CN110396525A - RNF213 gene completely knocks out the preparation method and purposes of homozygote zebra fish - Google Patents

RNF213 gene completely knocks out the preparation method and purposes of homozygote zebra fish Download PDF

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CN110396525A
CN110396525A CN201910592731.2A CN201910592731A CN110396525A CN 110396525 A CN110396525 A CN 110396525A CN 201910592731 A CN201910592731 A CN 201910592731A CN 110396525 A CN110396525 A CN 110396525A
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rnf213a
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盛文利
林晶
杨蜀岚
孙逊沙
温骏
梁杰
张晶晶
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First Affiliated Hospital of Sun Yat Sen University
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Abstract

The invention discloses preparations and its application that a kind of rnf213a gene completely knocks out zebra fish mutant；Design is directed to the TALEN sequence of zebra fish rnf213a gene；Its left and right arms plasmid is constructed, and corresponding mRNA is transcribed in vitro out；TALEN left and right arms mRNA is mixed and is injected into zebrafish embryo；Embryo after injection supports to F0 adult fish and mates with wild type adult fish, and the embryo for carrying rnf213a gene mutation is supported to F1 adult fish；F1 generation fish cuts coda gene type identification discovery, and there are two kinds of heterozygote genotypes :+2bp and -7bp；By heterozygote and wild type adult fish diplomatic 3 generations, after purifying its genetic background, then the heterozygote crosses of syngeneic type are obtained into two kinds of genotype of homozygote :+2bp and -7bp.The embryo that homozygote hybridizes with homozygote carries out Phenotypic Observation discovery and there is Moyamoya Disease phenotype similar with people and cvernous hemangioma phenotype.The present invention can rapidly and efficiently establish rnf213a gene and completely strike zebra fish strain by TALEN gene editing technology, can carry out the drug screening work for Moyamoya Disease or/and cvernous hemangioma using the model.

Description

RNF213 gene completely knocks out the preparation method and purposes of homozygote zebra fish
Technical field
The invention belongs to gene engineering technology fields, more particularly to are beaten using gene and technology is knocked out zebra fish rnf213a The method of gene.
Background technique
RNF213 gene is located at No. 17 chromosomes, and coding includes the ring finger protein of 5207 amino acid, and there are two the genes Important functional areas RING finger domain and AAA+ATPases domain.In recent years, RNF213 caused concern main It is because research discovery RNF213 is the tumor susceptibility gene of East Asia crowd's Moyamoya Disease, especially Japanese；The research in later period also found Gene point mutation p.R4810K is also related with asian population encephalic aortic stenosis；One research in Europe finds RNF213 Different loci mutation is also related to Caucasian's aneurysm.RNF213 gene may be related to a variety of cerebrovascular pathogenesis.
RNF213 p.R4810K is related to Moyamoya Disease, and there are obvious racial differences, are concentrated mainly on Asian, Japanese smog Patient's site mutation rate is 90.1%, South Korea 78.9%, China 23.1%；There is also the variation in the site, days by normal person I 2.5%, and Korean 2.7%, and Chinese 0.9%.Relative to Japan and South Korea, this mutation position of Chinese Han nationality's Moyamoya Disease patient Mutations in epithelial is lower.Caucasian's Moyamoya Disease disease incidence is Japanese 1/10, and Caucasian does not have found RNF213 p.R4810K Obviously related to Moyamoya Disease, this may be lower one of the reason of Caucasian's Moyamoya Disease disease incidence.In RNF213 p.R4810K It is found with the research of identical environment difference blood lineage Moyamoya Disease patient, p.R4810K is deposited in 56% Asian ancestry Moyamoya Disease patient Without p.R4810K point mutation in non-Asian ancestry Moyamoya Disease patient.Whether this prompt RNF213 p.R4810K participates in Moyamoya Disease Pathogenesis there are racial differences.
In the research of translational medicine, there is the life of part vascular bud, similar smog in zebra fish rnf213 gene knockdown The vascular bud of disease is raw；Utilize the phenotype of the 32 non-simulation smoke disease of exon of cre-loxp system knock-out mice Rnf213 gene； The phenotype of corresponding people RNF213c.14429G > A point mutation mouse also non-simulation smoke disease.Whether RNF213 takes part in Moyamoya Disease Pathogenesis still has dispute.
It found to strike using morpholino technology in recent years and subtracts related gene and be easy to appear phenotype, but phenotype is in mutant Can not repeat, research shows that morpholino technology be easy to appear miss the target knock out other genes to cause phenotype specificity compared with Difference.
