CN116218905B - Construction and application of point mutation rat epilepsy model - Google Patents
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Abstract
The invention provides construction and application of a point mutation rat epileptic model, and belongs to the technical field of animal modeling. The invention knocks PKHD1L1 gene into murine P.L867S based on CRISPR/Cas9 system, and the corresponding base is changed from TTA to TCA point mutation, thereby constructing and obtaining PKHD1L1 point mutation rat, observing male PKHD1L1 point mutation heterozygote (PKHD 1L 1) +/‑ ) Rats found no spontaneous epileptic behavior; but PKHD1L1 +/‑ Rats have a phenotype of increased neuronal excitability, and are shown to have significantly lower threshold concentration of epileptic seizures induced by intraperitoneal injection of Pentatetrazole (PTZ) than wild-type rats, and have significantly lower resting potential in brain slice electrical biology than wild-type rats, thus being capable of better simulating the phenotypes of FCMTE and other epileptic patients. The rat model can be further applied to various research scenes such as epileptic pathogenesis, design of novel anti-epileptic drugs and the like.
Description
Technical Field
The invention belongs to the technical field of animal modeling, and particularly relates to construction and application of a point mutation rat epileptic model.
Background
Epilepsy (epiepsy) is a serious chronic central nervous system disease, is a temporary brain dysfunction caused by repeated attacks and highly synchronized abnormal discharge of cerebral neurons, clinically manifested as different disorders of movement, sensation, consciousness, autonomic nerves, spirit and the like, is one of the more common diseases of the nervous system, and endangers over 6500 thousands of people worldwide. Disability, mortality and complications associated with epilepsy place a heavy burden on patients and society. At present, little is known about the occurrence mechanism of epilepsy, even the pathogenesis considered by different scholars still has obvious heterogeneity, so the established animal models for explaining the epileptic formation mechanism are various, and the epileptic animal models refer to the epileptic seizure tendency of a specific species with external induction or inheritance. Thus, in order to detect changes in the electroencephalogram and behavioral characteristics in the status of epileptic seizures, a number of epileptic model studies have used inducers to maintain epileptic seizures. Constructing TLE animal models by intraperitoneal administration of pilocarpine or pentyetrazine; injecting the sea acid into the lateral ventricle to establish a TLE animal model and the like. However, the existing epilepsy animal model can not fully meet a series of clinical demands for epileptic pathogenesis, pathophysiological process, treatment target screening and the like.
Disclosure of Invention
The invention aims to provide construction and application of a point mutation rat epileptic model, wherein the point mutation rat has a phenotype of increased neuron excitability, can better simulate the phenotypes of FCMTE and other epileptic patients, and can be further applied to various research scenes such as epileptic pathogenesis, design of novel antiepileptic drugs and the like.
The invention provides a construction method of a PKHD1L1 gene point mutation rat model, which comprises the following steps: designing sgRNA by using 22 th to 23 rd introns and 24 th to 25 th introns of PKHD1L1 gene as targeting sequences, annealing the designed sgRNA, connecting the annealed sgRNA into a plasmid vector with a T7 promoter, and carrying out in vitro transcription to obtain Cas9/sgRNA;
microinjection of Cas9/sgRNA and targeting vector into rat fertilized egg, placing gene edited fertilized egg into pseudopregnant mouse uterus, F 0 The generation contains PKHD1L1 gene point mutation chimeric rat.
Preferably, the method comprises the following steps: the primer pair for PCR amplification of the targeting sequence in the 22 th to 23 rd introns comprises PKHD1L1-5'MSD-F and PKHD1L1-5' MSD-R, wherein the nucleotide sequence of PKHD1L1-5'MSD-F is shown as SEQ ID NO.1, and the nucleotide sequence of PKHD1L1-5' MSD-R is shown as SEQ ID NO. 2;
the primer pair for amplifying the targeting sequence in the 24 th to 25 th introns comprises PKHD1L1-3'MSD-F and PKHD1L1-3' MSD-R, wherein the nucleotide sequence of the PKHD1L1-3'MSD-F is shown as SEQ ID NO.3, and the nucleotide sequence of the PKHD1L1-3' MSD-R is shown as SEQ ID NO. 4.
