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 the People's Republic of China on December 6, 2022, bearing application number 202211558984.6 and entitled "Establishment and application of a point mutation rat epilepsy model," and a Chinese patent application filed with the State Intellectual Property Office of the People's Republic of China on June 28, 2023, bearing application number 202310770803.4 and entitled "Method and application for identifying cortical excitability 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, chronic central nervous system disorder characterized by recurrent seizures and temporary cerebral dysfunction due to abnormal electrical activity of highly synchronized brain neurons. Clinical manifestations include various motor, sensory, autonomic, and conscious disorders. Epilepsy is a relatively common nervous system disorder, affecting more than 65 million people worldwide. The residual disability, mortality, and complications associated with epilepsy place a significant burden on patients and society. Currently, the mechanisms of epilepsy are poorly understood, and the etiological mechanisms proposed by different scholars vary widely. Therefore, various animal models have been developed to interpret the mechanisms of epilepsy. An animal model of epilepsy refers to a specific species that is prone to seizures induced by external factors or genetic factors. Therefore, in order to detect changes in electroencephalograms and behavioral characteristics during epilepsy, many epilepsy model studies use inducers to maintain epilepsy. For example, TLE animal models are constructed by intraperitoneally administering pilocarpine or pentylenediaminetetrazole, and by injecting kainic acid into the lateral ventricle. However, existing epilepsy animal models cannot fully meet a series of clinical needs, such as the mechanisms of epilepsy development, pathophysiological processes, and screening of therapeutic targets. Therefore, due to the variety of animal models constructed to interpret the mechanisms of epileptogenesis, it is still unknown whether the constructed models can truly represent the mechanisms of epilepsy development.

[0004] Although the validation of a constructed model is a necessary step, existing literature lacks a unified standard for determining whether an epilepsy model has been successfully constructed. For example, when constructing an epilepsy model using zebrafish, a zebrafish behavioral analyzer is generally used for detection, and the degree of epilepsy is reflected based on changes in the activity intensity of the zebrafish larvae. For example, in Chinese Patent CN201910244481.3 (A New Method for Drug-Induced Epilepsy Models), after drug induction, the animals' electroencephalograms are detected, the behavior of epileptic rats is classified, and primary rat hippocampal neurons are cultured in vitro, cell administration is performed, and the electrical activity of the primary rat hippocampal neurons is measured using patch clamp technology. Only after this can the final rat model be determined to be an epilepsy model.

[0005] As is known, electrophysiological signals are the gold standard for assessing 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 model of epilepsy and its application. The point mutation rat has a phenotype of enhanced neuronal excitability, which can closely mimic the phenotypes of FCMTE and other epilepsy patients. This rat model can be further applied to various research scenarios, such as the mechanism of epilepsy onset 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 abnormalities in epilepsy animal models, and to use the somatosensory evoked potential (SEP) method to further detect whether the constructed animal model has a phenotype of cortical excitability abnormalities, thereby determining 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 sgRNA using the 22-23 intron and the 24-25 intron of the PKHD1L1 gene as target sequences; annealing the designed sgRNA; and ligating it into a plasmid vector with a T7 promoter; and performing in vitro transcription to obtain Cas9 / sgRNA. The Cas9 / sgRNA and 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 contains chimeric rats with PKHD1L1 gene point mutations.

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

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

[0011] Preferably, the sequences of the sgRNAs 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 that 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 rats, When PCR identification was performed using PKHD1L1-L-GT-F, the nucleotide sequence of which is set forth in SEQ ID NO. 7, and PKHD1L1-L-GT-R, the nucleotide sequence of which is set forth in SEQ ID NO. 8, a 2662 bp product was amplified in the point mutation chimeric rat. When PCR identification was performed using PKHD1L1-R-GT-F, the nucleotide sequence of which is set forth in SEQ ID NO. 9, and PKHD1L1-R-GT-R, the nucleotide sequence of which is set forth in SEQ ID NO. 10, a 2697 bp product was amplified in the point mutation chimeric rat.

[0015] Preferably, the PCR identification step 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, and extending at 68°C for 1 kb / min, repeating 15 cycles with the annealing temperature decreasing by 0.7°C with each cycle; denaturing at 98°C for 10 seconds, annealing at 57°C for 30 seconds, and extending at 68°C for 1 kb / min, repeating 25 cycles; and extending at 68°C for 10 minutes.

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

[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 the method for detecting cortical excitability abnormalities in detecting phenotypes in animal models of epilepsy.

