WO2020228305A1 - Construction method for mutant gabrg2 transgenic zebrafish epilepsy model and applications - Google Patents

Abstract

Disclosed is a method constructing a mutant GABRG2 transgene-induced epileptic zebrafish model, with zebrafish serving as a model animal, a mutated human GABRG2 gene is transferred to the zebrafish for same to become a zebrafish system having an epileptic phenotype. The zebrafish system is applicable in researching relations between GABRG2 mutations and the pathogenesis of epilepsy and applicable in screening an antiepileptic medicament.

Description

Construction method and application of a mutant GABRG2 transgenic zebrafish epilepsy model Technical field

The invention belongs to the technical field of animal model preparation in biology and basic medical research, and specifically relates to a transgenic zebrafish epilepsy model constructed by using a mutant of the epilepsy-related gene GABRG2, and a method for constructing the epilepsy zebrafish model and its use in epilepsy Application in research work such as pathogenesis, drug screening and anti-epileptic drug target identification.

Background technique

Epilepsy is a common chronic neurological disease characterized by spontaneous unprovoked recurrent epileptic seizures. It is one of the most common disabling neurological diseases, affecting more than 50 million people worldwide, about 2.4 million each year The person is diagnosed with epilepsy. The increase in the frequency, duration and severity of epileptic seizures may increase the cognitive and emotional disorders of patients, lead to the occurrence of epileptic encephalopathy, and cause a series of sequelae such as cognitive impairment and emotional anxiety. Epilepsy is divided into primary and secondary according to the cause, and primary epilepsy is mostly related to genetic defects. In the past two decades, with the development of genome technology such as next-generation sequencing, nearly 1,000 gene mutations have been identified that are related to the occurrence of epilepsy, including many ion channel and non-ion channel gene mutations.

γ- aminobutyric acid A receptor (GABA _A R) is a major mediator of the central nervous system of fast inhibitory synaptic transmission, it has been repeatedly shown to have a key role in the seizure. GABA _A receptor is an isoprene dimer formed by the assembly of multiple subunits. It has been detected that the mutations of GABA _A receptor subunits α1, β1, β2, β3, γ2 and δ are closely related to the onset of epilepsy, and These mutations usually have autosomal dominant inheritance characteristics.

The GABA _A receptor γ2 subunit (GABRG2) is of great significance for GABA _A receptor transport and synaptic function. At present, more than ten GABRG2 mutations related to epilepsy have been found, including missense mutations, nonsense mutations, and frameshifts. Mutations and truncation mutations. These mutations reduce the function of ion channels to varying degrees through different mechanisms, such as degradation of mRNA and endoplasmic reticulum related proteins, affecting the normal function of receptors, and aggregation of mutant proteins. Febrile seizures, to more complicated inherited epilepsy with febrile seizures, and even severe Dravet syndrome. Studies have found a group of missense GABRG2 mutations that are highly related to epileptic encephalopathy in clinical children. The F343L mutation is located in the transmembrane region of GABRG2, which reduces the GABA current by about 30%, and children with this mutation have There is basically no sensitivity to antiepileptic drugs.

As a vertebrate, the zebrafish has a complex nervous system and its genetic structure is similar to that of humans. About 70% of its genes are shared with humans, and about 84% of genes related to human diseases are also expressed in zebrafish. Compared with rodents such as mice, zebrafish has simple genetic manipulation and is more suitable for preparing epilepsy models, and for high-throughput drug screening and new molecular target identification of antiepileptic drugs. GABA _A receptors in zebrafish usually exist in the form of α1β2γ2, α1β2δ, α2bβ3γ2, α2bβ3δ, α4β2γ2, α4β2δ, α6bβ2γ2 and α6bβ2δ. Although there has been a knockout model of GABA _A receptor α1 subunit (GABRA1), in which zebrafish juveniles exhibited epileptic phenotypes such as increased activity, active brain electrical activity, and increased mortality, there is still no GABRG2 mutant epileptic zebra Report of the fish line.