Preferably, the PCR amplification procedure comprises 94℃for 5min;94℃for 30s,62℃for 30s,72℃for 1kb/min,30 cycles; and at 72℃for 10min.
Preferably, the sgRNA has a sequence shown in SEQ ID NO.5 and SEQ ID NO. 6.
Preferably, the plasmid vector comprises a pCS-3G vector.
Preferably, the nucleotide sequence of the targeting vector is shown as SEQ ID NO. 27.
Preferably, get F 0 Identifying point mutation chimeric rats by PCR after generation;
the point mutant chimeric rat comprising:
when the PKHD1L1-L-GT-F and PKHD1L1-L-GT-R are used for PCR identification, a 2662bp product is amplified from the point mutation chimera rat; the nucleotide sequence of PKHD1L1-L-GT-F is shown as SEQ ID NO.7, and the nucleotide sequence of PKHD1L1-L-GT-R is shown as SEQ ID NO. 8;
and when the PKHD1L1-R-GT-F and PKHD1L1-R-GT-R are used for PCR identification, the point mutation chimera rat is amplified to obtain 2697bp product; the nucleotide sequence of PKHD1L1-R-GT-F is shown as SEQ ID NO.9, and the nucleotide sequence of PKHD1L1-R-GT-R is shown as SEQ ID NO. 10.
Preferably, the PCR identification procedure comprises: pre-denaturation at 94℃for 2min; denaturation at 98℃for 10s, annealing at 67℃for 30s, extension at 68℃for 1kb/min,15 cycles, annealing temperature-0.7℃per cycle; denaturation at 98℃for 10s, annealing at 57℃for 30s, extension at 68℃for 1kb/min,25 cycles; extending at 68℃for 10min.
The invention also provides a construction method of the stable inheritance PKHD1L1 gene point mutation rat model, which comprises the following steps: f obtained by the construction method 0 Substitution point mutant chimeric rat mated with wild rat, F 1 The heterozygote in the generation is a stable inheritance PKHD1L1 gene point mutation rat model.
The invention also provides application of the PKHD1L1 gene point mutation rat model obtained by the construction method or the stable inheritance PKHD1L1 gene point mutation rat model obtained by the construction method in screening and/or developing epileptic drugs.
The beneficial effects are that: the invention preliminarily confirms PKHD1L1 gene exon23:c.2602A through the pathogenicity research of familial adult myoclonus epileptic family>T heterozygous mutations are causative mutations of this family. The invention uses CRISPR/Cas9 system to knock PKHD1L1 gene into murine P.L867S, the corresponding base is changed from TTA to TCA point mutation, thus constructing and obtaining PKHD1L1 point mutation rat, taking 3 male PKHD1L1 point mutation heterozygotes (PKHD 1L 1) +/- ) Rats were observed for a continuous 5 day record, and no spontaneous epileptic behaviour was observed; but PKHD1L1 +/- Rats have a phenotype of increased neuronal excitability, and are shown to have significantly lower threshold concentration of epileptic seizures induced by intraperitoneal injection of Pentatetrazole (PTZ) than wild-type rats, and have significantly lower resting potential in brain slice electrical biology than wild-type rats, thus being capable of better simulating the phenotypes of FCMTE and other epileptic patients. The rat model can be further applied to various research scenes such as epileptic pathogenesis, design of novel anti-epileptic drugs and the like.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions of the prior art, the drawings that are needed in the embodiments will be briefly described below, it being obvious that the drawings in the following description are only some embodiments of the present invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a graph showing the sequencing result of PKDH1L1 gene mutation;
FIG. 2 is a schematic representation of a construction strategy for a targeting vector;
FIG. 3 is a precutpCS-3G plasmid map;
FIG. 4 is a map of a targeting vector;
FIG. 5 is a graph showing the results of the detection of the activity of sgRNA;
FIG. 6 is a graph showing the result of RNA electrophoresis using sg RNA, wherein sg1 refers to PKHD1L1-sgRNA1, and sg2 refers to PKHD1L1-sgRNA12;
FIG. 7 shows F of PKHD1L1-L-GT-F/PKHD1L1-L-GT-R 0 Generation of PCR identification result diagram;
