5 conditional knockout mice.

Fig. 2 is a schematic diagram of an in vivo electrophysiological recording system.

FIG. 3 is a recorded Emx1-cKO mouse electroencephalogram showing changes in the electroencephalogram of the mouse during a seizure (lower panel), and the number of seizures in the mouse can be easily seen by monitoring for a long time (upper panel).

FIG. 4 shows that the frequency of epileptic seizures can be reduced by using a novel antiepileptic drug, while the frequency of epileptic seizures in control mice treated with the drug solvent is not statistically different before and after administration.

FIG. 5 is a Timm staining of mouse brain slices showing that Emx1-cKO mouse granulosa cell axon fibers are distributed as bands in the dentate gyrus inner molecular layer (A-B, A '-B'), whereas VGAT-cKO mice (C-D, C '-D') and Knockout mice (E-F, E '-F') are not significantly abnormal compared to the control. Statistical results showed that the moss fiber sprouting phenomenon in Emx1-cKO mice was significantly different from that in control mice (G).

Detailed Description

The present inventors have conducted extensive and intensive studies and, for the first time, have unexpectedly developed a spontaneous intractable epilepsy animal model, and in particular, the present invention provides a neuronal cell (e.g., excitatory neuronal cell) specific knockout animal of Cdkl5, which can be used as a spontaneous intractable epilepsy animal model. The animal model of the invention has an epileptic phenotype, especially a refractory epileptic phenotype, and can be used for screening antiepileptic (especially refractory epilepsy) medicaments. On this basis, the present inventors have completed the present invention.

Term(s) for

Cdkl5 gene

The Cyclin-dependent kinase-like protein 5 (CDKL 5) gene is located at the position 22 of the X chromosome short arm and encodes a serine/threonine kinase expressed in human and mouse brains, and the kinase consists of an N '-terminal kinase catalytic region and a C' -terminal amino acid sequence. This gene was discovered by Montini et al in 1998 as a disease-related gene. Kalscheuer et al found disruption of the CDKL5 gene due to X-chromosomal and autosomal translocations in two infants with X-linked infantile spasms and intellectual disabilities in 2003, the identical symptoms of which strongly suggest that loss of the CDKL5 protein leads to neurological dysfunction. Statistically, the site of the pathogenic CDKL5 gene mutation is mainly concentrated in the N' end kinase catalytic region, and the symptoms of patients carrying kinase region mutation are more serious than those of patients carrying other region mutation.

Epilepsy

Epilepsy is a transient cerebral dysfunction caused by sudden abnormal discharge of neurons, and is a chronic disease. Epidemiological data show that the overall incidence rate of epilepsy in China is 7.0 per mill, and the prevalence rate of active epilepsy with attacks within 1 year is 4.6 per mill. Epilepsy has become the second most common disease in China, second only to headache in neurology. The causes of epilepsy are complex and various, including genetic factors, brain diseases and the like, and the pathogenesis is also very complex.

Inactivation of genes

Many methods are available for the study of genes of unknown function, such as inactivation of the gene to be studied, analysis of the resulting genetically modified phenotypic change, and subsequent acquisition of functional information about the gene. Another advantage of this approach is that it can correlate gene function with disease, thus obtaining both gene function and disease information and animal models of disease that the gene can treat as a potential drug or drug target. The gene inactivation method can be realized by means of gene knockout, gene interruption or gene insertion. Among them, gene knockout technology is a very powerful means for studying the function of human genes in the whole.

As used herein, the terms "gene inactivation", "gene knockout", and the like, are used interchangeably and refer to genetic manipulation such as disruption, knockout, etc. of a certain gene of interest, such that the expression and/or activity of the gene of interest is substantially reduced or even completely lost.

The invention utilizes a tissue-specific Cre-LoxP system to knock CDKL5 out of mice (Cdkl 5)^flox/flox) Hybridizing with excitatory neuron cell specific Cre expression mice (Emx1-Cre or CamK2 alpha-iCre mice) to obtain Cdkl5 specific knockout mice (Emx1-Cre or CamK2 alpha-iCre/Cdkl 5) of excitatory neuron cells^flox/floxMouse). Wherein loxp markers are inserted on two sides of exon 6 of a Cdkl5 gene in the genome of the Cdkl5 conditional knockout mouse; the genome of the excitable neuron cell-specific Cre expression mouse contains an exogenous Cre expression sequence,the exogenous Cre expression sequence comprises: (a) an excitatory neuronal cell-specific promoter (the promoter of Emx1 gene or the CamK2 α gene), and (b) a Cre gene located downstream of the excitatory neuronal cell-specific promoter. According to the invention, on an individual level, the two mice are crossed to obtain a progeny mouse which specifically expresses Cre recombinase in excitatory neuron cells, so that the Cdkl5 gene between LoxP sites in the genome of the excitatory neuron cells is excised, and the specific inactivation of the Cdkl5 gene in the excitatory neuron cells is realized.