Summary of the invention

The purpose of the present invention is to provide a method for constructing a zebrafish model of epilepsy induced by a mutant GABRG2 transgene. This method uses zebrafish as a model animal, and transforms the mutant GABRG2 gene into zebrafish to make it a zebrafish line with epileptic phenotype. This zebrafish line can be used to study the relationship between GABRG2 mutation and the pathogenesis of epilepsy, and for anti- Epilepsy drug screening.

In order to achieve the above objective, the technical solution of the present invention is as follows:

A method for constructing a mutant GABRG2 transgenic zebrafish epilepsy model. The mutant human GABRG2 gene is transferred into zebrafish to make it a zebrafish line with epileptic phenotype.

The mutant zebrafish is a zebrafish with a missense mutant GABRG2 gene.

The zebrafish model of epilepsy is caused by GABRG2 missense mutation.

The GABRG2 missense mutation is the mutation of base T to C (F343L) in the Phe codon of amino acid 343 of human GABRG2 gene.

The sequence of the transferred mutant human GABRG2 gene is shown in SEQ ID No:1.

Preferably, the construction method of the present invention uses the pDestTol2CG2 expression vector (shown in SEQ ID No: 2).

The pDestTol2CG2 expression vector is the excision of the gene encoding the transposase in the Tol2 transposon and replaced it with the CMLC2-EGFP-polyA expression sequence. This plasmid and the transposase mRNA are microinjected into zebrafish single-cell embryos, and the zebrafish heart can be observed Green fluorescent protein is specifically expressed in tissues. (Kwan KM, Fujimoto E, Grabher C, et al. The Tol2 kit: a multisite gateway-based construction kit for Tol2 transposon transgenesis constructs. Dev Dyn, 2007, 236(11): 3088-3099).

Preferably, the construction method of the present invention adopts gateway technology to construct the expression vector, including the following steps:

(1) Construction of GABRG2 mutant expression vector: Synthesize the mutant human GABRG2 gene, clone it into the pME-MCS plasmid vector, and construct a middle entry clone: pME-GABRG2 ^1027T>C (sequence shown in SEQ ID No: 3) ; Cloning contains the sequence of HuC neuron-specific promoter region, constructing vector 5'entry clone: p5E-HuC (sequence shown in SEQ ID No: 4) and 3'entry clone: p3E-polyA (sequence shown in SEQ ID No: 5); Recombinantly clone the 5'entry clone, middle entry clone and 3'entry clone vector into the expression vector pDestTol2CG2 by the Gateway LR reaction to obtain the expression vector: pDestTol2CG2; HuC:GABRG2 ^1027T>C -polyA (SEQ ID No : 6).

(2) Microinjection of zebrafish embryos: Microinject the above-mentioned GABRG2 mutant expression vector and Tol2 transposase/transposase mRNA into 1-2 cell stage wild-type zebrafish zygotes, and wait until the embryos develop to 24hpf Afterwards, the embryos expressing green fluorescence in the heart were screened to obtain the positive zebrafish containing GABRG2 mutant sequence.

The above-mentioned construction method further includes the steps of further identifying the genotype of the transgenic zebrafish and obtaining a stable genetic line: after the positive zebrafish embryos containing the GABRG2 mutant sequence are matured, the genotypes are identified by PCR, and the GABRG2 mutant sequence is selected. The positive zebrafish were crossed with wild-type zebrafish to obtain F1 generation zebrafish, and the GABRG2 mutant zebrafish line was finally screened through the green fluorescence expressed in the heart and genomic DNA detection methods.

The present invention observes the epilepsy phenotype of the GABRG2 mutant zebrafish line and verifies it by related experiments: the zebrafish juvenile high-throughput behavior screening box (DanioVision Chamber) and the animal motion trajectory tracking system (EthoVision XT) are used to record wild-type zebras. The trajectory of the fish and the mutant zebrafish under normal conditions is compared, and the swimming distance and average swimming speed are compared, and the zebrafish's excitement degree is analyzed by video tracking. Then PTZ and light stimulation were used to induce seizures, and the susceptibility of GABRG2 mutant fish lines to the related stimuli that induced seizures was analyzed. Furthermore, real-time quantitative PCR was used to detect the expression of c-fos gene in the brain of the GABRG2 mutant zebrafish line, which proved that the mutant zebrafish line had the characteristics of an epilepsy model from the perspective of molecular biology.