FIG. 8 shows PKHD1L1-R-GT-F/PKF of HD1L1-R-GT-R 0 Generation of PCR identification result diagram;
FIG. 9 shows F of PKHD1L1-L-GT-F/PKHD1L1-L-GT-R 1 Generation of PCR identification result diagram;
FIG. 10 shows F of PKHD1L1-R-GT-F/PKHD1L1-R-GT-R 1 Generation of PCR identification result diagram;
FIG. 11 is F 1 A southern blot detection result diagram of a substitution positive rat;
FIG. 12 positive F with correct recombination and no random insertion 1 Genotype sequencing analysis result diagram of the mice;
FIG. 13 shows PKHD1L1 P.L867S point mutation PKHD1L1 +/ -a graph of epileptic susceptibility studies of rats;
FIG. 14 is a graph of the effect of H89 on cortical pyramidal neurons sEPSC/sIPSC, wherein: (a) an exemplary plot of rat pyramidal neuron sEPSC current for each group; b) Changes in pyramidal neuron sEPSC amplitude; (C) a change in pyramidal neuron peepsc frequency; (D) an exemplary plot of rat pyramidal neuron sEPSC current for each group; (E) changes in pyramidal neuron sIPSC amplitude; (D) Changes in pyramidal neuronal sIPSC frequency (3 mice per group, 6 cells in control group, 6 cells in epileptic group, 6 cells in epileptic+h89 group), P <0.05, P <0.01;
FIG. 15 is a graph of H89 vs. cortical pyramidal neurons Na + /K + A graph of the effect of current, wherein: (A) Cone neuron Na + Is a variation of (2); (B) Cone neuron K + Is a variation of (2); (3 mice per group, 6 cells for control group, 6 cells for epileptic group, 6 cells for epileptic+h89 group), P<0.05；
Fig. 16 is a graph showing the effect of H89 on the change in action potential of cortical pyramidal neurons, wherein: (A) Action potential examples of pyramidal neurons in the cortex of each group of rats; (B) A single action potential example plot for each group of rat pyramidal neurons; (C) an action potential threshold of a pyramidal neuron; (D) a positive post-potential recovery period of action potential; (E) half-way duration of action potential; (F) Peak potential of action potential (3 mice per group, 6 cells of control group, 6 cells of epileptic + H89 group), P <0.05.
Detailed Description
The invention provides a construction method of a PKHD1L1 gene point mutation rat model, which comprises the following steps: designing sgRNA by using 22 th to 23 rd introns and 24 th to 25 th introns of PKHD1L1 gene as targeting sequences, annealing the designed sgRNA, connecting the annealed sgRNA into a plasmid vector with a T7 promoter, and carrying out in vitro transcription to obtain Cas9/sgRNA;
microinjection of Cas9/sgRNA and targeting vector into rat fertilized egg, placing gene edited fertilized egg into pseudopregnant mouse uterus, F 0 The generation contains PKHD1L1 gene point mutation chimeric rat.
The PKHD1L1 gene is preferably obtained by screening a familial adult myoclonus epileptic family, and specifically comprises the following steps: the family is composed of 30 persons in five generations and 6 persons suffering from the disease, and accords with autosomal dominant inheritance characteristics. All patients have myoclonus with or without systemic tonic clonus, with or without fine tremors at the distal ends of limbs, epileptic seizures are adult diseases, electroencephalogram examination prompts bilateral symmetrical ratchet slow wave release, evoked potential examination can see huge potential and C-reflex positive, and anti-epileptic drug treatment can effectively control seizures, and the course of the disease is benign. The family is definite in diagnosis and good in clinical phenotype consistency. Through whole genome exon sequencing and linkage analysis, it is found that PKHD1L1 gene exon23:c.2602A > T heterozygous mutation exists in 5 patients (1 of 6 patients die), and 11 family internal controls are homozygotes and have a co-segregation phenomenon. The invention further screens PKHD1L1 genes in 246 normal people matched with age, sex, region and ethnicity, and finds that the mutation site does not exist in the normal people. Thus, it was initially genetically confirmed that the heterozygous mutation of PKHD1L1 gene exon23:c.2602A > T was a pathogenic mutation of this family (FIG. 1).