[0019] Preferably, the method for detecting cortical excitability abnormalities includes 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 limbs of the epilepsy animal model in a prone position, applying percutaneous electrical stimulation to the posterior tibial nerve at the ankle of the right hind limb, and then inserting a recording needle electrode subcutaneously in the Cz region of the calvaria and a reference needle electrode subcutaneously above the nose; The method includes a step of filtering and amplifying the derived signal, inputting it into the system, measuring peak latency based on the output somatosensory evoked potential, evaluating cortical excitability, and determining whether or not it is an epilepsy model based on the cortical excitability.

[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. Finally, the somatosensory evoked potentials are displayed and printed, and their peak latency is measured.

[0023] Preferably, the animal model of epilepsy comprises a rat epilepsy model or a mouse epilepsy model. [Effects 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. This invention uses the CRISPR / Cas9 system to knock in mouse-derived P.L867S into the PKHD1L1 gene, resulting in a point mutation from TTA to TCA, thereby generating PKHD1L1 point mutant rats. Three male PKHD1L1 point mutation heterozygote (PKHD1L1+ / -) rats were continuously recorded and observed for five days. No spontaneous epileptic behavior was observed, but the PKHD1L1+ / - rats exhibited a phenotype of enhanced neuronal excitability, a significantly lower threshold concentration for epileptic seizure induction by intraperitoneal injection of pentylenetetrazole (PTZ) than wild-type rats, and a significantly lower resting potential in brain slice electrophysiology than wild-type rats, demonstrating that the PKHD1L1+ / - rats successfully mimic the phenotypes of FCMTE and other epileptic patients. This rat model can also be used in various research scenarios, such as the mechanisms of epilepsy development 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 an animal model of epilepsy is to use the SEP method to detect whether the constructed animal model has a phenotype of abnormal cortical excitability, thereby determining whether the construction was successful.The detection method of the present invention can be well applied to detecting cortical excitability in rats or mice, and the detection value can be used as a criterion for evaluating the effectiveness of drugs that suppress cortical excitability. [Brief explanation of the drawings]

[0026] [Figure 1] FIG. 1 shows the sequencing results of PKDH1L1 gene mutations. [Figure 2] FIG. 1 is a diagram of the construction policy of targeting vectors. [Figure 3] Precut pCS-3G plasmid profile. [Figure 4] 1 is a targeting vector map. [Figure 5] FIG. 1 shows the results of detecting sgRNA activity. [Figure 6] RNA electropherograms produced with sgRNAs. In the figure, sg1 refers to PKHD1L1-sgRNA1, and sg2 refers to PKHD1L1-sgRNA12. [Figure 7] FIG. 1 shows 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] FIG. 1 shows 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]Figure 1 shows the effects of H89 on sEPSC / sIPSC in cortical pyramidal neurons. (A) Illustrative images of sEPSC currents in pyramidal neurons from rats in each group. (B) Changes in sEPSC amplitude in pyramidal neurons. (C) Changes in sEPSC frequency in pyramidal neurons. (D) Illustrative images of sEPSC currents in pyramidal neurons from rats in each group. (E) Changes in sIPSC amplitude in pyramidal neurons. (F) Changes in sIPSC frequency in pyramidal neurons. (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**P<0.01. [Figure 15] Figure 1 shows 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 the control group, 6 cells in the epilepsy group, and 6 cells in the epilepsy + H89 group) *, P<0.05. [Figure 16] Figure 1 shows the effect of H89 on the action potential of cerebral cortical pyramidal neurons. (A) Illustrative images of action potentials 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 the positive after-potential of the action potential. (E) Half-length of the positive after-potential. (F) Peak potential of the action potential. (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. [Figure 17] FIG. 1 is a family tree diagram of a family in an embodiment of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0027] The method for constructing a PKHD1L1 gene point mutation rat model according to the present invention includes the steps of: designing sgRNA using the 22-23 intron and the 24-25 intron of the PKHD1L1 gene as target sequences; annealing the designed sgRNA; and ligating it into 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 contains chimeric rats with a PKHD1L1 gene point mutation.