Cre recombinase was found from P1 phage in 1981 and belongs to lambda Int enzyme supergene family. The Cre recombinase gene coding region sequence has a full length of 1029bp (EMBL database accession number X03453) and codes 38kDa protein. The Cre recombinase is a monomeric protein consisting of 343 amino acids. It not only has catalytic activity, but also recognizes specific DNA sequences, i.e., loxP sites, similarly to restriction enzymes, so that gene sequences between loxP sites are deleted or recombined. The Cre recombinase has 70 percent of recombination efficiency, and can act on DNA substrates with various structures, such as linear, circular and even supercoiled DNA, without any auxiliary factors. It is a site-specific recombinase, which can mediate the specific recombination between two LoxP sites (sequences) to delete or recombine the gene sequences between the LoxP sites.

LoxP (locus of X-overP1) sequence: derived from the P1 phage, the gene is composed of two 13bp inverted repeat sequences and an 8bp sequence with a middle interval, and the 8bp interval sequence also determines the orientation of LoxP. Cre is covalently bound to DNA during catalytic DNA strand exchange, and the 13bp inverted repeat sequence is the binding domain of Cre enzyme.

Animal model

Animal models of human diseases (animal models of human diseases) refer to animals with human disease-mimicking manifestations established in various medical science studies, and are classified into spontaneous animal models and induced or experimental animal models according to the cause of the disease.

Spontaneous Animal Models (Spontaneous Animal Models) refer to diseases that occur in natural conditions in experimental animals without any conscious artificial treatment. Including genetic diseases of mutant lines and tumor disease models of inbred lines. The biggest advantage of utilizing the animal disease model to research human diseases is that the occurrence and development of the diseases are similar to the corresponding diseases of human beings, and the diseases are all diseases occurring under natural conditions and have higher application value, but the model is difficult to source.

In the present invention, there is provided an animal model of spontaneous epilepsy in a non-human mammal, the animal model being as described in the fifth aspect of the invention.

Specifically, the present invention utilizes a genome having: (E1) an endogenous CDKL5 gene, and (E2) a mouse (Cdkl 5) operably linked to the CDKL5 gene and used for conditional knock-out of a conditional knock-out element of the CDKL5 gene (such as the Cre recombinase targeting sequence loxp sequence)^flox/flox) A conditional knockout mouse of Cdkl5 in excitatory neuron cells is constructed by hybridizing with excitatory neuron cell-specific Cre recombinase expression mice (Emx1-Cre or CamK2 alpha-iCre mice), and the expression of Cdkl5 is knocked out specifically in the excitatory neuron cells of the mice, so that the function of the Cdkl5 in the excitatory neuron cells can be researched by the inventor in a horizontal tissue specificity manner.

The invention takes a Cdkl5 gene knockout animal model with refractory epilepsy phenotype as a research object, judges the frequency of the mouse epileptic seizure by a method of recording animal (such as mouse) electroencephalogram, researches the pathological morphology of the mouse brain, and preliminarily judges whether the medicament has the effect of resisting the CDKL5 related epileptic seizure. In view of the clear phenotypic advantages of the Cdkl5 conditional knockout animal model, the method disclosed by the invention can be used for rapidly and efficiently carrying out preliminary evaluation on the effectiveness of the medicament, thereby promoting the development and marketing of the medicament.

The main advantages of the invention include:

(a) the invention discovers for the first time that conditional knockout of Cdkl5 in excitatory neuronal cells can be used for preparing animal models of refractory epilepsy.

(b) The refractory epilepsy animal model can be used for screening antiepileptic drugs.

(c) The invention takes a Cdkl5 gene knockout mouse with spontaneous intractable epileptic phenotype as a research object, judges the frequency of epileptic seizure of the mouse by a method of recording electroencephalogram of the mouse, researches the pathological form of brain of the mouse, and preliminarily judges whether the medicament has the effect of resisting epileptic seizure related to CDKL 5. In view of the clear phenotypic advantages of the Cdkl5 conditional knockout mice, the method can rapidly and efficiently perform initial evaluation on the effectiveness of the drug, and further promote the development and marketing of the drug.

