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CN111944835B - Construction method and application of mutant GABRG2 transgenic zebra fish epilepsy model

Detailed Description

The present invention will be described in further detail with reference to examples, but the method of carrying out the present invention is not limited thereto.

The terms "wild type" or "WT" as used herein both refer to wild type zebrafish. Other related terms, unless otherwise specified, generally have the meanings that are commonly understood by those of ordinary skill in the art.

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

Example 1 construction of GABRG2(F343L) transgenic zebrafish model line for epilepsy.

This example 1 provides a protocol for transferring a mutant GABRG2(F343L) into fertilized eggs of zebra fish by transgenic means and constructing a zebra fish model of epilepsy. The method specifically comprises the following steps:

firstly, constructing a transgenic expression vector: searching human GABRG2 gene sequence by using Pubmed database, synthesizing GABRG2 open reading frame sequence, mutating 1027 th base T to C (SEQ ID No: 1), cloning into pME-MCS plasmid vector, 5 'cloning site to EcoRI, 3' cloning site to XbaI, constructing intermediate vector middle entry clone pME-GABRG2^1027T>C(SEQ ID No: 3). Cloning the sequence containing the HuC neuron specific promoter region, and constructing a 5' entry clone: p5E-HuC vector (SEQ ID No: 4). Clone 3' entry clone: p3E-polyA vector (SEQ ID No: 5). Recombining and cloning the 5 'entry clone, the midle entry clone and the 3' entry clone vectors into an expression vector pDestTol2CG2 through LR reaction to construct a transgenic mutation expression vector pDestTol2CG2; HuC GABRG2^1027T>CpolyA (SEQ ID No: 6) (shown in FIG. 1), the reaction system of which is as follows: TE buffer 4. mu.L, p5E-HuC (30ng) 1. mu.L, pME-GABRG2^1027T>C1 Î¼ L (30ng), 1 Î¼ L p3E-polyA (30ng), 1 Î¼ L pDesttol2CG2(60ng), 2 Î¼ L LR clonase II, in a total volume of 10 Î¼ L. The reaction is carried out for 20h at 25 â. The constructed plasmid is identified by agarose gel electrophoresis, and the result is shown in figure 2, and a positive band is detected near 20kb, namely the GABRG2 gene mutation expression vector.

Preparation of Tol2 transposase mRNA (pCS2FA-transposase, DNA sequence shown in SEQ ID No: 7): the enzyme digestion system is as follows: 10 XBuffer 5. mu.L, DNA 1. mu.g, NotI-HF restriction enzyme 1. mu.L, add ddH2O to a total volume of 50. mu.L. 15min at 37 â and 15min at 65 â. The enzyme products were electrophoresed on agarose gel, and the gel was recovered and then transcribed into Tol2 transposase mRNA using an RNA in vitro transcription kit.

And secondly, microinjecting zebrafish embryos: the transgenic mutation expression vector pDesttol2CG2; HuC GABRG2^1027T>CAnd-simultaneously microinjecting polyA and Tol2 transposase mRNA into wild zebra fish fertilized eggs at a 1-2 cell stage, after the embryos develop to 24hpf, screening embryos with green fluorescence expressed by hearts for subsequent experiments, and after the zebra fish mature, identifying the genotypes of the zebra fish through PCR and genome sequencing. As shown in FIG. 3, mutant zebrafish embryos were obtained whose hearts expressed green fluorescence.

Thirdly, identifying the genotype of the transgenic zebra fish and obtaining a stable genetic strain: extracting zebra fish genome DNA: extracting zebra fish genome DNA by using a genome DNA extraction kit (All-DNA-Out) one-step method, adding 20ul of All-DNA-Out solution, and heating at 85 â for 5 min.

And (3) genotype identification: the PCR reaction is adopted to identify the genotype of the zebra fish, and the reaction system is as follows: 2 XKOD FX Buffer 10. mu.L, dNTP 4. mu.L, forward Primer (Primer F) 0.6. mu.L, reverse Primer (Primer R) 0.6. mu.L, KOD FX enzyme 0.4. mu.L, DNA template 2. mu.L, total volume 20. mu.L. The reaction conditions are as follows: 5min at 95 â; 98 â 10s, 50 â 30s, 68 â 30s (35 cycles); 5min at 68 â. Wherein the primer sequence is as follows: f: 5'-GCACAGGAAGCTCAGTCTAC-3' (SEQ ID No: 8), R: 5'-GGAGTCCATTTTGGCAATGC-3' (SEQ ID No: 9).

Obtaining a stable genetic line of the transgenic zebra fish: and (3) selecting the zebra fish with positive gene identification result to be hybridized with wild zebra fish to obtain F1 generation zebra fish, and obtaining the mutant GABRG2 transgenic zebra fish line by heart expression green fluorescence and genomic DNA detection screening. As a result of genomic DNA sequencing, as shown in FIG. 4, it was confirmed that the mutation of the 1027 th base from T to C resulted in the determination that the zebrafish contained the mutation GABRG2 (F343L).