The PKHD1L1 gene is on the positive strand of chromosome 7, the full length is about 172.46kb,Gene ID:314917, the invention uses a Pkhd1L1-201 transcript (ENSRNOT 00000005958.7, NM_001034931, PKHD1L1 gene for short) to carry out point mutation research on rats, and specifically comprises the step of mutating 867 th amino acid Lys of the PKHD1L1 gene of the rats into Ser through a CRISPR/Cas9 system, the corresponding base is changed from TTA into TCA, and sgRNA is designed in intron22-23 and intron24-25 (figure 2).
In the embodiment of the invention, in order to ensure the efficiency of the designed Cas9/sgRNA, firstly, PCR amplification and sequencing verification are required to be carried out on the target site sequence of the SD rat tail so as to ensure that the sgRNA recognition sequence is completely consistent with the DNA sequence of the SD rat tail, and the primer information is as follows: PKHD1L1-5'MSD-F and PKHD1L1-5' MSD-R, wherein the nucleotide sequence of PKHD1L1-5'MSD-F is shown as SEQ ID NO.1, the nucleotide sequence of PKHD1L1-5' MSD-R is shown as SEQ ID NO.2, and the amplified product is 754bp; and PKHD1L1-3'MSD-F and PKHD1L1-3' MSD-R, wherein the nucleotide sequence of PKHD1L1-3'MSD-F is shown as SEQ ID NO.3, the nucleotide sequence of PKHD1L1-3' MSD-R is shown as SEQ ID NO.4, and the amplified product is 569bp. The PCR amplification procedure of the present invention preferably comprises 94℃for 5min;94℃for 30s,62℃for 30s,72℃for 1kb/min,30 cycles; and at 72℃for 10min. The invention carries out sequencing on the PCR product, and the result proves that: SD rat tail target sequences are identical to those given by Genebank and Ensembl and can be used for the target gene of point mutant sgRNA.
TABLE 1 primers for large PCR amplification of the sequence of the target site in the rat tail
The invention designs two sgRNAs based on the target genes, wherein the sequences of the sgRNAs are shown in SEQ ID NO.5 and SEQ ID NO.6, and the sequences are specifically shown in PKHD1L1-sgRNA1 and PKHD1L1-sgRNA12 in Table 2. The invention preferably links the sgRNA into the pCS-3G carrier by way of annealing polymerization, and the connection product (figure 3) is converted and then sent to sample sequencing to verify that the sequencing is correct, thus obtaining the Cas9/sgRNA capable of carrying out microinjection. The annealing polymerization according to the invention preferably comprises annealing at 65℃for 5min.
TABLE 2 sgRNA sequences
The plasmid map of the targeting vector required by microinjection is shown in figure 4, and the nucleotide sequence of the targeting vector is shown as SEQ ID NO. 27.
The invention microinjects Cas9/sgRNA and targeting vector into fertilized eggs of rats, F after injection 0 The rat is born, and F is obtained because the embryo early cleavage speed is very fast 0 The rat is chimeric, thus F is obtained 0 Post-generation preferably also includes identifying point mutant chimeric rats using PCR. Wherein, the point mutation chimeric rat needs to simultaneously meet the positive requirement of PKHD1L1-L-GT-F/PKHD1L1-L-GT-R (2662 bp) and PKHD1L1-R-GT-F/PKHD1L1-R-GT-R (2697 bp). The PCR identification is preferably carried out by adopting a TouchDown mode, and a reaction system is constructed according to the instruction of KOD-FX enzyme, and the PCR identification program preferably comprises the following steps: pre-denaturation at 94℃for 2min; denaturation at 98℃for 10s, annealing at 67℃for 30s, extension at 68℃for 1kb/min,15 cycles, annealing temperature-0.7℃per cycle; denaturation at 98℃for 10s, annealing at 57℃for 30s, extension at 68℃for 1kb/min,25 cycles; extending at 68℃for 10min.
TABLE 3 Point mutation chimera identification primers
The invention also provides a construction method of the stable inheritance PKHD1L1 gene point mutation rat model, which comprises the following steps: f obtained by the construction method 0 Substitution point mutant chimeric rat mated with wild rat, F 1 The heterozygote in the generation is a stable inheritance PKHD1L1 gene point mutation rat model.