(d) The invention discovers for the first time that a mouse with the Cdkl5 gene conditionally knocked out by excitatory neurons reproduces the refractory epilepsy phenotype of a patient with CDKL5 deficiency, so that the mouse model constructed by the method is used for developing and screening disease treatment drugs, the drug research and development efficiency and accuracy are greatly improved, and the risk of clinical research failure is reduced.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, molecular cloning is generally performed according to conventional conditions such as Sambrook et al: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are by weight. The test materials and reagents used in the present invention are commercially available without specific reference.

General procedure

Breeding method of epileptic mice related to Cdkl5 gene

By means of the Cre-LoxP technique, through Cdkl5^flox/floxMice (donated by Joe Zhou laboratory) and Emx1-Cre mice (purchased from Jackson laboratories, USA) or CamK2 α -iCre mice (donated by the German center for cancer research) were bred to produce the conditional knock-out mice of the invention. Female Cdkl5 of suitable age^flox/floxMating the mice with male mice specifically expressing Cre recombinase to generate conditional knockout mice with Cdkl5 gene knockout in excitatory neurons, and after the mice become adult, the conditional knockout mice can be used for observing epilepsia and screening antiepileptic drugs.

2. Method for accurately recording epileptic seizure frequency by electroencephalogram

The homemade electrode is installed above the mouse pia mater through an operation, after the mouse recovers for one week, the homemade electrode is connected to a long-time electroencephalogram recording system, and the electroencephalogram activity condition of the mouse is monitored for 24 hours. The frequency of the epilepsy of the mice is judged according to the electroencephalogram, and the frequency of the mouse attack before and after administration is compared to judge the treatment effect of the medicament on the intractable epilepsy.

Method for judging seizure epilepsy of mouse by Timm staining method

The granular cell axon fibers of the CDKL5 conditional knockout mouse with the epilepsia are distributed in a band shape in the dentate gyrus inner molecular layer, and whether the epilepsia occurrence process can be stopped by the drug treatment or not is judged by the method.

EXAMPLE 1 preparation of seizure mice

Female Cdkl5 of suitable age^flox/floxMating the mouse with male mouse expressing Cre recombinase specifically, cutting toes of the mouse after the newborn mouse is born seven days, numbering, taking tail about 3 mm, and carrying out genotype identification. The mouse tail was immersed in 150ul of rat tail lysate (containing 2ul of 5% proteinase K solution) and incubated overnight at 55 ℃; placing the centrifuge tube containing the lysis solution on a metal bath at 95 ℃ and heating for 15 min; after cooling to room temperature, centrifuging at 12000rpm/min for 5 min; the supernatant was used for genotyping by PCR. Preparing a PCR reaction system, wherein the formula is as follows:

the PCR cycling conditions were:

35 cycles of 95 ℃ for 15s, 60 ℃ for 15s and 72 ℃ for 45 s.

The PCR product and 2 Xloading buffer were mixed well and added to 1.5% agarose gel, electrophoresed for 35min in a constant voltage electric field of 120mV, and the results were observed and photographed in a gel imager. According to the gel electrophoresis result, a conditional knockout mouse with the Cdkl5 gene knocked out in excitatory neurons is identified, and the conditional knockout mouse can be used for observing the occurrence of epilepsy and screening antiepileptic drugs after the conditional knockout mouse is grown up.

Emx1-Cre mediates recombination of LoxP in excitatory neurons of the forebrain at day 12.5 of the mouse embryo, while CamK2 α -iCre specifically knocks out the gene of interest in excitatory neurons from the postnatal time of the mouse.

By long-term video recording, Emx1-Cre driven CDKL5 conditional knockout mice were observed to develop symptoms of spontaneous epilepsy in succession from 2 months, and the progression of epilepsy from low to high was evident according to the Racine epilepsy grading criteria (FIG. 1A). It was further statistically found that 80% of Emx 1-Cre-mediated conditional knockdown mice (n-15) exhibited symptoms of grand mal seizures (fig. 1B), with a frequency of seizures ranging from 1-23 times/day (fig. 1C). While 66.7% of CDKL5 conditioned knockdown mice (n-15) driven by CamK2 α -cre produced grand mal seizures (fig. 1B), with a frequency of seizures ranging from 0-3 times/day (fig. 1C), even directly resulting in mouse death. It is demonstrated that mice with refractory seizures can be generated by the method of excitatory neuron knockout of the mouse CDKL5 gene.