Fourthly, the observation of the epilepsy phenotype of the GABRG2(F343L) mutant zebra fish line and the verification of related experiments: observation of epilepsy behavioral phenotype: selecting zebra fish juvenile fish with 5dpf, adopting a zebra fish juvenile fish high-throughput behavior screening box (Danio Vision Chamber) and an animal motion trajectory tracking system (EthoVision XT 13), recording the motion trajectories of the wild zebra fish and the mutant zebra fish under normal conditions, recording for 30min totally, analyzing the excitation degree of the zebra fish juvenile fish through the swimming distance and the average swimming speed, and finding that the excitation of the mutant fish is increased under normal culture conditions and the swimming distance is remarkably increased as shown in a result of figure 5.

Swimming behavior changes in zebrafish in PTZ: adding a seizure inducer PTZ with the final concentration of 15mM into the zebra fish culture solution, and recording the swimming movement behavior of the zebra fish by adopting an EthoVision XT 13 zebra fish behavior video analysis system, wherein the swimming movement behavior is recorded once every 1min for 30 min. Comparing the behavior difference of the wild type and the transgenic zebra fish. Results as shown in fig. 5, PTZ can cause significant increase in excitability of wild-type zebrafish and transgenic zebrafish, but swimming distance of transgenic zebrafish is still significantly higher than wild-type.

Swimming behavior change of zebra fish in light change stimulation: under the condition of alternating illumination and darkness (5s), a zebra fish behavior video analysis system is adopted to record the movement behavior of the zebra fish in 100 cycles, every 5s is a parameter, and the average moving distance difference of every 5s between the wild zebra fish and the transgenic zebra fish is counted. The results are shown in fig. 6, the normal light movement distance of the transgenic zebra fish is significantly higher than that of the wild type, but the light and dark stimuli have no significant effect on the wild type and the transgenic zebra fish.

Molecular biological phenotype verification of epilepsy: and (3) detecting c-fos gene expression by real-time fluorescent quantitative PCR: respectively extracting wild type zebra fish total RNA and transgenic zebra fish total RNA of 24h, 48h, 72h, 96h and 120h after the birth, performing reverse transcription to obtain cDNA, and performing real-time fluorescent quantitative PCR analysis by adopting a SYBR green dye method, wherein the reaction conditions are as follows: 6min at 95 â; 95 â 10s, 60 â 30s, 72 â 10s (41 cycles); the temperature is increased to 95 â in a gradient way at 65 â and is maintained for 5s every 0.5 â. Wherein the primer sequence is as follows: f: 5'-AACTGTCACGGCGATCTCTT-3' (SEQ ID No: 10), R: 5-CTTGCAGATGGGTTTGTGTG-3' (SEQ ID No: 11). The results are shown in fig. 7, and at 72h after birth, the expression of the c-fos of the transgenic zebrafish reaches the highest peak and is obviously higher than that of the wild type, and then the expression is gradually reduced.

Example 2 application of GABRG2(F343L) transgenic zebrafish epilepsy model in antiepileptic drug screening.

Selecting clinically common antiepileptic drugs, such as sodium valproate, carbamazepine, clonazepam, levetiracetam and the like, adding the drugs into a zebrafish culture solution, wherein the use concentrations are respectively 50 mu M, 100 mu M, 50 mu M and 30mM, recording for 30min by adopting a video analysis system after 24h of adding the drugs, and collecting parameters once per minute. The medicine with the treatment effect on the epilepsy phenotype of the transgenic zebra fish is screened by means of swimming behavior analysis and the like. The results are shown in fig. 8, the four drugs can significantly reduce the swimming distance of the transgenic zebra fish, so that the excitability of the transgenic zebra fish is reduced, and the drugs have an inhibitory effect on the seizures of the transgenic zebra fish of GABRG2 (F343L).

Example 3 construction of transgenic Zebra fish line of GABRG2(R177G)

As described in example 1, different mutant GABRG2 gene expression sequences were designed, the mutation site is G (SEQ ID No: 12) from 529 th base C of human GABRG2 gene. Constructing a GABRG2 mutation expression vector pDesttol2CG2; HuC GABRG2^529C>G-polyA, by microinjection of zebrafish embryos, obtaining transgenic zebrafish gene lines.