Another object of the present invention is to provide an application of a zebrafish epilepsy model that can be used in basic scientific research and clinical application research. Based on the GABRG2 mutant zebrafish epilepsy model, it can study the biochemical, molecular biology and genetics related to the onset of epilepsy. It can also be used for the screening and research of clinical anti-epileptic drugs, which will be commonly used clinically anti-epileptic drugs. Such as valproic acid, carbamazepine, clonazepam, Levetiracetam, etc., directly added to the zebrafish culture solution, and then through the zebrafish swimming behavior Analysis and other means to screen effective drugs for the treatment of epilepsy, and to study the mechanism of drug action.

The invention provides an effective method basis for the establishment of a zebrafish epilepsy model, and also provides an important tool for epilepsy pathological research and anti-epileptic drug screening research, and has greater basic and clinical research application value.

Description of the drawings

Figure 1 is a schematic diagram of using Gateway technology to construct expression plasmids required for transgenic fish. Take the F343L mutant as an example, construct 5'entry clone (p5E-HuC), 3'entry clone (p3E-polyA) and middle entry clone (pME-GABRG2 ^1027T>C ) respectively, and obtain a plasmid expressing mutant GABRG2 by LR reaction pDestTol2CG2; HuC:GABRG2 ^1027T>C -polyA.

Figure 2 is a graph of agarose gel electrophoresis of the constructed GABRG2 (F343L) mutant plasmid. M is the DNA molecular weight Marker.

Figure 3 shows the obtained F1 generation positive mutant zebrafish. Among them, WT is a wild-type zebrafish and F343L is a mutant zebrafish. The heart can be seen to express green fluorescence.

Figure 4 shows zebrafish genomic DNA sequencing to identify genotypes.

Figure 5 shows the swimming behavior analysis of GABRG2 (F343L) mutant zebrafish induced by PTZ. Picture A is a visualization of swimming trajectories of wild-type and mutant zebrafish in normal medium or 15mM PTZ-containing medium (1min); Picture B is wild-type and mutant zebrafish in normal medium or 15mM PTZ, respectively Analysis of movement distance in PTZ culture medium (30min); C is a statistical analysis graph (*P<0.05, **P<0.01).

Figure 6 shows the swimming behavior analysis of GABRG2 (F343L) mutant zebrafish induced by light stimulation. Picture A is a visualization of swimming trajectories of wild-type and mutant zebrafish; Picture B is an analysis of swimming behavior of wild-type and mutant-type zebrafish at different time periods when the lights are turned on or off; C is a statistical analysis chart (**P< 0.01).

Figure 7 shows the real-time fluorescence quantitative PCR detection of wild-type and GABRG2 (F343L) mutant zebrafish at different times (24h, 48h, 72h, 96h, 120h) c-fos gene expression (**P<0.01, similar to WT ratio).

Figure 8 shows the swimming behavior analysis of GABRG2 (F343L) mutant zebrafish in different antiepileptic drugs. Add 50μM, 100μM, 50μM, 30mM sodium valproate, carbamazepine, clonazepam, and levetiracetam to the medium respectively. After 24h, use the video analysis system to record for 30min, collect the parameters every minute, and analyze Zebrafish movement distance (*P<0.05, **P<0.01).

Figure 9 shows the changes in the circadian rhythm of GABRG2 (R177G) mutant zebrafish swimming. Under the condition of normal light time, the swimming behavior of 5dpf zebrafish juveniles is recorded by the motion track tracking system. The recording starts at 4 pm for a total of 24 hours (14 hours of light and 10 hours of darkness). The behavior parameters are recorded every 1 hour, and each time is compared. The difference in total movement distance between wild-type and transgenic zebrafish was analyzed, and circadian rhythm changes were analyzed.

Detailed ways

The present invention will be further described in detail below in conjunction with embodiments, but the implementation method of the present invention is not limited to this.

The term "wild type" or "WT" used in the present invention refers to wild type zebrafish. Unless otherwise specified, other related terms generally have the meanings commonly understood by those of ordinary skill in the art.