However, no epileptogenesis was observed before 6 months in VGAT-Cre mediated inhibitory neuron-specific Cdkl5 gene knockout mice and Cdkl5 systemic knockout mice (fig. 1B), suggesting that the mechanism of Cdkl5 compensation is likely to be present early in embryonic development.

Example 2EEG recording mode to accurately determine seizure frequency in transgenic mice

The homemade electrode was mounted on the rat pia mater by surgery, and during surgery, the rat was anesthetized with 1.5% isoflurane and placed above stereotaxic position with the skull in horizontal position. The self-made electrode is composed of two stainless steel screws (diameter 1mm) and is used as an electroencephalogram recording electrode, and the position of inserting into the skull is determined according to the brain atlas of a mouse (the coordinate taking bregma as the origin point is +1.0mm front and back, the middle and outer side is +1.5mm, the front and back are-3.2 mm, and the middle and outer side is +1.5 mm). Two insulated silver wires (Coonerwire, # AS633) AS myoelectric recording electrodes were placed in the trapezius muscles on both sides, respectively. The electrodes were attached to a mini-connector and fixed to the skull with dental cement. The skin of the mice was then sutured over with surgical sutures, reducing direct contact with the outside world, and then the mice were placed in a warm environment until they returned to normal activity.

After the mouse recovers for one week, the mouse is connected to a long-time electroencephalogram recording system (figure 2), the mouse is allowed to continuously adapt for two days, and then the electroencephalogram activity condition of the mouse is monitored for 24 hours. The activity of the free-moving mouse is recorded when the electroencephalogram and the electromyogram of the mouse are synchronously recorded. The set of long-time electroencephalogram recording system comprises: a dual channel amplifier (a-M SYSTEMS, Model 1800), a digital to analog converter (CED Ltd., Micro1401mk ii) and Spike2 software (CED Ltd.).

The frequency of the epilepsy of the mice is judged according to the electroencephalogram, and the frequency of the mouse attack before and after administration is compared to judge the treatment effect of the medicament on the intractable epilepsy. Fig. 3 shows an electroencephalogram at the time of epileptic seizure, and fig. 4 shows the change in epileptic frequency of a mouse before and after administration of a certain drug.

Example 3 method of Timm staining to determine the onset of CDKL 5-associated epilepsy in mice

The budding of the moss fibers of the hippocampal dentate gyrus is a characteristic morphological change in patients and mice with epilepsy in the limbic system. A hippocampal section of a CDKL5 conditioned knockout mouse with epilepsy was subjected to Timm staining, and granular cell axon fibers were found to be distributed in a band shape in the dentate gyrus inner molecular layer (FIG. 5, B-B '), while a control mouse without epilepsy (FIG. 5, A-A', C-C ', E-E') and a VGAT-Cre-mediated cKO mouse (FIG. 5, D-D '), a CDKL5 whole body knockout mouse (FIG. 5, F-F') did not show the projection of granular cell axon fibers to the dentate gyrus inner molecular layer. Statistical results also showed that Emx1-Cre mediated CDKL5 conditional knockdown resulted in a significant statistical difference in granulocytic ectopic projection compared to the control group (fig. 5G). Therefore, the method can be used for judging whether the drug treatment can prevent the occurrence process of the mouse epilepsy of knocking out CDKL5 in excitatory neuron cells.

Mice were anesthetized by intraperitoneal injection with 8% chloral hydrate and then perfused with 1xPBS buffer, sulfide solution (1.2% (wt/vol) Na2S · 9H2O, 1% (wt/vol) NaH2PO4 distilled water) and 4% paraformaldehyde sequentially through the heart. The mouse brains were then removed and placed in 4% PFA and fixed overnight, and frozen sections were taken after 15% and 30% sucrose gradient dehydration. The rat brain was horizontally sliced to a brain slice thickness of 30 um. The brain piece was then mounted on a glass slide and air dried. After rehydration with gradient ethanol, brain pieces were treated with a mixed solution of gum arabic (50%, wt/vol), hydroquinone (5.67%, wt/vol), citric acid-sodium citrate buffer (pH 3.6), and silver nitrate (17%, wt/vol) at a volume ratio of 12:6:2:1, and left to stand at room temperature in the dark for 45 minutes. After the brain slices are obviously colored, dehydrating by using gradient ethanol, sealing the slices by using neutral gum after dimethylbenzene is transparent, and drying in the air. Brightfield images were taken using an olympus VS120 microscope, followed by quantification of staining in Image J software and assessment of moss fibre budding using a Timm index (total area of Timm particles divided by length of dentate gyrus). For each animal, the Timm index is the average of at least three calculated values of adjacent brain slices.

