PKHD1L1 gene point mutation rat model and its construction method and detection method

Abstract

The present invention relates to the technical field of animal modeling, and specifically discloses a PKHD1L1 gene point mutation rat model and its construction and detection method. The present invention is to construct a PKHD1L1 point mutation rat by knocking in mouse-derived P.L867S into the PKHD1L1 gene based on the CRISPR / Cas9 system, and the corresponding base becomes a TTA to TCA point mutation, and the rat model can be further applied to various research scenes such as epilepsy development mechanism and design of new antiepileptic drugs. The present invention provides an application of the method for detecting cortical excitability abnormality in detecting the phenotype of an epilepsy animal model. After constructing an animal model, the present invention uses the method of somatosensory evoked potential to detect whether there is a phenotype of cortical excitability abnormality to confirm whether the model construction is successful. The present invention can be used to detect cortical excitability abnormality and can also be used to verify the effectiveness of related medicines or treatments.

Description

[Technical field]

[0001] This application claims priority to a Chinese patent application filed with the State Intellectual Property Office of China on December 6, 2022, bearing application number 202211558984.6 and entitled "Construction and Application of Point Mutation Rat Epilepsy Model", and a Chinese patent application filed with the State Intellectual Property Office of China on June 28, 2023, bearing application number 202310770803.4 and entitled "Method and Application for Identifying Cortical Excitation Abnormalities in Epilepsy Animal Models", the entire contents of which are incorporated herein by reference.

[0002] The present invention relates to the technical field of animal modeling, in particular to the construction and application of point mutation rat epilepsy models, and methods and applications for identifying cortical excitability abnormalities in epilepsy animal models. [Background technology]

[0003] Epilepsy is a severe and chronic central nervous system disease, which is characterized by repeated seizures and temporary cerebral dysfunction due to abnormal electrical activity of highly synchronized brain neurons. Its clinical manifestations include various disorders of movement, sensation, autonomic nerves, and consciousness. It is one of the relatively common diseases of the nervous system, with more than 65 million people suffering from epilepsy worldwide. The residual disabilities, death, and complications associated with epilepsy place a heavy burden on patients and society. At present, the mechanism of epilepsy seizures is not well understood, and the pathogenesis mechanisms proposed by different scholars are significantly heterogeneous, so various types of animal models have been constructed to interpret the mechanism of epilepsy formation. An animal model of epilepsy refers to a certain species that has a tendency to seizures induced by external environment or genetically. Therefore, in order to detect the changes in electroencephalogram and behavioral characteristics during the epilepsy-induced seizure state, many epilepsy model studies use inducers to maintain epilepsy-induced seizures. For example, pilocarpine or pentylenetetrazole is administered intraperitoneally to construct a TLE animal model, and kainic acid is injected into the lateral ventricle to construct a TLE animal model. However, existing epilepsy animal models cannot fully meet a series of clinical needs, such as screening of epilepsy development mechanism, pathophysiological process and therapeutic target. Therefore, due to the variety of types of animal models constructed to interpret epileptogenesis mechanism, it is still unknown whether the constructed model can truly represent the epilepsy development mechanism.

[0004] Although detecting the constructed model is a necessary step, there is no unified standard in the existing materials for whether an epilepsy model has been successfully constructed. For example, when constructing an epilepsy model using zebrafish in general, it is necessary to use a zebrafish behavioral analyzer to perform detection, and the degree of epilepsy is reflected based on the change in the activity intensity of the zebrafish larvae. For example, in Chinese Patent CN201910244481.3 (a new method for drug-induced epilepsy model), after drug induction, the electroencephalogram is used to detect the animal's electroencephalogram, the behavior of epilepsy rats is classified and evaluated, primary rat hippocampal neurons are cultured in vitro, cell administration is performed, and the electrical activity function of primary rat hippocampal neurons is measured using patch clamp technology, only after which it can be determined whether the rat model finally obtained is an epilepsy model.

[0005] As is known, electrophysiological signals are the gold standard for evaluating the onset of epilepsy or the enhancement of cortical excitability and the efficacy of drug therapy, but how to perform the corresponding detection in animal models is an urgent technical challenge to be solved. Summary of the Invention [Problem to be solved by the invention]

[0006] The objective of the present invention is to provide a point mutation rat epilepsy model and its application. The point mutation rat has a phenotype of enhanced neuronal excitability and can well mimic the phenotypes of FCMTE and other epilepsy patients. This rat model can be further applied to various research scenes such as the mechanism of epilepsy development and the design of new antiepileptic drugs.

[0007] Another object of the present invention is to provide a method and application for identifying cortical excitability abnormality in epilepsy animal models, and using the method of somatosensory evoked potential (SEP) to further detect whether the constructed animal model has a phenotype of cortical excitability abnormality to determine whether the construction of the epilepsy animal model is successful. [Means for solving the problem]

[0008] The method for constructing a PKHD1L1 gene point mutation rat model according to the present invention includes the steps of: designing an sgRNA using the 22-23 intron and the 24-25 intron of the PKHD1L1 gene as a target sequence; annealing the designed sgRNA and then linking it to a plasmid vector with a T7 promoter; and performing in vitro transcription to obtain Cas9 / sgRNA; The Cas9 / sgRNA and the targeting vector are microinjected into rat fertilized eggs, and the gene-edited fertilized eggs are placed into the uterus of pseudopregnant mice, so that the F0 generation includes chimeric rats with PKHD1L1 gene point mutations.