In the following examples, various processes and methods that are not described in detail are conventional methods known in the art.

Example 1 Construction of GABRG2 (F343L) transgenic zebrafish epilepsy model strain.

This Example 1 provides a scheme for transferring mutant GABRG2 (F343L) into zebrafish fertilized eggs by transgenic means and constructing a zebrafish model of epilepsy. Specifically include the following steps:

The first step is to construct the transgene expression vector: use Pubmed database to search for the human GABRG2 gene sequence, synthesize the GABRG2 open reading frame sequence, mutate the 1027th base T to C (SEQ ID No:1), and clone it into pME- MCS plasmid vector, the ^5'cloning site is EcoRI, the ^3'cloning site is XbaI, and the middle entry clone: pME-GABRG2 ^1027T>C (SEQ ID No: 3) is constructed. The clone contains the sequence of the HuC neuron-specific promoter region to construct the 5'entry clone: p5E-HuC vector (SEQ ID No: 4). Clone 3'entry clone: p3E-polyA vector (SEQ ID No: 5). Recombinantly clone the 5'entry clone, middle entry clone and 3'entry clone vector into the expression vector pDestTol2CG2 by LR reaction to construct the transgenic mutation expression vector pDestTol2CG2; HuC:GABRG2 ^1027T>C- polyA (SEQ ID No:6) (such as Figure 1), the reaction system is as follows: TE buffer 4μL, p5E-HuC(30ng) 1μL, pME-GABRG2 ^1027T>C (30ng)1μL, p3E-polyA(30ng)1μL, pDestTol2CG2(60ng)1μL, LR clonase II 2μL, total volume 10μL. React at 25°C for 20h. The constructed plasmid was identified by agarose gel electrophoresis. As shown in Figure 2, a positive band was detected near 20 kb, which is the desired GABRG2 gene mutation expression vector.

Tol2 transposase mRNA (pCS2FA-transposase, the DNA sequence is shown in SEQ ID No: 7): The digestion system is as follows: 10×Buffer 5μL, DNA 1μg, NotI-HF restriction endonuclease 1μL, add ddH2O to The total volume is 50μL. 37℃ for 15min, 65℃ heat inactivation for 15min. The digested product was subjected to agarose gel electrophoresis, and the Tol2 transposase mRNA was transcribed with RNA in vitro transcription kit after gel recovery.

The second step, microinjection of zebrafish embryos: the above transgenic mutation expression vector pDestTol2CG2; HuC:GABRG2 ^1027T>C- polyA and Tol2 transposase mRNA were simultaneously microinjected into wild-type zebrafish zygotes at the 1-2 cell stage Inside, after the embryo develops to 24hpf, the embryos with green fluorescence in the heart are screened for follow-up experiments. After the zebrafish matures, the genotype is identified by PCR and genome sequencing. As shown in Figure 3, a mutant zebrafish embryo with a heart expressing green fluorescence was obtained.

The third step is to identify the genotype of transgenic zebrafish and obtain stable genetic strains: zebrafish genomic DNA extraction: use the genomic DNA extraction kit (All-DNA-Out) to extract zebrafish genomic DNA in one step, and add 20ul All-DNA- Heat the Out solution at 85°C for 5 min.

Genotype identification: PCR reaction was used to identify zebrafish genotype. The reaction system is as follows: 2×KOD FX Buffer 10 μL, dNTP 4 μL, upstream primer (Primer F) 0.6 μL, downstream primer (Primer R) 0.6 μL, KOD FX enzyme 0.4 μL , DNA template 2μL, total volume 20μL. The reaction conditions are: 95°C for 5min; 98°C for 10s, 50°C for 30s, 68°C for 30s (35 cycles); 68°C for 5min. The primer sequence is: F: 5'-GCACAGGAAGCTCAGTCTAC-3' (SEQ ID No: 8), R: 5'-GGAGTCCATTTTGGCAATGC-3' (SEQ ID No: 9).