Summary of the invention
It provides and is completely knocked out using TALEN technology it is an object of the invention to overcome above-mentioned the deficiencies in the prior art place Zebra fish rnf213a gene, the target spot of design are 2 exons of zebra fish rnf213a, and pass through the interior friendship again of diplomatic 3 generations Form homozygote zebra fish.By observing the phenotype of homozygous mutation body, caused by avoidable injection and morpholino miss the target False phenotype increases the specificity of phenotype.
To achieve the above object, the technical scheme adopted by the invention is as follows:
A kind of RNF213 gene completely knocks out the preparation method of homozygote zebra fish, comprising the following steps:
S1, design are directed to the TALEN sequence of 2 exon target sequence of zebra fish RNF213 gene；
S2, the building left and right arm plasmid of TALEN, and the left and right arm mRNA of TALEN is transcribed in vitro out respectively；
S3, the left and right arm mRNA of TALEN is mixed and is injected into transgenic zebrafish embryo, extract embryo's DNA sequencing hair The mRNA of existing TALEN can act on 2 exon of rnf213a gene；
S4, it mates by the Embryo Culture after injection to F0 generation, and with wild type adult fish, obtains F1 generation adult fish；
S5, identification F1 whether there is rnf213a gene mutation, and identify rnf213a gene mutation type through DNA sequencing, By the heterozygote containing rnf213a gene mutation and wild type diplomatic 3 generations, its genetic background is purified；
S6, the heterozygote crosses of syngeneic type are obtained to two kinds of genotype of homozygote :+2bp and -7bp.
Preferably, the 2 exon target sequence of zebra fish RNF213 gene in the step S1 is SEQ ID NO.1 institute Show.
Preferably, the left arm sequence of the TALEN sequence in the step S1 is as shown in SEQ ID NO.2；Right arm sequence is such as Shown in SEQ ID NO.3.
Preferably, the TALEN sequence in the step S1 further includes the nucleotide sequence and its volume that can identify different bases The amino acid sequence of code, wherein the amino acid sequence that the nucleotide sequence of identification base A as shown in SEQ ID NO.4, encodes As shown in SEQ ID NO.5；The nucleotide sequence of base T is identified as shown in SEQ ID NO.6, the amino acid sequence of coding is such as Shown in SEQ ID NO.7；The nucleotide sequence of base C is identified as shown in SEQ ID NO.8, the amino acid sequence of coding is such as Shown in SEQ ID NO.9；The nucleotide sequence of bases G is identified as shown in SEQ ID NO.10, the amino acid sequence of coding is such as Shown in SEQ ID NO.11.
Preferably, further include the competent cell for the plasmid conversion low-copy that will be constructed in the step S2, expanded After culture, plasmid is extracted, is identified through restricted interior digestion DNA sequencing, determines plasmid dna sequence.
Preferably, the forward primer for plasmid order-checking design is as shown in SEQ ID NO.12；Reverse primer such as SEQ Shown in ID NO.13.
Preferably, the identified adult fish F1 heterozygote containing rnf213a gene mutation is+2bp mutation in the step S5 Body and -7bp mutant
Preferably, homozygote is respectively+2bp mutant and -7bp mutant in the step S6.
The homozygous preparation side of RNF213 gene knockout zebra fish mutant that the present invention also provides a kind of according to The RNF213 gene knockout zebra fish mutant homozygosis submodel that method is established is as Moyamoya Disease or/and cvernous hemangioma Medicaments sifting model in purposes.
Beneficial effects of the present invention: the present invention completely knocks out zebra fish rnf213a base by TALEN gene editing technology It is lower to knock out the probability that misses the target relative to CRISPR/Cas9 system gene for cause.Generally there are transgene backgrounds for rnf213a mutant Tg (flk1:eGFP/gata1:DsRed) has red green fluorescence, such as: red-label red blood cell, Green Marker blood vessel endothelium, But through the invention diplomatic 3 generations again in hand over the homozygous mutation body obtained, reduce genetic background.The present invention obtains Two kinds of genotype that rnf213a is completely knocked out, phenotype can be verified in two kinds of genotype simultaneously, increase the specificity of phenotype. The rnf213a gene mutation body that the present invention constructs can be used as the zebra fish animal model of Moyamoya Disease and cvernous hemangioma.
Detailed description of the invention
Fig. 1 is that (wherein A is TALEN2 exon nucleotide for the sequencing result of F1 generation+2bp of the present invention and -7bp mutant Sequence and target spot；B is+2bp mutant sequencing result；C is -7bp mutant sequencing result)；
Fig. 2 is to pass through laser lamella microscope and the micro- sem observation rnf213a of single photon-/-The observation result of phenotype is (wherein A-C expression+2bp, -7bp mutation type surface are similar to Moyamoya Disease；D-F expression+2bp, -7bp mutation type surface are similar to the spongy of people Hemangioma).