[0009] Preferably, the primer pair for PCR amplifying the target sequence in the 22-23 intron comprises PKHD1L1-5'MSD-F, the nucleotide sequence of which is shown in SEQ ID NO.1, and PKHD1L1-5'MSD-R, the nucleotide sequence of which is shown in SEQ ID NO.2; The primer pair for PCR amplifying the target sequence in the 24th to 25th introns includes PKHD1L1-3'MSD-F, whose nucleotide sequence is shown in SEQ ID NO.3, and PKHD1L1-3'MSD-R, whose nucleotide sequence is shown in SEQ ID NO.4.

[0010] Preferably, the PCR amplification step includes the steps of 94°C for 5 min, 30 cycles of 94°C for 30 s, 62°C for 30 s, and 72°C at 1 kb / min, and amplifying at 72°C for 10 min.

[0011] Preferably, the sequences of the sgRNA are as shown in SEQ ID NO.5 and SEQ ID NO.6.

[0012] Preferably, the plasmid vector comprises the pCS-3G vector.

[0013] Preferably, the nucleotide sequence of the targeting vector is as shown in SEQ ID NO.27.

[0014] Preferably, the method further comprises the step of identifying point mutation chimeric rats by PCR after obtaining the F0 generation; Regarding the point mutation chimeric rat, When PCR identification was performed using PKHD1L1-L-GT-F, the nucleotide sequence of which is shown in SEQ ID NO. 7, and PKHD1L1-L-GT-R, the nucleotide sequence of which is shown in SEQ ID NO. 8, a 2662 bp product was amplified in the point mutation chimeric rat; When PCR identification is performed using PKHD1L1-R-GT-F, the nucleotide sequence of which is shown in SEQ ID NO.9, and PKHD1L1-R-GT-R, the nucleotide sequence of which is shown in SEQ ID NO.10, a 2697 bp product is amplified in the point mutation chimeric rat.

[0015] Preferably, the PCR identification step includes the steps of pre-denaturing at 94°C for 2 min, denaturing at 98°C for 10 s, annealing at 67°C for 30 s, extending at 68°C for 1 kb / min, and performing 15 cycles with the annealing temperature decreasing by 0.7°C after each cycle; denaturing at 98°C for 10 s, annealing at 57°C for 30 s, extending at 68°C for 1 kb / min, and performing 25 cycles; and extending at 68°C for 10 min.

[0016] The method of the present invention for constructing a rat model that stably inherits a PKHD1L1 gene point mutation includes the step of mating the F0 generation point mutant chimeric rat obtained by the above construction method with a wild-type rat, so that the heterozygote in the F1 generation is a rat model that stably inherits the PKHD1L1 gene point mutation.

[0017] The present invention further provides the application of the PKHD1L1 gene point mutation rat model obtained by the above construction method or the rat model stably inheriting the PKHD1L1 gene point mutation obtained by the above construction method in the screening and / or research and production of antiepileptic drugs.

[0018] The present invention provides an application of methods for detecting cortical excitability abnormalities in detecting phenotypes in animal models of epilepsy.

[0019] Preferably, the method for detecting cortical excitability abnormalities comprises somatosensory evoked potentials.

[0020] The method for detecting a phenotype of an epilepsy animal model according to the present invention includes the steps of: fixing the head and four limbs of an epilepsy animal model in a prone position; performing percutaneous electrical stimulation on the posterior tibial nerve at the ankle of the right hind limb; and then inserting a recording needle electrode subcutaneously into the Cz region of the skull and a reference needle electrode subcutaneously above the nose; The derived signal is filtered and amplified, and then input into the system, and based on the output somatosensory evoked potential, peak latency is measured, cortical excitability is evaluated, and based on the cortical excitability, it is determined whether or not it is an epilepsy model.

[0021] Preferably, the parameters of the electrical stimulation are a constant voltage square wave with a wave width of 0.1 ms, a frequency of 3 Hz, and an intensity sufficient to cause slight movement of the hind toes, with the needles being grounded subcutaneously on the back.

[0022] Preferably, the derived signals are filtered and amplified, then input into a computer operating system for averaging, with the number of additions being 1024 and the analysis time being 56 ms, and finally the somatosensory evoked potentials are displayed and printed, and their peak latency is measured.

[0023] Preferably, said animal model of epilepsy comprises a rat epilepsy model or a mouse epilepsy model. Effect of the Invention

[0024] The present invention initially demonstrated, through pathogenicity studies on a family with familial adult myoclonic epilepsy (FIG. 17), that a heterozygous mutation in exon 23:c.2602A>T of the PKHD1L1 gene was the pathogenic mutation in the family. The present invention uses the CRISPR / Cas9 system to knock in mouse-derived P.L867S into the PKHD1L1 gene, and the corresponding base becomes a TTA to TCA point mutation to construct a PKHD1L1 point mutation rat. Three male PKHD1L1 point mutation heterozygote (PKHD1L1+ / -) rats were continuously recorded and observed for 5 days, and no spontaneous epilepsy behavior was observed. However, the PKHD1L1+ / - rats have a phenotype of enhanced neuronal excitability, and the threshold concentration of pentylenetetrazole (PTZ) injected intraperitoneally to induce epilepsy seizures is significantly lower than that of wild-type rats, and the resting potential in the rat brain slice electrophysiology is significantly lower than that of wild-type rats, which can successfully mimic the phenotype of FCMTE and other epilepsy patients. This rat model can also be applied to various research scenes such as the mechanism of epilepsy onset and the design of new antiepileptic drugs.