Obtaining stable genetic lines of transgenic zebrafish: select the zebrafish with positive results of the above gene identification and cross with wild-type zebrafish to obtain F1 generation zebrafish, and obtain the mutant GABRG2 transgenic zebrafish line through the heart expression green fluorescence and genomic DNA detection . After genomic DNA sequencing, the result is shown in Figure 4, it can be seen that the 1027th base has been mutated from T to C, confirming that the zebrafish contains GABRG2 (F343L) mutation.

The fourth step is the observation of the epilepsy phenotype of the GABRG2 (F343L) mutant zebrafish line and related experimental verification: observation of the epileptic behavior phenotype: select 5dpf zebrafish juveniles, and use the zebrafish juvenile high-throughput behavior screening box (DanioVision) Chamber) and animal motion trajectory tracking system (EthoVision XT 13), record the motion trajectories of wild zebrafish and mutant zebrafish under normal conditions for a total of 30 minutes, and analyze the excitement of zebrafish juveniles by swimming distance and average swimming speed The results are shown in Figure 5. It was found that the excitability of the mutant fish increased under normal culture conditions and the swimming distance increased significantly.

Changes in zebrafish swimming behavior in PTZ: add the seizure inducer PTZ with a final concentration of 15 mM to the zebrafish culture medium, and use EthoVision XT 13 zebrafish behavior video analysis system to record zebrafish swimming behavior, every 1 minute A parameter is recorded for 30 minutes. Compare the behavioral differences between wild-type and transgenic zebrafish. The results are shown in Figure 5. PTZ can cause a significant increase in excitability of wild-type zebrafish and transgenic zebrafish, but the swimming distance of transgenic zebrafish is still significantly higher than that of wild-type zebrafish.

Changes in zebrafish swimming behavior under the stimulation of light changes: under alternating light and dark conditions (5s), the zebrafish behavior video analysis system is used to record the zebrafish’s movement behavior in 100 cycles, every 5s is a parameter, statistics The difference in average moving distance between wild-type zebrafish and transgenic zebrafish every 5s. The results are shown in Figure 6. The distance of movement of transgenic zebrafish under normal light is significantly higher than that of wild type, but light and dark stimulation have no significant effect on wild type and transgenic zebrafish.

Phenotype verification of epilepsy molecular biology: real-time fluorescence quantitative PCR to detect c-fos gene expression: extract the total RNA of wild-type and transgenic zebrafish 24h, 48h, 72h, 96h, 120h after birth, and reverse transcribed into cDNA, using SYBR green The dye method is used for real-time fluorescent quantitative PCR analysis. The reaction conditions are as follows: 95°C for 6min; 95°C for 10s, 60°C for 30s, 72°C for 10s (41 cycles); 65°C to 95°C with a gradient of temperature for 5s every 0.5°C. The primer sequence is: F: 5'-AACTGTCACGGCGATCTCTT-3' (SEQ ID No: 10), R: 5-CTTGCAGATGGGTTTGTGTG-3' (SEQ ID No: 11). The results are shown in Figure 7. At 72 hours after birth, the expression of c-fos in the transgenic zebrafish reached the highest peak, which was significantly higher than that of the wild type, and then the expression gradually decreased.

Example 2 Application of GABRG2 (F343L) transgenic zebrafish epilepsy model in the screening of antiepileptic drugs.

Select commonly used anti-epileptic drugs in clinical practice, such as sodium valproate, carbamazepine, clonazepam, levetiracetam, etc., and add them to the zebrafish culture medium at concentrations of 50μM, 100μM, 50μM, 30mM, and add 24 hours after the drug was administered, the video analysis system was used to record for 30 minutes, and the parameters were collected every minute. Through swimming behavior analysis and other methods, we can screen drugs that have therapeutic effects on the epileptic phenotype of transgenic zebrafish. The results are shown in Figure 8. The above four drugs can significantly reduce the swimming distance of transgenic zebrafish and reduce their excitability, suggesting that the above drugs have inhibitory effects on the seizures of GABRG2 (F343L) transgenic zebrafish.