Specific embodiment
For more concise displaying technical solution of the present invention, objects and advantages, combined with specific embodiments below And its attached drawing is described in further detail the present invention.
Embodiment
1, the shot design of TALEN
(1) sequence of 2 exon of zebra fish rnf213a gene is obtained in Pubmed database.It is designed according to TALEN former Then, it using TALEN sequence special designing software, is designed for 2 exon of rnf213a gene.Share 8 kinds of TALEN bones Frame carrier, left and right arms each 4, they contain NLS, N-terminal, the basic structures such as C-terminal and Fok I.In the serial carrier, in advance It assembles in TALE repetitive structure domain, last 0.5 repetitive unit at 3 ' ends, also known as half site (Half-site).It can be special Identify to property the last one base of TALEN target spot.According to the difference of identification A/T/C/G base, it is divided into left arm L1/L2/L3/ L4, right arm L1/L2/L3/L4.
(2) for the TALEN left arm sequence of 2 exon of zebra fish rnf213a gene design are as follows: 5 '- CTGCAGTGAGTGTGGACAT-3 ', TALEN right arm sequence: 5 '-TACGAGACAACACAAGGT-3 '；Zebra fish RNF213 base Because of the target sequence of 2 exons are as follows: 5 '-TAAACTGCAATCTCAA-3 '.
2, the building of TALEN expression vector
(1) it is completed according to block coupled in series building block principle using TALEN kit (Shanghai Si Dansai biology Co., Ltd) The building of TALEN expression vector, specific steps are as follows: by identification last T of the sequence removal of TALEN left and right arms, with one or two A base composition is as a module, first label, the last one is labeled as 9, and so on be marked.
(2) 9 modules accordingly numbered are taken out, each module takes 1.5ul, and is added into the same PCR pipe.It is shared 8 kinds of TALEN skeleton carriers, left and right arms each 4, they contain NLS, N-terminal, the basic structures such as C-terminal and Fok I.The series carries In body, in pre-assembly TALE repetitive structure domain, last 0.5 repetitive unit at 3 ' ends, also known as half site (Half- site).It can specifically identify the last one base of TALEN target spot.According to the difference of identification A/T/C/G base, it is divided into a left side Arm L1/L2/L3/L4, right arm R1/R2/R3/R4, structure are as shown in table 1.
Table 1: the structural representation of left arm L1/L2/L3/L4 and right arm R1/R2/R3/R4
(3) it is loaded by 2 reaction system of table, corresponding TALEN skeleton carrier is first added, then by solution 3, solution 1, solution 2 sequentially add in PCR pipe, and finally plus water is mended to 20ul, mix, gently get rid of.
Table 2: left and right arm TALEN coupled reaction system
Above-mentioned reaction condition is as follows
(4) it takes out PCR pipe and 4 1ul of solution, 5 0.5ul of solution (solution 5 asks 37 DEG C to melt in advance) is added, mix, In It is incubated for one hour for 37 DEG C in PCR instrument or water-bath.
(5) it by the EP pipe of final product conversion competent cell containing low-copy, mixes, places 30min on ice, then It is put into heat shock 45s in 42 DEG C of water-baths, then is placed in 3min on ice rapidly.500ulSOC solution is added into EP pipe, is placed in 37 DEG C, 30min is cultivated in the shaking table of 250rpm.After taking out EP pipe, 5min is centrifuged in the centrifuge of 4000r/min.Discard EP pipe In most of supernatant, leave about 100ul, and will precipitating gently piping and druming mixes, be spread evenly across card and receive the plate of chloramphenicol resistance In, it is inverted in 37 DEG C of incubators and is incubated overnight 12-16 hours.
3, plasmid extracts
(1) it is operated using small amount plasmid extraction kit, single colonie in picking culture plate, is inoculated into that is mould containing card In the 5ml LB culture medium of element, 37 DEG C of constant-temperature table 250rpm shaken overnight cultures.