[0025] The application of the method for detecting abnormal cortical excitability of the present invention in detecting the phenotype of epilepsy animal models is to detect whether the animal model constructed by the method of SEP has a phenotype of abnormal cortical excitability, and judge whether the construction is successful.The detection method of the present invention can be well applied to detect the cortical excitability of rats or mice, and the detection value can be used as an evaluation standard for the effectiveness of drugs that suppress cortical excitability. [Brief description of the drawings]

[0026] [Figure 1] FIG. 1 shows the sequencing results of PKDH1L1 gene mutations. [Diagram 2] FIG. 1 is a diagram of the construction policy of a targeting vector. [Diagram 3] Precut pCS-3G plasmid profile. [Figure 4] 1 is a targeting vector map. [Diagram 5] FIG. 1 shows the results of detecting sgRNA activity. [Figure 6] RNA electropherograms produced with sgRNAs, where sg1 in the figure refers to PKHD1L1-sgRNA1 and sg2 refers to PKHD1L1-sgRNA12. [Figure 7] This is a diagram showing the results of PCR identification of the F0 generation of PKHD1L1-L-GT-F / PKHD1L1-L-GT-R. [Figure 8] FIG. 1 shows the results of PCR identification of the F0 generation of PKHD1L1-R-GT-F / PKHD1L1-R-GT-R. [Figure 9] This is a graph showing the results of PCR identification of the F1 generation of PKHD1L1-L-GT-F / PKHD1L1-L-GT-R. [Figure 10] FIG. 1 shows the results of PCR identification of the F1 generation of PKHD1L1-R-GT-F / PKHD1L1-R-GT-R. [Figure 11] FIG. 1 shows the results of Southern blot detection of positive rats of the F1 generation. [Figure 12] This shows the results of gene sequencing analysis of positive mice of the F1 generation that were correctly recombined and had no random insertions. [Figure 13] FIG. 1 shows the results of a study on epilepsy susceptibility in PKHD1L1 P.L867S point mutation PKHD1L1+ / - rats. [Figure 14]4 shows the results of the effect of H89 on cortical pyramidal neuron sEPSC / sIPSC. (A) Illustrative diagram of sEPSC current of pyramidal neurons of rats in each group; (B) Changes in sEPSC amplitude of pyramidal neurons; (C) Changes in sEPSC frequency of pyramidal neurons; (D) Illustrative diagram of sEPSC current of pyramidal neurons of rats in each group; (E) Changes in sIPSC amplitude of pyramidal neurons; (F) Changes in sIPSC frequency of pyramidal neurons (3 mice per group, 6 cells in control group, 6 cells in epilepsy group, 6 cells in epilepsy+H89 group). *P<0.05**P<0.01. [Figure 15] Results of the effect of H89 on cortical pyramidal neuron Na+ / K+ currents. (A) Changes in pyramidal neuron Na+; (B) Changes in pyramidal neuron K+. (3 mice per group, 6 cells in control group, 6 cells in epilepsy group, 6 cells in epilepsy+H89 group) *, P<0.05. [Figure 16] 4 shows the results of the effect of H89 on the change of action potential of cerebral cortical pyramidal neurons. (A) Illustrative images of the action potential of cerebral cortical pyramidal neurons of rats in each group. (B) Illustrative images of a single action potential of a pyramidal neuron of rats in each group. (C) Action potential threshold of pyramidal neurons. (D) Recovery period of positive after-potential of action potential. (E) Half-length of positive after-potential. (F) Peak potential of action potential. (3 mice per group, 6 cells in control group, 6 cells in epilepsy group, 6 cells in epilepsy+H89 group). *P<0.05. [Figure 17] FIG. 2 is a family tree diagram of a family in an embodiment of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] The method for constructing a PKHD1L1 gene point mutation rat model according to the present invention includes the steps of: designing an sgRNA using the 22-23 intron and the 24-25 intron of the PKHD1L1 gene as a target sequence; annealing the designed sgRNA and then linking it to a plasmid vector with a T7 promoter; and performing in vitro transcription to obtain Cas9 / sgRNA; The method includes microinjecting the Cas9 / sgRNA and the targeting vector into rat fertilized eggs, and then placing the gene-edited fertilized eggs into the uterus of pseudopregnant mice, so that the F0 generation includes chimeric rats with PKHD1L1 gene point mutations.

[0028] The PKHD1L1 gene of the present invention is preferably screened from one familial adult myoclonic epilepsy family, and the family tree diagram is shown in Figure 17. Specifically, the family has a total of 30 people in 5 generations, 6 people are affected, and conforms to autosomal dominant genetic characteristics. All patients have myoclonus with or without generalized tonic-clonic seizures, with or without distal limb fine tremor, all epileptic seizures begin in adulthood, electroencephalogram examination shows symmetric spike-and-wave discharges on both sides, evoked potential examination shows giant potential and C-reflex positive, seizures can be effectively controlled by antiepileptic drug treatment, and the disease course is benign. All of the families are clearly diagnosed, and the clinical phenotype is highly consistent. By combining whole genome and exome sequencing with linkage analysis, it was found that all five patients (one of the six patients died) had a heterozygous mutation in the PKHD1L1 gene exon23:c.2602A>T, and all 11 family controls were homozygous, indicating the existence of a co-segregation phenomenon. The present invention further screened the PKHD1L1 gene from a group of 246 healthy individuals matched for age, sex, region, and ethnicity, and found that the mutation site was not present in the healthy individuals. This initially demonstrated that the heterozygous mutation in the PKHD1L1 gene exon23:c.2602A>T was the pathogenic mutation in the family (Figure 1).