Example 3 Construction of GABRG2 (R177G) transgenic zebrafish strain

According to the method described in Example 1, different mutant GABRG2 gene expression sequences were designed, and the mutation site was the mutation of G at base 529 of the human GABRG2 gene (SEQ ID No: 12). The GABRG2 mutant expression vector pDestTol2CG2; HuC:GABRG2 ^529C>G- polyA was constructed, and the transgenic zebrafish gene line was obtained by microinjecting zebrafish embryos.

Phenotype analysis of GABRG2(R177G) transgenic zebrafish strains:

Select 5dpf zebrafish juveniles, use the high-throughput zebrafish juvenile behavior screening box (DanioVision Chamber) and animal motion trajectory tracking system (EthoVision XT 13) to record the movement of wild zebrafish and mutant zebrafish under normal conditions The track record starts at 4 pm for a total of 24 hours (14 hours of light and 10 hours of darkness). The behavioral parameters are recorded every 1 hour. The difference in the total movement distance of wild-type and transgenic zebrafish in each time period is compared, and the circadian rhythm changes are analyzed. The excitement degree of zebrafish juveniles was analyzed by swimming distance, and the results are shown in Figure 9. It was found that under normal culture conditions, GABRG2 (R177G) transgenic zebrafish had no significant increase in swimming distance compared with wild-type zebrafish. The results showed that: GABRG2 (R177G) transgenic zebrafish had no epileptic phenotype.

The above-mentioned embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above-mentioned embodiments, and any other changes, modifications, substitutions, combinations, etc. made without departing from the spirit and principle of the present invention Simplified, all should be equivalent replacement methods, and they are all included in the protection scope of the present invention.

Claims (10)

A method for constructing a mutant GABRG2 transgenic zebrafish epilepsy model is characterized in that the mutant human GABRG2 gene is transferred into zebrafish to make it a zebrafish line with epileptic phenotype. The construction method according to claim 1, wherein the mutant human GABRG2 gene is a mutation of base T to C in the Phe codon of amino acid 343 of human GABRG2. The construction method according to claim 1, characterized in that the sequence of the transferred mutant human GABRG2 gene is shown in SEQ ID No:1. The construction method of claim 3, wherein the expression vector is pDestTol2CG2. The construction method according to claim 4, characterized in that the gateway technology is used to construct the expression vector. The construction method according to claim 5, characterized by comprising the following steps: (1) Construction of GABRG2 mutant expression vector: Synthesize the mutant human GABRG2 gene, clone it into the pME-MCS plasmid vector, construct a middle entry clone: pME-GABRG2 ^1027T>C ; clone the HuC neuron-specific promoter region Sequence, construct the vector 5'entry clone: p5E-HuC and 3'entry clone: p3E-polyA; through the Gateway LR reaction, clone the 5'entry clone, middle entry clone and 3'entry clone vector into the expression vector pDestTol2CG2 to obtain Expression vector: pDestTol2CG2; HuC: GABRG2 ^1027T>C -polyA; (2) Microinjection of zebrafish embryos: Microinject the above-mentioned GABRG2 mutant expression vector and Tol2 transposase/transposase mRNA into 1-2 cell stage wild-type zebrafish zygotes, and wait until the embryos develop to 24hpf Afterwards, the embryos expressing green fluorescence in the heart were screened to obtain the positive zebrafish containing GABRG2 mutant sequence. The construction method according to claim 6, characterized in that the sequence of pDestTol2CG2; HuC:GABRG2 ^1027T>C- polyA is shown in SEQ ID No:6. The construction method according to any one of claims 1-7, which is characterized in that it further comprises the steps of identifying the genotype of the transgenic zebrafish and obtaining a stable genetic strain. The construction method according to claim 8, characterized in that after the positive zebrafish embryos containing the GABRG2 mutant sequence are matured, their genotypes are identified by PCR, and the positive zebrafish and wild-type zebrafish containing the GABRG2 mutant sequence are selected Hybridization, F1 generation zebrafish was obtained, and the GABRG2 mutant zebrafish line was finally screened through the green fluorescence expressed in the heart and genomic DNA detection methods. Application of the mutant GABRG2 transgenic zebrafish epilepsy model constructed by the method of any one of claims 1-9 in epilepsy pathological research and anti-epileptic drug screening.

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