In the present invention, F is selected 0 Positive rats in the rat tail genotype identification result are mated with wild type to obtain F with stable genotype 1 Rats were replaced. The invention is to obtain F 1 The generation is subjected to genotyping, and the genotyping method preferably comprises PCR (polymerase chain reaction) identification, southern blot and sequencing identification, wherein the PCR identification is preferably the same as detection of the F0 generation, and is not repeated herein. By means ofEcoRV and SpeI served as southern blot cleavage sites. The 3' probe-A was used to detect if correct recombination occurred, and if so, both wild-type and mutant bands would occur. LR Probe-A was used to detect whether random inserts were included, and if not, both wild-type and mutant bands were present.
TABLE 4 3'Probe-A and 3' Probe-A primer information
For F1 generation for which point mutation is determined, heterozygote/homozygote genotype detection can also be performed by PCR verification and sequencing, wherein primer information for detection comprises PKHD1L1-R-GT-F and PKHD1L1-L-GT-R, and the procedure is preferably at 94 ℃ for 5min;94℃for 30s,62℃for 30s,72℃for 1kb/min,30 cycles; at 72℃for 10min, the final homozygous, heterozygous, wild-type genotypes were confirmed by sequencing, since both the mutant and wild-type products were 625 bp.
The invention also provides application of the PKHD1L1 gene point mutation rat model obtained by the construction method or the stable inheritance PKHD1L1 gene point mutation rat model obtained by the construction method in screening and/or developing epileptic drugs.
For further explanation of the present invention, the construction and application of a point mutation rat epilepsy model provided in the present invention will be described in detail with reference to the accompanying drawings and examples, which should not be construed as limiting the scope of the present invention.
Example 1
Preparation of PKHD1L1 Gene knock-in Pattern rats
1. Cas9/sgRNA design and construction
1.1 design of 1Cas9/sgRNA
Based on the design principle of sgrnas, 7 sgrnas were designed in the 5 'target site and 3' target site regions, respectively (table 1).
1.2 construction of the Cas9/sgRNA plasmid
Primers were designed for sgRNA sequence synthesis according to Table 1, ligated into pCS-3G vector by annealing polymerization (5 min at 65 ℃), and the ligation products (FIG. 3) were transformed and sequenced to verify correct.
The result of the activity detection of sgRNA by using the CRISPR/Cas9 activity detection method developed by the Baioci corporation, UCATM mode, is shown in FIG. 5, and thus PKHD1L1-sgRNA1 (Guide # 1) and PKHD1L1-sgRNA12 (Guide # 12) are selected in combination for the next experiment.
1.3 RNA preparation of sgRNA
PKHD1L1-sgRNA1 and PKHD1L1-sgRNA12 were ligated into a T7 promoter-harboring plasmid vector and transcribed in vitro to give microinjected RNA (FIG. 6).
1.4 construction of targeting vectors as described in FIG. 4.
1.5 microinjection of Cas9/sgRNA
Microinjection of Cas9/sgRNA and targeting vector into fertilized ovum of rat, F after injection 0 The birth of the rats is shown in Table 5.
TABLE 5F 0 Rat birth statistics
| Date of day | Family system | Number of transfer zygotes | Expiration date | Birth number | Number of positives |
| 2018/01/12 | SD | 210 | 2018/02/03 | 41 | 0 |
| 2018/03/30 | SD | 250 | 2018/04/21 | 35 | 2 |
| 2018/04/08 | SD | 328 | 2018/04/30 | 44 | 5 |
1.6 F 0 Genotype detection for rats
PCR was performed using the primers PKHD1L1-L-GT-F/PKHD1L1-L-GT-R (Mut: 2662bp, WT:2650 bp) and PKHD1L1-R-GT-F/PKHD1L1-R-GT-R (Mut: 2697bp, WT:2680 bp), and the results are shown in FIG. 7 and FIG. 8, and the results are shown by the PCR products and sequencing that EY55-072 and EY55-073 were positive F 0 Rats.
1.7F 1 Identification of genotype and southern blot of rats
Select F 0 Positive rats in the rat tail genotype identification result are mated with wild type to obtain F with stable genotype 1 The rats were replaced and the mating results are shown in table 6.