[0029] The PKHD1L1 gene is located in the sense strand of chromosome 7, has a total length of approximately 172.46 kb, and is Gene ID: 314917. The present invention uses the Pkhd1l1-201 transcript (ENSRNOT00000005958.7, NM_001034931, abbreviated as PKHD1L1 gene) to study point mutations in rats. Specifically, the amino acid Lys at position 867 of the rat PKHD1L1 gene was mutated to Ser using the CRISPR / Cas9 system, and the corresponding base was mutated from TTA to TCA, and the sgRNA was designed in intron22-23 and intron24-25 (Figure 2).

[0030] In an embodiment of the present invention, in order to ensure the efficiency of the designed Cas9 / sgRNA, the target site sequence of SD rat tail is first PCR amplified, sequenced and verified to ensure that the sgRNA recognition sequence is completely consistent with the DNA sequence of SD rat tail, and the primer information includes PKHD1L1-5'MSD-F whose nucleotide sequence is shown in SEQ ID NO.1 and PKHD1L1-5'MSD-R whose nucleotide sequence is shown in SEQ ID NO.2, and the amplification product is 754bp, and also includes PKHD1L1-3'MSD-F whose nucleotide sequence is shown in SEQ ID NO.3 and PKHD1L1-3'MSD-R whose nucleotide sequence is shown in SEQ ID NO.4, and the amplification product is 569bp. The PCR amplification step of the present invention preferably includes a step of performing 30 cycles of 94°C for 5 min, 94°C for 30 s, 62°C for 30 s, and 72°C for 1 kb / min, and a step of amplifying at 72°C for 10 min. The present invention sequences the above PCR product, and the results show that the target sequence of SD rat tail is completely consistent with the sequence provided by Genebank and Ensembl, and can be used to point mutate the target gene of sgRNA.

[0031] Table 1 Primers for PCR amplification of target site sequences in rat tail [Table 1]

[0032] The present invention designs two sgRNAs based on the target gene, and the sequences of the sgRNAs are as shown in SEQ ID NO.5 and SEQ ID NO.6, specifically PKHD1L1-sgRNA1 and PKHD1L1-sgRNA12 in Table 2. The present invention preferably ligates the sgRNAs to the pCS-3G vector (FIG. 3) by annealing polymerization, converts the ligation product, and then sends out a sample for sequencing. If it is verified to be accurate, a microinjectable Cas9 / sgRNA is obtained. The annealing and polymerization of the present invention preferably includes a step of annealing at 65°C for 5 min.

[0033] Table 2 sgRNA sequences [Table 2]

[0034] The present invention relates to a targeting vector having a plasmid profile as shown in FIG. 4 upon microinjection, and the nucleotide sequence of said targeting vector is shown in SEQ ID NO.27.

[0035] The present invention further includes the step of microinjecting Cas9 / sgRNA and targeting vector into rat fertilized eggs, and F0 rats are born after injection, and the F0 rats are chimeric due to the fast cleavage rate in early embryonic stage, and thus, after obtaining F0 generation, preferably, identifying point mutation chimeric rats by PCR. The point mutation chimeric rats should simultaneously satisfy PKHD1L1-L-GT-F / PKHD1L1-L-GT-R (2662bp) and PKHD1L1-R-GT-F / PKHD1L1-R-GT-R (2697bp) positivity. In the present invention, PCR identification is preferably performed using the Touchdown method, and the reaction system is constructed based on the instructions for the KOD-FX enzyme. The PCR identification process of the present invention preferably includes the steps of pre-denaturing at 94°C for 2 minutes, denaturing at 98°C for 10 seconds, annealing at 67°C for 30 seconds, elongating at 68°C for 1 kb / min, and performing 15 cycles with the annealing temperature being reduced by 0.7°C for each cycle, denaturing at 98°C for 10 seconds, annealing at 57°C for 30 seconds, elongating at 68°C for 1 kb / min, and performing 25 cycles, and elongating at 68°C for 10 minutes.

[0036] Table 3. Identification primers for point mutation chimeras [Table 3]

[0037] The method of the present invention for constructing a rat model that stably inherits a PKHD1L1 gene point mutation includes the step of mating the F0 generation point mutant chimeric rat obtained by the above construction method with a wild-type rat, so that the heterozygote in the F1 generation is a rat model that stably inherits the PKHD1L1 gene point mutation.

[0038] In the present invention, F1 generation rats with stable genotypes obtained by mating positive rats with wild-type rats in the genotype identification results of the tails of F0 generation rats are selected. In the present invention, genotype identification is performed on the obtained F1 generation, and the genotype identification method preferably includes PCR identification, Southern blot and sequencing identification, and PCR identification is preferably the same as the detection of the F0 generation, and the description is omitted here. EcoRV and SpeI are used as Southern blot restriction enzyme cleavage sites. 3'Probe-A is used to detect whether or not recombination has been performed correctly, and if recombination has been performed correctly, two bands, one for the wild type and one for the mutant type, appear. LR Probe-A is used to detect whether or not there is random insertion, and if there is no random insertion, two bands, one for the wild type and one for the mutant type, appear.