[0028] The PKHD1L1 gene of the present invention was preferably screened from a family with familial adult myoclonic epilepsy, the family tree of which is shown in Figure 17. Specifically, the family consists of 30 members across 5 generations, with 6 affected individuals, consistent with autosomal dominant inheritance. All patients had myoclonus with or without generalized tonic-clonic seizures and distal extremity fine tremors. All epileptic seizures began in adulthood. Electroencephalograms showed bilateral symmetric spike-and-wave discharges, evoked potentials showed giant potentials and positive C-reflexes. Antiepileptic drug treatment effectively controlled seizures, and the disease course was benign. All members of the family were clearly diagnosed, and the clinical phenotypes were highly consistent. Whole-genome and exome sequencing combined with linkage analysis revealed that five patients (one of the six patients died) all had a heterozygous mutation in exon 23:c.2602A>T of the PKHD1L1 gene, while all 11 family controls were homozygous, demonstrating cosegregation. Furthermore, the present inventors screened the PKHD1L1 gene in 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 provided initial genetic evidence that the heterozygous mutation in exon 23:c.2602A>T of the PKHD1L1 gene was the pathogenic mutation in this family (Figure 1).

[0029] The PKHD1L1 gene is located on the sense strand of chromosome 7, is approximately 172.46 kb long, and has Gene ID: 314917. The present invention used the Pkhd1l1-201 transcript (ENSRNOT00000005958.7, NM_001034931, abbreviated as the PKHD1L1 gene) to study point mutations in rats. Specifically, the CRISPR / Cas9 system was used to mutate the amino acid Lys at position 867 of the rat PKHD1L1 gene to Ser, and the corresponding base was mutated from TTA to TCA. sgRNAs were designed in introns 22-23 and 24-25 (Figure 2).

[0030] In one embodiment of the present invention, to ensure the efficiency of the designed Cas9 / sgRNA, the target site sequence of SD rat tail was first PCR-amplified and sequenced to verify that the sgRNA recognition sequence perfectly matched the DNA sequence of SD rat tail. The primer information included PKHD1L1-5'MSD-F, whose nucleotide sequence is set forth in SEQ ID NO. 1, and PKHD1L1-5'MSD-R, whose nucleotide sequence is set forth in SEQ ID NO. 2, resulting in a 754-bp amplification product. The primer information also included PKHD1L1-3'MSD-F, whose nucleotide sequence is set forth in SEQ ID NO. 3, and PKHD1L1-3'MSD-R, whose nucleotide sequence is set forth in SEQ ID NO. 4, resulting in a 569-bp amplification product. The PCR amplification step of the present invention preferably includes the following steps: 5 min at 94°C, 30 s at 94°C, 30 s at 62°C, 30 s at 72°C, and 1 kb / min at 72°C for 30 cycles; and 10 min at 72°C for 10 cycles. The present invention sequenced the PCR product and found that the target sequence of SD rat tail was completely consistent with the sequences provided by Genebank and Ensembl, and can be used to mutate the target gene of sgRNA.

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

[0032] The present invention designs two sgRNAs based on the target gene. The sequences of the sgRNAs are shown in SEQ ID NO. 5 and SEQ ID NO. 6, specifically PKHD1L1-sgRNA1 and PKHD1L1-sgRNA12 in Table 2. The present invention preferably uses annealing polymerization to ligate the sgRNAs into the pCS-3G vector (Figure 3). After converting the ligation product, a sample is sent for sequencing to verify accuracy and obtain a Cas9 / sgRNA suitable for microinjection. The annealing and polymerization process of the present invention preferably includes a 5-minute annealing step at 65°C.

[0033] Table 2 sgRNA sequences [Table 2]

[0034] In the present invention, the targeting vector plasmid profile upon microinjection is shown in Figure 4, and the nucleotide sequence of the targeting vector is shown in SEQ ID NO. 27.

[0035] The present invention further includes a step of identifying point mutation chimeric rats by PCR after microinjecting Cas9 / sgRNA and a targeting vector into fertilized rat eggs, which results in F0 rats being born. Due to the rapid cleavage rate in the early fetal stage, the resulting F0 rats are chimeric. The point mutation chimeric rats must simultaneously satisfy the PKHD1L1-L-GT-F / PKHD1L1-L-GT-R (2662 bp) and PKHD1L1-R-GT-F / PKHD1L1-R-GT-R (2697 bp) positivity criteria. In the present invention, PCR identification is preferably performed using the touchdown method, and the reaction system can be 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, and extending at 68°C for 1 kb / min, repeating 15 cycles with the annealing temperature reduced by 0.7°C with each cycle; denaturing at 98°C for 10 seconds, annealing at 57°C for 30 seconds, and extending at 68°C for 1 kb / min, repeating 25 cycles; and extending at 68°C for 10 minutes.