TABLE 6 mating result statistics
| Mouse OD | Date of mating | Expiration date | Birth number | Number of positives |
| EY55-073(♀) | 2018/05/24 | 2018/06/14 | 24 | 10 |
1.7.1 F 1 Genotyping of generations (primer design e.g.F 0 Shown):
primer design principle and F 0 As with the genotyping method, the partial results of the genotyping are shown in FIGS. 9 and 10, and the results of the PCR identification and the point mutation site sequencing indicate that 1EY55-025, 1EY55-027, 1EY55-029, 1EY55-030, 1EY55-031, 1EY55-032, 1EY55-034, 1EY55-035, 1EY55-036, 1EY55-037 and 1EY55-038 are F 1 Instead of PCR positive rats.
1.7.2 F 1 Southern blot detection of rats with positive substitution
Extraction of F identified as positive by the PCR 1 Southern blot and sequencing of rat tail DNA was performed, and the results of the detection are shown in FIG. 11, 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 were not randomly inserted.
1.7.3 pair of Positive F with correct recombination and no random insertion 1 Genotyping of mice
PCR was performed using the primers PKHD1L1-R-GT-F and PKHD1L1-L-GT-R, and sequencing, and the results are shown in FIG. 12, wherein mut/mut is homozygous, mut/+ is heterozygous, and +/- + is wild type.
Example 2
Epileptic behavioral phenotypic analysis of PKHD1L1 Point mutant rats constructed in example 1
First, 3 male PKHD1L1 point mutation heterozygotes (PKHD 1L 1) +/- ) Spontaneous seizures were observed in rats with recordings for 5 consecutive days, however, no onset of spontaneous seizures was observed.
Subsequently, PKHD1L1 was studied using the PTZ epilepsy model +/ Rat seizure susceptibility study: 10 male PKHD1L1 were taken each +/- The rats were matched to the weight of the week-old wild-type (WT) rats and seizure induction was performed with PTZ (40 mg/kg) in a lower than conventional modeling dose, as a result, it was found that only 2 out of 10 wild-type rats (20%) were found, and 10 PKHD1L1 was found +/- In rats, 7 (70%) were induced with 4-5 grade seizures, which reached a maximum epileptic grade that was also significantly higher than in the WT group (see fig. 13). Further, additional injection of PTZ (5 mg/kg, every 15 min interval) in rats not reaching grade 4-5 inhibited the onset of epileptic seizure, and it was found that the average dose of PTZ required for WT was also significantly higher than PKHD1L1 +/- The dosage required for the rats. Description PKHD1L1 c.2602A found according to FAME family genomics>PKHD1L1 constructed by T point mutation +/- Rats have characteristics of seizure susceptibility.
Example 3
Results of transgenic animal brain slice electrophysiological experiments
1. Transgenic animal group (PKHD 1L 1) +/- Rat) neuronal excitability is increased, H89 can decrease the frequency of pyramidal neurons sEPSC in the cerebral cortex
sEPSC and sIPSC of the pyramidal neurons of the computer cortex are recorded by adopting a patch clamp technology, and the change of excitatory synaptic transmission and inhibitory synaptic transmission of each group of pyramidal neurons is observed to reflect the change of excitability of the neurons. The study found that the amplitude and frequency of the epileptic group sEPSC were higher than those of the control group, and the amplitude and frequency were reduced after H89 treatment. The experimental results show that H89 can be used to reduce the amplitude and frequency of cortical peescs, reducing their excitability, and thus acting therapeutically in epileptic group rats (fig. 14).
H89 does not affect pyramidal neurons Na in the cerebral cortex + And K + The excitability of the current neurons is such that,
neuronal excitability and Na + And K + The current was related, therefore the present invention records Na in each group of rat cortical pyramidal neurons + And K + A current. The results are shown in FIG. 15, with each group of rat pyramidal neurons Na + And K + No difference in current (3 mice per group, 6 cells in control group, 6 in epileptic group, 6 cells in epileptic + H89 group), P<0.05。
H89 can reduce the excitability of cortical pyramidal neurons in epileptic rats
To further confirm the excitability of pyramidal neurons in the cortex, the present invention also recorded the action potential of pyramidal neurons. Action potentials can intuitively reflect the excitability of neurons. Cone neuron action potential recordings showed a decrease in action potential threshold (P < 0.05) for epileptic group rats and an increase in threshold (P < 0.05) after H89 treatment. Positive potential was decreased after epileptic group compared to control group and neuronal potential was increased after H89 treatment (P > 0.05). The peak values and half-way durations of the action potentials of the two groups of rats are not different. The decrease in postpyramidal potential of epileptic group rat neurons indicates reduced function of sodium ion pump. Indicating that a decrease in the neuronal action potential threshold of epileptic rats indicates a decrease in pyramidal neuronal excitability, H89 reversed this trend and ameliorated the epileptic symptoms in rats (fig. 16).
Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.
Claims (8)
1. The method comprises the following steps ofPKHD1L1The construction method of the gene point mutation rat model is characterized by comprising the following steps: by means ofPKHD1L1Designing sgRNA by taking 22 th to 23 rd introns and 24 th to 25 th introns of genes as targeting sequences, and annealing the designed sgRNAConnecting into a plasmid vector with a T7 promoter, and performing in vitro transcription to obtain Cas9/sgRNA;
microinjection of Cas9/sgRNA and targeting vector into rat fertilized egg, placing gene edited fertilized egg into pseudopregnant mouse uterus, F 0 The generation containsPKHD1L1Gene point mutation chimeric rats;
the sequence of the sgRNA is shown as SEQ ID NO.5 and SEQ ID NO. 6;
the nucleotide sequence of the targeting vector is shown as SEQ ID NO. 27;
PKHD1L1the gene point mutation chimera rat knocks in the point mutation P.L 867S.
2. The method of construction according to claim 1, comprising the steps of: the primer pair for PCR amplification of the target sequence in the 22 th-23 rd introns comprises PKHD1L1-5'MSD-F and PKHD1L1-5' MSD-R, wherein the nucleotide sequence of PKHD1L1-5'MSD-F is shown as SEQ ID NO.1, and the nucleotide sequence of PKHD1L1-5' MSD-R is shown as SEQ ID NO. 2;
the primer pair for amplifying the targeting sequence in the 24 th to 25 th introns comprises PKHD1L1-3'MSD-F and PKHD1L1-3' MSD-R, wherein the nucleotide sequence of the PKHD1L1-3'MSD-F is shown as SEQ ID NO.3, and the nucleotide sequence of the PKHD1L1-3' MSD-R is shown as SEQ ID NO. 4.
3. The method of claim 2, wherein the PCR amplification procedure comprises 94℃for 5min;94℃for 30s,62℃for 30s,72℃for 1kb/min,30 cycles; and at 72℃for 10min.
4. The method of claim 1, wherein the plasmid vector comprises a pCS-3G vector.
5. The method of claim 1, wherein F is obtained 0 Identifying point mutation chimeric rats by PCR after generation;
the point mutant chimeric rat comprising:
when the PKHD1L1-L-GT-F and PKHD1L1-L-GT-R are used for PCR identification, a 2662bp product is amplified from the point mutation chimera rat; the nucleotide sequence of PKHD1L1-L-GT-F is shown as SEQ ID NO.7, and the nucleotide sequence of PKHD1L1-L-GT-R is shown as SEQ ID NO. 8;
and when the PKHD1L1-R-GT-F and PKHD1L1-R-GT-R are used for PCR identification, the point mutation chimera rat is amplified to obtain 2697bp product; the nucleotide sequence of PKHD1L1-R-GT-F is shown as SEQ ID NO.9, and the nucleotide sequence of PKHD1L1-R-GT-R is shown as SEQ ID NO. 10.
6. The method of claim 5, wherein the PCR identification procedure comprises: pre-denaturation at 94℃for 2min; denaturation at 98℃for 10s, annealing at 67℃for 30s, extension at 68℃for 1kb/min,15 cycles, annealing temperature-0.7℃per cycle; denaturation at 98℃for 10s, annealing at 57℃for 30s, extension at 68℃for 1kb/min,25 cycles; extending at 68℃for 10min.
7. Stable inheritancePKHD1L1The construction method of the gene point mutation rat model is characterized by comprising the following steps: f obtained by the construction method according to any one of claims 1 to 6 0 Substitution point mutant chimeric rat mated with wild rat, F 1 Heterozygotes in the generation are stably inheritedPKHD1L1Gene point mutation rat model.
8. The construction method according to claim 1 to 6PKHD1L1Stable inheritance obtained by gene point mutation rat model or construction method according to claim 7PKHD1L1The application of the gene point mutation rat model in screening and/or developing epileptic drugs.
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