[0039] Table 4. 3'Probe-A and LR-Probe-A primer information [Table 4]

[0040] For the F1 generation determined to have a point mutation, the heterozygote / homozygote genotype can be further detected by PCR verification and sequencing, the primer information for detection includes PKHD1L1-R-GT-F and PKHD1L1-L-GT-R, and the process is preferably 94°C for 5 min, 94°C for 30 s, 62°C for 30 s, 72°C for 1 kb / min for 30 cycles, and 72°C for 10 min. Since both the mutation and wild product are 625 bp, the final homozygote, heterozygote, and wild type genotype need to be determined by sequencing.

[0041] The present invention further provides the application of the PKHD1L1 gene point mutation rat model obtained by the above construction method or the rat model stably inheriting the PKHD1L1 gene point mutation obtained by the above construction method in the screening and / or research and production of antiepileptic drugs.

[0042] The present invention provides an application of methods for detecting cortical excitability abnormalities in detecting phenotypes in animal models of epilepsy.

[0043] The method for detecting abnormalities in cortical excitability of the present invention preferably includes somatosensory evoked potentials (SEPs). The present invention does not particularly limit the specific construction method and animal type of the epilepsy animal model, and preferably includes rats, mice, or zebrafish. In the examples of the present invention, SD rats are described as an example, but this alone cannot be considered as the entire scope of protection of the present invention. The present invention initially demonstrated that the heterozygous mutation of PKHD1L1 gene exon23:c.2602A>T is a pathogenic mutation in the family through pathogenicity research of the familial adult myoclonic epilepsy (FAME) family shown in Figure 17, and constructed a PKHD1L1 point mutation rat by knocking in P.L867S into the PKHD1L1 gene based on the CRISPR / Cas9 system, and the corresponding base becomes a point mutation from TTA to TCA. Through detection, electrophysiological tests on epilepsy seizure patients in this family showed improved cortical excitability, and antiepileptic drug treatment could effectively control seizures, and the disease process was benign.In addition, the constructed PKHD1L1 gene point mutation rats showed significantly shorter SEP latency and increased amplitude compared with weight-matched wild-type rats, and the cortical excitability of PKHD1L1+ / - rats was significantly higher than that of wild-type rats.The improved cortical excitability characteristics of PKHD1L1+ / - rats were further verified, and the phenotypes of FAME and other epilepsy patients could be well simulated, which could be further applied to various research scenarios such as epilepsy onset mechanism and design of new antiepileptic drugs.

[0044] The method for detecting a phenotype of an epilepsy animal model according to the present invention includes the steps of: fixing the head and four limbs of an epilepsy animal model in a prone position; performing percutaneous electrical stimulation on the posterior tibial nerve at the ankle of the right hind limb; and then inserting a recording needle electrode subcutaneously into the Cz region of the skull and a reference needle electrode subcutaneously above the nose; The derived signal is filtered and amplified, and then input into the system, and based on the output somatosensory evoked potential, peak latency is measured, cortical excitability is evaluated, and based on the cortical excitability, it is determined whether or not it is an epilepsy model.

[0045] The parameters of the electrical stimulation of the present invention are preferably a constant voltage square wave, with a wave width of 0.1 ms, a frequency of 3 Hz, and an intensity based on the level of causing slight movement of the hind toes, with the needle grounded subcutaneously on the back.The present invention preferably filters and amplifies the derived signal, then inputs it into a computer operating system for averaging, with the number of averaging being 1024, and the analysis time being 56 ms, and finally displays and prints out the somatosensory evoked potential map, and measures its peak latency.

[0046] In order to further explain the present invention, the construction and application of the point mutation rat epilepsy model according to the present invention will be described in detail below with reference to the drawings and examples, but these should not be construed as limiting the scope of protection of the present invention.

[0047] Example 1 1. Screening for pathogenic mutations The family (Figure 17) includes 30 people from 5 generations, 6 of whom are affected, and is consistent with autosomal dominant inheritance characteristics. All patients have myoclonus with or without generalized tonic-clonic seizures and with or without distal limb fine tremor, epileptic seizures all began in adulthood, electrophysiological tests showed improved cortical excitability, antiepileptic drug treatment could effectively control seizures, and the disease course was found to be benign. All patients in the family were clearly diagnosed, and the clinical phenotype was highly consistent. Through whole genome and exome sequencing combined with linkage analysis, it was found that 5 patients (one patient out of 6 patients has died) all had the PKHD1L1 gene exon23:c.2602A>T heterozygous mutation, and 11 controls all had the homozygous mutation, and cosegregation phenomenon exists.

[0048] The PKHD1L1 gene was screened from a group of 246 healthy individuals matched for age, sex, region, and ethnicity, and the mutation site was found to be absent in the healthy individuals, providing initial genetic evidence that the site in PKHD1L1 is the pathogenic mutation in this family.