Comparative example

With the method of the present embodiment, the difference is that male and female Cdkl5 expressing Cre recombinase by germ cells^flox/floxMouse mating produced Cdkl5 systemic knockout mice.

The results show that the mice had no overt epileptic symptoms, and that no typical grand-mal behavior occurred six months ago, nor were epileptiform discharges detected in the brain.

Discussion of the related Art

According to the existing research report and the recent research result in the laboratory, the CDKL5 systemic knockout model has obvious gene compensation effect, so that a Cdkl5 knockout mouse cannot show obvious epileptic symptoms. Although it has been reported that mouse spasms can be recorded in older female heterozygous mice, the model mice lack high amplitude and high frequency of epileptiform discharges and have a late onset time that is not suitable for screening for antiepileptic drugs. After CDKL5 is knocked out specifically by the excitatory neuron cells, the mice can generate typical epileptiform behaviors and epileptiform brain waves from 3 months, so that the method is more suitable for screening intractable epileptic drugs, and is an important application direction of the method.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims (10)

1. A method of making a non-human mammal animal model of refractory epilepsy, comprising the steps of:

(1) providing a non-human mammal A and a non-human mammal B expressed by a neuron cell-specific Cre recombinase of the same species;

wherein the genome of said 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 said conditional knockout element conditionally knocks out the Cdkl5 gene of the genome of the neuronal cell in the presence of said Cre recombinase, thereby inactivating the Cdkl5 gene;

(2) mating and breeding the animal A and the animal B to obtain a progeny non-human mammal C with the CDKL5 gene specifically knocked out in neuron cells;

(3) culturing said progeny in a non-human mammal C, thereby obtaining said animal model of refractory epilepsy.

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

3. The method of claim 1, wherein the animal model of refractory epilepsy 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, and gradually increases with the progress of epilepsy;

the animal model of the invention is spontaneous and high frequency compared to previous models of epilepsy.

4. The method of claim 1, wherein the refractory epilepsy comprises spontaneous refractory epilepsy, infantile early onset epilepsy, or epilepsy with CDKL5 syndrome.

5. The method of claim 1, wherein the neuronal cells comprise: an excitatory neuronal cell.

6. The method of claim 1, wherein the animal model of refractory epilepsy has one or more of the following characteristics compared to littermate or wild-type control animals:

(a) epilepsy progresses from low to high according to the Racine epilepsy rating scale;

(b) granular cell axon fibers are distributed in a band shape in a dentate gyrus inner molecular layer;

(c) an increased frequency of epileptic seizures;

(d) epilepsy eventually leads to death of the animal;

(e) characteristic epileptic brain waves are generated;

(f) the behavior of typical grand mal seizures;

(g) epileptiform discharges of the brain occur.

7. Use of a non-human mammalian model prepared by the method of claim 1, in an animal model for studying the pathogenesis of refractory epilepsy.

8. Use of a non-human mammalian model prepared by the method of claim 1 to screen or identify substances (therapeutic agents) that reduce or treat refractory epilepsy.

9. A method of screening for or identifying potential therapeutic agents for treating or ameliorating refractory epilepsy, comprising the steps of:

(a) administering a test compound to the non-human mammalian model prepared by the method of claim 1 in the presence of the test compound in a test panel, and analyzing the phenotype of said animal model in the test panel; and analyzing the phenotype of said animal model in a control group not administered said test compound and otherwise identical;

(b) comparing the behavior of the test and control animal models, wherein an improvement in the phenotype characterizing refractory epilepsy in the animal model to which the test compound is administered as compared to the control group indicates that the test compound is a potential therapeutic agent for refractory epilepsy.

10. Use of a cell in which a conditional knock-out of the CDKL5 gene occurs for the preparation of a biologic for the construction of an animal model of refractory epilepsy in a non-human mammal, and wherein the cell is a neuronal cell.

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