[0036] Table 3. Primers for identifying point mutation chimeras [Table 3]

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

[0038] In the present invention, F1 generation rats with stable genotypes are selected by mating a positive rat with a wild-type rat based on the genotyping results of the tails of F0 generation rats. In the present invention, the resulting F1 generation is genotyped. The genotyping method preferably includes PCR, Southern blot, and sequencing. PCR is preferably the same as that used in the F0 generation, and will not be described here. EcoRV and SpeI are used as restriction enzyme cleavage sites for Southern blot. 3' Probe-A is used to detect whether or not recombination has occurred; if recombination has occurred, two bands, one for the wild type and one for the mutant, will appear. LR Probe-A is used to detect whether or not random insertion has occurred; if no random insertion has occurred, two bands, one for the wild type and one for the mutant, will 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, further PCR verification and sequencing can be used to detect the heterozygote / homozygote genotype. The primer information for detection includes PKHD1L1-R-GT-F and PKHD1L1-L-GT-R. The process preferably involves 30 cycles of 94°C for 5 minutes, 94°C for 30 seconds, 62°C for 30 seconds, and 72°C at 1 kb / min, followed by 72°C for 10 minutes. Since the mutant and wild-type products are both 625 bp, the final homozygote, heterozygote, and wild-type genotypes must 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 the method for detecting cortical excitability abnormalities in detecting phenotypes in animal models of epilepsy.

[0043] The method for detecting cortical excitability abnormalities of the present invention preferably involves somatosensory evoked potentials (SEPs). The present invention is not limited to specific methods for constructing the animal model of epilepsy or animal species, and preferably includes rats, mice, or zebrafish. While SD rats are used in the examples of the present invention, this alone should not be construed as the entire scope of protection of the present invention. Based on pathogenicity studies of the familial adult myoclonic epilepsy (FAME) family shown in Figure 17, the present invention initially demonstrated that a heterozygous mutation in exon 23:c.2602A>T of the PKHD1L1 gene is the pathogenic mutation in the family. Using the CRISPR / Cas9 system, P.L867S was knocked into the PKHD1L1 gene, resulting in a point mutation of the corresponding base from TTA to TCA, thereby constructing PKHD1L1 point mutant rats. Through this detection, electrophysiological tests on patients with epilepsy in this family showed improved cortical excitability, and antiepileptic drug treatment effectively controlled seizures, demonstrating a benign disease course. Furthermore, the constructed PKHD1L1 gene point mutation rats tended to have significantly shorter SEP latencies and increased amplitudes compared with weight-matched wild-type rats, indicating that the cortical excitability of PKHD1L1+ / - rats was significantly higher than that of wild-type rats. Further verification of the improved cortical excitability of PKHD1L1+ / - rats demonstrated that they could closely mimic the phenotypes of FAME and other epilepsy patients, and could be further applied in various research scenarios, such as the mechanism of epilepsy onset and the 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 limbs of the epilepsy animal model in a prone position, applying percutaneous electrical stimulation to the posterior tibial nerve at the ankle of the right hind limb, and then inserting a recording needle electrode subcutaneously in the Cz region of the calvaria and a reference needle electrode subcutaneously above the nose; The method includes a step of filtering and amplifying the derived signal, inputting it into the system, measuring peak latency based on the output somatosensory evoked potential, evaluating cortical excitability, and determining whether or not it is an epilepsy model based on the cortical excitability.

[0045] The electrical stimulation parameters of the present invention are preferably a constant voltage square wave with a pulse width of 0.1 ms, a frequency of 3 Hz, and an intensity sufficient to induce slight movement of the hind toes, with the needles grounded under the skin on the back. The present invention preferably filters and amplifies the derived signals, then inputs them into a computer operating system for averaging, with 1024 additions and an analysis time of 56 ms, and finally displays and prints out the somatosensory evoked potentials and measures their peak latency.

[0046] To further illustrate 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 mutation genes This family (Figure 17) consisted of 30 individuals across five generations, six of whom were affected, consistent with autosomal dominant inheritance. All patients had myoclonus with or without generalized tonic-clonic seizures and distal extremity fine tremors. All epileptic seizures began in adulthood. Electrophysiological testing demonstrated improved cortical excitability. Antiepileptic drug treatment effectively controlled seizures, suggesting a benign disease course. All patients in this family were clearly diagnosed, and the clinical phenotype was highly consistent. Whole-genome and exome sequencing combined with linkage analysis revealed that five patients (one of the six patients has died) all carried the PKHD1L1 exon 23:c.2602A>T heterozygous mutation, while 11 controls were all homozygous, demonstrating cosegregation.