[0049] Table 5. Clinical data of the patients in the family [Table 5]

[0050] 2. Preparation of rats by knock-in of PKHD1L1 gene (Gene ID: 314917) (The present invention uses Pkhd1l1-201 transcript (ENSRNOT00000005958.7, NM_001034931, abbreviated as PKHD1L1 gene))

[0051] 1. Design and construction of Cas9 / sgRNA 1.1. Cas9 / sgRNA design Based on the sgRNA design principles, design seven sgRNAs for the 5' target site and 3' target site regions, respectively (Table 1).

[0052] 1.2. Construction of Cas9 / sgRNA plasmid Based on Table 1, design the sgRNA sequence synthesis primer and ligate it into the pCS-3G vector (Figure 3) by annealing and polymerization (at 65°C for 5 min). After converting the ligation product, send out the sample for sequencing to verify whether it is accurate.

[0053] The activity of sgRNA was detected using the CRISPR / Cas9 activity detection method-UCATM method developed by Biocytogen, and the results are shown in Figure 5. Therefore, PKHD1L1-sgRNA1 (Guide#1) and PKHD1L1-sgRNA12 (Guide#12) were comprehensively selected for the following experiment.

[0054] 1.3. Preparation of sgRNA RNA PKHD1L1-sgRNA1 and PKHD1L1-sgRNA12 are ligated into a plasmid vector with a T7 promoter and in vitro transcription is performed to obtain microinjectable RNA (Figure 6).

[0055] 1.4. Construction of the targeting vector shown in FIG. 1.5. Microinjection of Cas9 / sgRNA Cas9 / sgRNA and targeting vector were microinjected into rat fertilized eggs, and the birth status of F0 rats after injection is shown in Table 6.

[0056] Table 6. Birth statistics of F0 rats [Table 6]

[0057] 1.6. Detection of F0 generation rat genotype PCR identification was performed using primers PKHD1L1-L-GT-F / PKHD1L1-L-GT-R (Mut: 2662bp, WT: 2650bp) and PKHD1L1-R-GT-F / PKHD1L1-R-GT-R (Mut: 2697bp, WT: 2680bp). The results are shown in Figures 7 and 8. The PCR products and sequencing demonstrated that EY55-072 and EY55-073 were positive F0 rats.

[0058] 1.7. Genotype and Southern blot identification of F1 generation rats Based on the results of identifying the genotype of the tails of F0 generation rats, positive rats were mated with wild-type rats to select F1 generation rats with stable genotypes. The mating results are shown in Table 7.

[0059] Table 7 Mating result statistics [Table 7]

[0060] 1.7.1. F1 Generation Genotyping (Primer designs are as shown for F0) The primer design principle was the same as the F0 generation genotype identification method. Some of the identification results are shown in Figures 9 and 10. Based on the PCR identification and point mutation site sequencing results, it was demonstrated that 1EY55-025, 1EY55-027, 1EY55-029, 1EY55-030, 1EY55-031, 1EY55-032, 1EY55-034, 1EY55-035, 1EY55-036, 1EY55-037 and 1EY55-038 were F1 generation PCR positive rats.

[0061] 1.7.2 Southern blot detection of F1 generation positive rats Tail DNA was extracted from the F1 generation rats with the above PCR identification positive for Southern blot and sequencing detection. The detection results, as shown in Figure 11, show that 1EY55-025, 1EY55-027, 1EY55-029, 1EY55-030, 1EY55-031, 1EY55-032, 1EY55-034, 1EY55-036, 1EY55-037 and 1EY55-038 have been accurately recombined and there is no random insertion.

[0062] 1.7.3. Genotyping of positive mice of the F1 generation with correct recombination and no random insertions PCR verification was performed using primers PKHD1L1-R-GT-F and PKHD1L1-L-GT-R, and sequencing was performed. The results are shown in Figure 12: mut / mut is homozygous, mut / + is heterozygous, and + / + is wild type.

[0063] Example 2 1. Analysis of epilepsy behavior phenotype of PKHD1L1 point mutant rats constructed in Example 1 First, spontaneous epilepsy in three male PKHD1L1 point mutation heterozygous (PKHD1L1+ / -) rats was continuously recorded and observed for 5 days, but no spontaneous epileptiform behavior was observed.

[0064] Next, we used the PTZ epileptogenic lesion model to study the epilepsy susceptibility of PKHD1L1+ / - rats. We took 10 male PKHD1L1+ / - rats and 10 wild-type (WT) rats matched for age and weight, and induced epilepsy using PTZ (40 mg / kg), which is lower than the general model construction dose. As a result, only 2 of the 10 wild-type rats (20%) and 7 of the 10 PKHD1L1+ / - rats (70%) were induced to have 4-5 level grand mal seizures, and their maximum epilepsy levels were also significantly higher than those of the WT group (see Figure 13). Furthermore, we further injected PTZ (5 mg / kg, every 15 minutes) into rats that did not reach the 4-5 level to suppress the occurrence of grand mal seizures, and statistically found that the average dose of PTZ required for WT was also significantly higher than that required for PKHD1L1+ / - rats. PKHD1L1+ / - rats, constructed based on the PKHD1L1 c.2602A>T point mutation discovered through FAME family genomics, are characterized by high susceptibility to epilepsy.

[0065] Second, the constructed PKHD1L1+ / - rats were further examined using SEP method to detect whether they had a phenotype of abnormal cortical excitability.