Phenotypic analysis of GABRG2(R177G) transgenic zebrafish lines:

selecting zebra fish juvenile fish with 5dpf, adopting a zebra fish juvenile fish high-throughput behavior screening box (Danio Vision Chamber) and an animal motion trail tracking system (EthoVision XT 13), recording the motion trail of the wild zebra fish and the mutant zebra fish under normal conditions, recording 24h (illumination 14h and darkness 10h) from 4 o/m, recording behavior parameters once every 1h, comparing the total motion distance difference of the wild zebra fish and the transgenic zebra fish in each time period, and analyzing the circadian rhythm change. The excitation degree of the zebra fish juvenile fish was analyzed by the swimming distance, and as a result, as shown in fig. 9, it was found that under normal culture conditions, GABRG2(R177G) transgenic zebra fish had no significant increase in the swimming distance compared to wild zebra fish. The results show that: GABRG2(R177G) transgenic zebrafish did not have an epileptic phenotype.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Sequence listing

<110> university of southeast Tong

<120> construction method and application of mutant GABRG2 transgenic zebra fish epilepsy model

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cacatcttgc gaatatatgt gtagaaactg ccggaaatcg tcgtggtatt cactccagag 1260

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gaccatgtga tcgcgcttct cgttggggtc tttgctcagg gcggactggg tgctcaggta 2400

gtggttgtcg ggcagcagca cggggccgtc gccgatgggg gtgttctgct ggtagtggtc 2460

ggcgagctgc acgctgccgt cctcgatgtt gtggcggatc ttgaagttca ccttgatgcc 2520

gttcttctgc ttgtcggcca tgatatagac gttgtggctg ttgtagttgt actccagctt 2580

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cagggtgtcg ccctcgaact tcacctcggc gcgggtcttg tagttgccgt cgtccttgaa 2700

gaagatggtg cgctcctgga cgtagccttc gggcatggcg gacttgaaga agtcgtgctg 2760

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gatctgcgaa gatacggcca cgggtgctct tgatcctgtg gctgattttg gactgtgctg 3960

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atttccctaa tttccaggtc aaggtgctgt gcattgtggt aatagatgtg acatgacgtc 4080

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taggcagaat aaataacatt taaattaaac tgggcatcag cgcaattcaa ttggtttggt 4200

aatagcaagg gaaaatagaa tgaagtgatc tccaaaaaat aagtactttt tgactgtaaa 4260

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aaagtattga tttttaattg tactcaagta aagtaaaaat ccccaaaaat aatacttaag 4380

tacagtaatc aagtaaaatt actcaagtac tttacacctc tggttcttga ccccctacct 4440

tcagcaagcc cagcagatcc actagttcta gagcggccgc caccgcggtg gagctccagc 4500

ttttgttccc tttagtgagg gttaattgcg cgcttggcgt aatcatggtc atagctgttt 4560

cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag 4620

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cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg 4740

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tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc 4860

acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg 4920

aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat 4980

cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag 5040

gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga 5100

tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg 5160

tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt 5220

cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac 5280

gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc 5340

ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt 5400

ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc 5460

ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc 5520

agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg 5580

aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag 5640

atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg 5700

tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt 5760

tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca 5820

tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca 5880

gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc 5940

tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt 6000

ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg 6060

gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc 6120

aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg 6180

ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga 6240

tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga 6300

ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta 6360

aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg 6420

ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact 6480

ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata 6540

agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt 6600

tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa 6660

ataggggttc cgcgcacatt tccccgaaaa gtg 6693

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<213> Artificial Sequence (Artificial Sequence)

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ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga 60

taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga 120

gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca 180

cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaata cgcgtaccgc 240

tagccaggaa gagtttgtag aaacgcaaaa aggccatccg tcaggatggc cttctgctta 300

gtttgatgcc tggcagttta tggcgggcgt cctgcccgcc accctccggg ccgttgcttc 360

acaacgttca aatccgctcc cggcggattt gtcctactca ggagagcgtt caccgacaaa 420

caacagataa aacgaaaggc ccagtcttcc gactgagcct ttcgttttat ttgatgcctg 480

gcagttccct actctcgcgt taacgctagc atggatgttt tcccagtcac gacgttgtaa 540

aacgacggcc agtcttaagc tcgggcccca aataatgatt ttattttgac tgatagtgac 600

ctgttcgttg caacacattg atgagcaatg cttttttata atgccaactt tgtacaaaaa 660

agcaggcttg taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat 720

tgggtaccgg gccccccctc gaggtcgacg gtatcgataa gcttgatatc gaattcgcca 780

ccatgagttc gccaaatata tggagcacag gaagctcagt ctactcgact cctgtatttt 840

cacagaaaat gacggtgtgg attctgctcc tgctgtcgct ctaccctggc ttcactagcc 900

agaaatctga tgatgactat gaagattatg cttctaacaa aacatgggtc ttgactccaa 960

aagttcctga gggtgatgtc actgtcatct taaacaacct gctggaagga tatgacaata 1020

aacttcggcc tgatatagga gtgaagccaa cgttaattca cacagacatg tatgtgaata 1080

gcattggtcc agtgaacgct atcaatatgg aatacactat tgatatattt tttgcgcaaa 1140

cgtggtatga cagacgtttg aaatttaaca gcaccattaa agtcctccga ttgaacagca 1200

acatggtggg gaaaatctgg attccagaca ctttcttcag aaattccaaa aaagctgatg 1260

cacactggat caccaccccc aacaggatgc tgagaatttg gaatgatggt cgagtgctct 1320

acaccctaag gttgacaatt gatgctgagt gccaattaca attgcacaac tttccaatgg 1380

atgaacactc ctgccccttg gagttctcca gttatggcta tccacgtgaa gaaattgttt 1440

atcaatggaa gcgaagttct gttgaagtgg gcgacacaag atcctggagg ctttatcaat 1500

tctcatttgt tggtctaaga aataccaccg aagtagtgaa gacaacttcc ggagattatg 1560

tggtcatgtc tgtctacttt gatctgagca gaagaatggg atactttacc atccagacct 1620

atatcccctg cacactcatt gtcgtcctat cctgggtgtc tttctggatc aataaggatg 1680

ctgttccagc cagaacatct ttaggtatca ccactgtcct gacaatgacc accctcagca 1740

ccattgcccg gaaatcgctc cccaaggtct cctatgtcac agcgatggat ctctttgtat 1800

ctgtttgtct catctttgtc ttctctgctc tggtggagta tggcaccttg cattattttg 1860

tcagcaaccg gaaaccaagc aaggacaaag ataaaaagaa gaaaaaccct cttcttcgga 1920

tgttttcctt caaggcccct accattgata tccgcccaag atcagcaacc attcaaatga 1980

ataatgctac acaccttcaa gagagagatg aagagtacgg ctatgagtgt ctggacggca 2040

aggactgtgc cagttttttc tgctgttttg aagattgtcg aacaggagct tggagacatg 2100

ggaggataca tatccgcatt gccaaaatgg actcctatgc tcggatcttc ttccccactg 2160

ccttctgcct gtttaatctg gtctattggg tctcctacct ctacctgtga tctagagcgg 2220

ccgccaccgc ggtggagctc cagcttttgt tccctttagt gagggttaat tgcgcgcttg 2280

gcgtaatcat ggtcatagca cccagctttc ttgtacaaag ttggcattat aagaaagcat 2340

tgcttatcaa tttgttgcaa cgaacaggtc actatcagtc aaaataaaat cattatttgc 2400

catccagctg atatccccta tagtgagtcg tattacatgg tcatagctgt ttcctggcag 2460

ctctggcccg tgtctcaaaa tctctgatgt tacattgcac aagataaaat aatatcatca 2520

tgaacaataa aactgtctgc ttacataaac agtaatacaa ggggtgttat gagccatatt 2580

caacgggaaa cgtcgaggcc gcgattaaat tccaacatgg atgctgattt atatgggtat 2640

aaatgggctc gcgataatgt cgggcaatca ggtgcgacaa tctatcgctt gtatgggaag 2700

cccgatgcgc cagagttgtt tctgaaacat ggcaaaggta gcgttgccaa tgatgttaca 2760

gatgagatgg tcagactaaa ctggctgacg gaatttatgc ctcttccgac catcaagcat 2820

tttatccgta ctcctgatga tgcatggtta ctcaccactg cgatccccgg aaaaacagca 2880

ttccaggtat tagaagaata tcctgattca ggtgaaaata ttgttgatgc gctggcagtg 2940

ttcctgcgcc ggttgcattc gattcctgtt tgtaattgtc cttttaacag cgatcgcgta 3000

tttcgtctcg ctcaggcgca atcacgaatg aataacggtt tggttgatgc gagtgatttt 3060

gatgacgagc gtaatggctg gcctgttgaa caagtctgga aagaaatgca taaacttttg 3120

ccattctcac cggattcagt cgtcactcat ggtgatttct cacttgataa ccttattttt 3180

gacgagggga aattaatagg ttgtattgat gttggacgag tcggaatcgc agaccgatac 3240

caggatcttg ccatcctatg gaactgcctc ggtgagtttt ctccttcatt acagaaacgg 3300

ctttttcaaa aatatggtat tgataatcct gatatgaata aattgcagtt tcatttgatg 3360

ctcgatgagt ttttctaatc agaattggtt aattggttgt aacactggca gagcattacg 3420

ctgacttgac gggacggcgc aagctcatga ccaaaatccc ttaacgtgag ttacgcgtcg 3480

ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt 3540

ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg 3600

ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata 3660

ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca 3720

ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag 3780

tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc 3840

tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga 3900

tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg 3960

tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac 4020

gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg 4080

tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg 4140

ttcctggcct tttgctggcc ttttgctcac atgtt 4175

<210> 4

<211> 11596

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4