[0048] The PKHD1L1 gene was screened in 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. This provided initial genetic evidence that the site in PKHD1L1 was the pathogenic mutation in the family.

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

[0050] 2. Construction of rats using the PKHD1L1 gene (Gene ID: 314917) knock-in method (the present invention uses the 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, seven sgRNAs were designed for the 5' and 3' target site regions, respectively (Table 1).

[0052] 1.2. Construction of Cas9 / sgRNA plasmid Based on Table 1, synthetic primers for sgRNA sequences were designed and ligated into the pCS-3G vector (Figure 3) by annealing and polymerization (at 65°C for 5 min). The ligation product was converted and the sample was sent for sequencing to verify accuracy.

[0053] The activity of the sgRNA was detected using the UCATM method, a CRISPR / Cas9 activity detection 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 experiments.

[0054] 1.3. sgRNA RNA Preparation 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 Figure 4 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. Determination 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. PCR products and sequencing demonstrated that EY55-072 and EY55-073 were positive F0 rats.

[0058] 1.7. Genotyping of F1 generation rats and Southern blot identification Based on the results of genotyping of the tails of F0 generation rats, positive rats were mated with wild-type rats to obtain 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 principles were the same as those of the F0 generation genotyping method. Some of the identification results are shown in Figures 9 and 10. Based on the PCR identification and point mutation site sequencing results, 1EY55-025, 1EY55-027, 1EY55-029, 1EY55-030, 1EY55-031, 1EY55-032, 1EY55-034, 1EY55-035, 1EY55-036, 1EY55-037 and 1EY55-038 were PCR-positive F1 generation rats.

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

[0062] 1.7.3 Genotyping of F1 generation positive mice 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, as shown in Figure 12, show that mut / mut is homozygous, mut / + is heterozygous, and + / + is wild type.

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

[0064] Next, we used the PTZ epileptogenic lesion model to study the susceptibility of PKHD1L1+ / - rats. Ten male PKHD1L1+ / - rats and ten age- and weight-matched wild-type (WT) rats were induced with PTZ (40 mg / kg), a lower dose than the typical model construction dose. Only two of the ten wild-type rats (20%) and seven of the ten PKHD1L1+ / - rats (70%) developed grand mal seizures of level 4-5, and the maximum seizure level was significantly higher than that of the WT group (Figure 13). Furthermore, rats that did not develop a level 4-5 were injected with additional PTZ (5 mg / kg, every 15 minutes) to prevent the occurrence of grand mal seizures. Statistical analysis revealed that the average PTZ dose required for WT rats was 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 epileptic seizures.

[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 PKHD1L1+ / - rats were immobilized in the prone position, and transcutaneous electrical stimulation (TES) was administered to the posterior tibial nerve at the ankle of the right hind limb. The electrical stimulation parameters were a constant-voltage square wave with a pulse width of 0.1 ms and a frequency of 3 Hz, and the intensity was determined to elicit slight movement of the hind toes. The needle was placed under the skin on the back of the rat. A recording needle electrode was inserted under the skin in the Cz region of the skull, and a reference needle electrode was inserted under the skin above the nose. The derived signals were filtered and amplified, then input into a computer operating system for averaging. The number of averaging steps was 1024, and the analysis time was 56 ms. Finally, the somatosensory evoked potentials (SEPs) were displayed and printed, and their peak latencies were measured. Each wave was named by polarity and order of appearance, e.g., P1, P2, ... N1, N2, where P is the positive wave, N is the negative wave, and the numbers indicate the order of appearance of the wave.

[0068] The results, as shown in Table 8, showed that PKHD1L1 + / - rats (MU) had significantly shorter SEP latencies (10.17 ± 1.17 vs. 12.32 ± 1.65, P = 0.0071) and tended to have increased amplitudes (3.95 ± 1.72 vs. 2.87 ± 1.6, P = 0.1794) compared with weight-matched wild-type rats (WT), suggesting that cortical excitability in PKHD1L1 + / - rats was significantly higher than that in wild-type rats. Further validation of the enhanced cortical excitability in PKHD1L1 + / - rats demonstrated that they closely mimic the phenotypes of FAME and other epilepsy patients. This rat model can also be used in various research scenarios, such as the mechanisms of epilepsy development and the design of new antiepileptic drugs.

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

[0070] Example 3 Electrophysiological experiments using brain slices from genetically modified animals 1. H89 enhances neuronal excitability in transgenic animals (PKHD1L1+ / - rats), and reduces the frequency of sEPSCs in cortical pyramidal neurons.