[0066] The specific method is as follows:

[0067] The head and limbs of a PKHD1L1+ / - rat are fixed in a prone position, and percutaneous electrical stimulation is performed on the posterior tibial nerve at the malleolus of the right hind leg. The parameters of the electrical stimulation are a constant voltage square wave, with a wave width of 0.1 ms, a frequency of 3 Hz, and an intensity that causes the hind toes to move slightly, and the needle is grounded subcutaneously on the back. A recording needle electrode is inserted subcutaneously in the Cz region of the skull, and a reference needle electrode is inserted subcutaneously above the nose. The derived signal is filtered and amplified, then input into a computer operating system for averaging, the number of additions is 1024, the analysis time is 56 ms, and finally the somatosensory evoked potential diagram is displayed and printed, and its peak latency is measured. Each wave is named by polarity and appearance order, for example, P1, P2...N1, N2, where P is a positive wave, N is a negative wave, and the numbers are the numbers of the appearance order of the waves.

[0068] The results are shown in Table 8. Compared with the weight-matched wild-type rat group (WT), the PKHD1L1+ / - rat group (MU) had a significantly shorter SEP latency (10.17±1.17 vs 12.32±1.65, P=0.0071) and a tendency to increase the amplitude (3.95±1.72 vs 2.87±1.6, P=0.1794), suggesting that the cortical excitability of PKHD1L1+ / - rats is significantly higher than that of wild-type rats. The improved cortical excitability characteristics of PKHD1L1+ / - rats can be further verified and can well mimic the phenotypes of FAME and other epilepsy patients. This rat model can also be applied to various research scenarios such as epilepsy development mechanism and design of new antiepileptic drugs.

[0069] Table 8. SEP latency and amplitude values ​​for WT and MU groups [Table 8]

[0070] Example 3 Electrophysiological results of brain slices from genetically modified animals 1. Neuronal excitability was increased in transgenic animals (PKHD1L1+ / - rats), and H89 reduced the frequency of sEPSCs in pyramidal neurons of the cerebral cortex.

[0071] Patch clamp technique was used to record sEPSC and sIPSC of cerebral cortical pyramidal neurons, and the changes in excitatory and inhibitory synaptic transmission of pyramidal neurons in each group were observed to reflect the changes in neuronal excitability. The study found that the amplitude and frequency of sEPSC in epilepsy group were both higher than that of control group, and both amplitude and frequency were reduced after H89 treatment. The experimental results showed that the excitability of epilepsy group rats was improved, and H89 could reduce the amplitude and frequency of cortical sEPSC, reduce its excitability, and play a therapeutic role (Figure 14).

[0072] 2. H89 does not affect the excitability of Na+ and K+ current neurons in pyramidal neurons of the cerebral cortex. Because neuronal excitability is related to Na+ and K+ currents, the present invention records the Na+ and K+ currents of the rat cerebral cortex pyramidal neurons in each group. The results are shown in Figure 15, that there is no difference in the Na+ and K+ currents of the rat pyramidal neurons in each group (3 mice per group, 6 cells in the control group, 6 cells in the epilepsy group, and 6 cells in the epilepsy+H89 group)*, P<0.05.

[0073] 3. H89 can reduce the excitability of cerebral cortical pyramidal neurons in epileptic rats.

[0074] In order to further confirm the excitability of pyramidal neurons in the cerebral cortex, the present invention further recorded the action potential of pyramidal neurons. The action potential can intuitively reflect the excitability of neurons. The results of recording the action potential of pyramidal neurons show that the action potential threshold of the rats in the epilepsy group is reduced (P<0.05), and the threshold increases after H89 treatment (P<0.05). Compared with the control group, the positive afterpotential of the epilepsy group is reduced, and the neuronal potential increases after H89 treatment (P>0.05). There is no difference in the peak and half-length of the action potential of the rats in the two groups. The decrease in the positive afterpotential of pyramidal neurons in the epilepsy group rats shows that the function of their sodium ion pump is reduced. The decrease in the neuronal action potential threshold of the epilepsy rats shows the decrease in the excitability of pyramidal neurons, and H89 reverses this trend and improves the epilepsy symptoms of the rats (Figure 16).

[0075] Although the above embodiments have described the present invention in detail, these are only some of the embodiments of the present invention and not all of the embodiments. Those skilled in the art can obtain other embodiments without performing creative work based on the embodiments, and all of these embodiments are included in the protection scope of the present invention.

Claims

1. Using the 22-23 intron and the 24-25 intron of the PKHD1L1 gene as the target sequence to design sgRNA, annealing the designed sgRNA and then linking it to a plasmid vector with a T7 promoter, and performing in vitro transcription to obtain Cas9 / sgRNA; The Cas9 / sgRNA and the targeting vector are microinjected into a rat fertilized egg to obtain a gene-edited fertilized egg, and the gene-edited fertilized egg is placed into the uterus of a pseudopregnant mouse to contain a chimeric rat with a PKHD1L1 gene point mutation in the F0 generation. Method for constructing a PKHD1L1 gene point mutation rat model.

2. The primer pair for PCR amplifying the 22-23 intron comprises PKHD1L1-5'MSD-F, the nucleotide sequence of which is shown in SEQ ID NO. 1, and PKHD1L1-5'MSD-R, the nucleotide sequence of which is shown in SEQ ID NO. 2; The primer pair for PCR amplification of the 24-25 intron comprises PKHD1L1-3'MSD-F, the nucleotide sequence of which is shown in SEQ ID NO. 3, and PKHD1L1-3'MSD-R, the nucleotide sequence of which is shown in SEQ ID NO. 4; 2. The method of claim 1 .