[0071] Patch clamp techniques were used to record sEPSCs and sIPSCs from cortical pyramidal neurons, and changes in excitatory and inhibitory synaptic transmission in pyramidal neurons in each group were observed to reflect changes in neuronal excitability. The study found that the amplitude and frequency of sEPSCs in the epilepsy group were both higher than in the control group, and that both amplitude and frequency decreased after treatment with H89. The experimental results showed that the excitability of the epilepsy group was improved, and that H89 could reduce the amplitude and frequency of cortical sEPSCs, thereby reducing excitability and providing a therapeutic effect (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 recorded the Na+ and K+ currents of the cerebral cortical pyramidal neurons of rats in each group. The results, as shown in Figure 15, show that there was no difference in the Na+ and K+ currents of 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] To further confirm the excitability of pyramidal neurons in the cerebral cortex, the present inventors also recorded action potentials of pyramidal neurons. Action potentials can intuitively reflect neuronal excitability. The results of pyramidal neuron action potential recordings showed that the action potential threshold of the epilepsy group rats was reduced (P<0.05), and the threshold increased after H89 treatment (P<0.05). Compared with the control group, the positive afterpotential of the epilepsy group was reduced, and the neuronal potential increased after H89 treatment (P>0.05). There was no difference in the peak and half-length of the action potential between the two groups of rats. The reduced positive afterpotential of pyramidal neurons in the epilepsy group rats indicated a decrease in the function of their sodium ion pump. The reduced neuronal action potential threshold of the epilepsy rats indicated a decrease in the excitability of pyramidal neurons, and H89 reversed this trend, improving the rats' epileptic symptoms (Figure 16).

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

Claims

1. Designing sgRNA using the region spanning from intron 22 to intron 23 and the region spanning from intron 24 to intron 25 of the PKHD1L1 gene as target sequences, annealing the double-stranded DNA that produces the designed sgRNA, and then ligating it into 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 to obtain gene-edited fertilized eggs, and the gene-edited fertilized eggs are placed into the uterus of pseudopregnant mice to produce chimeric rats with PKHD1L1 gene point mutations during the F0 generation; The sequence of the sgRNA has the nucleotide sequence shown in SEQ ID NO. 5 and the nucleotide sequence shown in SEQ ID NO. 6; The nucleotide sequence of the targeting vector has the nucleotide sequence shown in SEQ ID NO. 27, The method further comprises the step of identifying point mutation chimeric rats using PCR after obtaining the F0 generation; Regarding the point mutation chimeric rats, When PCR identification was performed using PKHD1L1-L-GT-F having the nucleotide sequence shown in SEQ ID NO. 7 and PKHD1L1-L-GT-R having the nucleotide sequence shown in SEQ ID NO. 8, if a 2662 bp product was amplified, the rat was identified as a point mutation chimeric rat. When PCR identification was performed using PKHD1L1-R-GT-F having the nucleotide sequence shown in SEQ ID NO. 9 and PKHD1L1-R-GT-R having the nucleotide sequence shown in SEQ ID NO. 10, if a 2697 bp product was amplified, the rat was identified as a point mutation chimeric rat. mating the identified chimeric rats with PKHD1L1 gene point mutations of the F0 generation with wild-type rats to obtain F1 generation; and The method further comprises identifying heterozygotes for the PKHD1L1 gene point mutation from the obtained F1 generation by a genotyping method; A heterozygote of the PKHD1L1 gene point mutation is used as an epilepsy rat model. Method for constructing a PKHD1L1 gene point mutation heterozygous F1 generation epilepsy rat model.

2. In constructing the targeting vector, the region spanning from the 22nd intron to the 23rd intron is amplified by PCR, and the primer pair for amplifying this region includes PKHD1L1-5'MSD-F having the nucleotide sequence shown in SEQ ID NO. 1 and PKHD1L1-5'MSD-R having the nucleotide sequence shown in SEQ ID NO. 2; In constructing the targeting vector, the region spanning from the 24th intron to the 25th intron is amplified by PCR, and the primer pair for amplifying this region includes PKHD1L1-3'MSD-F having the nucleotide sequence shown in SEQ ID NO. 3 and PKHD1L1-3'MSD-R having the nucleotide sequence shown in SEQ ID NO.

4.

2. The method of claim 1.

3. The amplification step using the PCR method includes: 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 extending at 72°C for 10 minutes.

3. The method of claim 2.

4. The PCR identification step 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, with the annealing temperature decreasing 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 minutes.

2. The method of claim 1.

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