3. The PCR amplification step comprises: 30 cycles of 94° C. for 5 min, 94° C. for 30 s, 62° C. for 30 s, and 72° C. at 1 kb / min; and amplifying at 72° C. for 10 min.

3. The method of claim 2.

4. The sequences of the sgRNA are shown in SEQ ID NO. 5 and SEQ ID NO.

6.

2. The method of claim 1 .

5. The plasmid vector with a T7 promoter includes a pCS-3G vector.

2. The method of claim 1 .

6. The sgRNA is ligated into the pCS-3G vector by annealing polymerization, and the ligation product is subjected to in vitro transcription to obtain the Cas9 / sgRNA; 6. A method for constructing a semiconductor device according to claim 1, 4 or 5.

7. The nucleotide sequence of the targeting vector is shown in SEQ ID NO.

27.

2. The method of claim 1 .

8. The method further comprises the step of identifying point mutation chimeric rats by PCR after obtaining the F0 generation; Regarding the point mutation chimeric rat, When PCR was performed using PKHD1L1-L-GT-F, the nucleotide sequence of which is shown in SEQ ID NO. 7, and PKHD1L1-L-GT-R, the nucleotide sequence of which is shown in SEQ ID NO. 8, a 2662 bp product was amplified in the point mutation chimeric rat. When PCR identification is performed using PKHD1L1-R-GT-F, the nucleotide sequence of which is shown in SEQ ID NO. 9, and PKHD1L1-R-GT-R, the nucleotide sequence of which is shown in SEQ ID NO. 10, a 2697 bp product is amplified in the point mutation chimeric rat; 2. The method of claim 1 .

9. The step of PCR identification comprises: pre-denaturation at 94°C for 2 min, denaturation at 98°C for 10 s, annealing at 67°C for 30 s, and extension at 68°C at 1 kb / min, for 15 cycles, decreasing the annealing temperature by 0.7°C with each cycle; 25 cycles of denaturation at 98° C. for 10 s, annealing at 57° C. for 30 s, and extension at 68° C. at 1 kb / min; and extending at 68° C. for 10 min.

9. The method of claim 8.

10. A PKHD1L1 gene point mutation rat model obtained by the construction method according to any one of claims 1 to 9.

11. A method for constructing a rat model that stably inherits a PKHD1L1 gene point mutation, comprising the steps of: crossing a PKHD1L1 gene point mutation rat model obtained by the construction method according to any one of claims 1 to 9 with a wild-type rat as a point mutation chimeric rat of F0 generation, and a heterozygote in the F1 generation is a rat model that stably inherits a PKHD1L1 gene point mutation; How to build it:

12. further comprising identifying heterozygotes in the F1 generation by genotyping methods. The method of claim 11 .

13. The method of genotyping includes PCR identification, Southern blot or sequencing identification; 13. The method of claim 12.

14. A rat model stably inheriting a point mutation in the PKHD1L1 gene obtained by the construction method according to any one of claims 11 to 13.

15. Application of the PKHD1L1 gene point mutation rat model according to claim 10 or the rat model stably inheriting the PKHD1L1 gene point mutation according to claim 14 in screening and / or research and production of antiepileptic drugs.

16. The application of the method for detecting abnormalities in cortical excitability in detecting the phenotype of the PKHD1L1 gene point mutation rat model described in claim 10 or the rat model stably inheriting the PKHD1L1 gene point mutation described in claim 14.

17. The method for detecting cortical excitability abnormalities includes somatosensory evoked potentials; 17. The application according to claim 16 .

18. A method for detecting a phenotype of an animal model of epilepsy, comprising: Fixing the head and limbs of the epilepsy animal model in a prone position, and performing percutaneous electrical stimulation on the posterior tibial nerve in the ankle of the right hind leg, and then inserting a recording needle electrode subcutaneously in the Cz region of the skull and a reference needle electrode subcutaneously above the nose; The derived signal is filtered and amplified, and then input to the system, and based on the output somatosensory evoked potential, a peak latency is measured, cortical excitability is evaluated, and based on the cortical excitability, whether or not it is an epilepsy model is determined. A method for detecting a phenotype of an animal model of epilepsy, comprising:

19. The parameters of the electrical stimulation were a constant voltage square wave, with a wave width of 0.1 ms and a frequency of 3 Hz, and the intensity was set to cause slight movement of the hind toes, with the needle grounded subcutaneously on the back. The phenotype detection method according to claim 18.

20. The step of filtering and amplifying the derived signal, inputting it into the system, and measuring the peak latency based on the output somatosensory evoked potential is to filter and amplify the derived signal, input it into the computer operating system for averaging, the number of additions is 1024, and the analysis time is 56 ms; and finally display and print the somatosensory evoked potential map, and measure its peak latency. The phenotype detection method according to claim 18.

21. The epilepsy animal model includes a rat epilepsy model or a mouse epilepsy model; The phenotype detection method according to claim 18.

22. The epilepsy animal model is a PKHD1L1 gene point mutation rat model according to claim 10 or a PKHD1L1 gene point mutation stably inherited rat model according to claim 14. The phenotype detection method according to claim 21.

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