(2) to reduce recombination fraction, the competent bacteria of low-copy is selected in this research.When extracting plasmid, each clone is at least Extract 4ml bacterium solution.After bacterium solution to be poured into the EP pipe of 1.5ml, 12000rpm is centrifuged 30 seconds at 4 DEG C, collects precipitating.Supernatant is removed, The solution P1 of 250ul pre-cooling is added, is vibrated in vortex oscillator, until bacterial sediment is broken up completely.250ul solution is added P2 mildly reverse centrifuge tube 5 times, mixes content, is sure not to vibrate.The solution P3 for adding 350ul to be pre-chilled is overturned for several times repeatedly, can See that White Flocculus occurs.12000rpm is centrifuged 10 minutes at 4 DEG C, abandons precipitating.Supernatant is transferred in adsorption column CP3, is stood 10 minutes.4 DEG C of 12000rpm are centrifuged 1 minute, outwell the waste liquid in collecting pipe.500l protein liquid removal PD is added into adsorption column, 12000rpm is centrifuged 30-60sec, outwells the waste liquid in collecting pipe, adsorption column CP3 is put back in collecting pipe.Add to adsorption column CP3 Enter 600l rinsing liquid PW, 12,000rpm centrifugation 30-60sec outwell the waste liquid in collecting pipe, adsorption column CP3 is put into collecting pipe In, it washes repeatedly 1 time.Last 12,000rpm is centrifuged 2min, and rinsing liquid remaining in adsorption column is removed.Adsorption column CP3 is opened Lid, is placed in and is placed at room temperature for several minutes, dry rinsing liquid remaining in adsorption column.It places it in clean centrifuge tube, to absorption 50-100l elution buffer EB is added dropwise among film.It is placed at room temperature for 10min, 12,000rpm centrifugation 2min collect plasmid solution and arrive In centrifuge tube.
(3) digestion is identified, endonuclease reaction system is as shown in table 3；
Table 3: endonuclease reaction system
It 37 DEG C, reacts 10 minutes.Digestion products 5ul is taken, 6 × loading Buffer 1ul is added, mixes, is loaded onto In 1.5% Ago-Gel, whether constant pressure 120V electrophoresis 30 minutes, identification digestion are correct at room temperature.
(4) it is sequenced: sending the correct plasmid of digestion to sequencing, and be compared with standard sequence, obtain final correct matter Grain, by Shanghai, Invitrogen Corp. is completed.Sequencing forward primer is 5 '-CTCCCCTTCAGCTGGACAC-3 ', and reverse primer is 5'-AGCTGGGCCACGATTGAC-3'.The corresponding TALEN DNA sequence dna of different bases and amino acid sequence such as 4 institute of table:
Table 4: corresponding TALEN DNA sequence dna and amino acid sequence with base
4, the left and right arm mRNA of TALEN matter is transcribed in vitro
(1) preparation of template is transcribed in vitro
1) linearize TALEN plasmid (restriction enzyme Not I), using as be transcribed in vitro template.Digestion system such as table 5 It is shown:
Table 5: template reaction system is transcribed in vitro
It 37 DEG C, reacts 2 hours.3 μ l are taken to carry out 1.5% agarose gel electrophoresis, whether digestion is complete with clear plasmid.
2) digestion products purify: Beijing Tiangeng biology Co., Ltd；
Using common purified product kit, product after purification and recovery digestion, detailed step is as follows: into adsorption column CB2 500ul equilibrium liquid BL is added, 12,000rpm centrifugation 1min outwell the waste liquid in collecting pipe, adsorption column CB2 is put back to collecting pipe In.Into endonuclease reaction liquid, 5 times of volume combination liquid PB are added, mix well.Acquired solution is added in adsorption column CB2, room temperature 10min is placed, 12,000rpm centrifugation 30-60sec outwell the waste liquid in collecting pipe, adsorption column CB2 is put into collecting pipe.To 600ul rinsing liquid PW is added in adsorption column CB2,12,000rpm centrifugation 30-60sec abandon waste liquid, adsorption column CB2 is put into collection Guan Zhong.Repeated washing is primary.Adsorption column CB2 is put back in collecting pipe, 12,000rpm centrifugation 2min, as far as possible removing rinsing liquid.It will Adsorption column CB2 is placed several minutes in room temperature, is thoroughly dried.Finally adsorption column CB2 is put into clean centrifuge tube, to suction The hanging 30ulDEPC that is added dropwise handles water among membrane, is placed at room temperature for 5min.12,000rpm is centrifuged 2min and collects solution.
3) measurement of template plasmid concentration
The eluent TE for taking sample 2ul that 98ul is added is detected under ssDNA mode with Nano drop spectrophotometer. Testing result shows that OD260/OD280 between 1.7-1.9, illustrates that purity is preferable.
(2) it is transcribed in vitro: Life Techologies company；
Using mMessagemMachine SP6kit kit, mRNA synthetic reaction is carried out.Reaction system is as shown in table 6:
Table 6:mRNA synthetic reaction system
According to plasmid concentration adjust additional amount, guarantee plasmid template total amount 1ug, after mixing gently, 37 DEG C water-bath 2 hours. Add 1ul TURBO Dnase, 37 DEG C of water-bath 15min is mixed, to eliminate DNA profiling.
(3) purifying (lithium chloride precipitating) of transcription product
Using mMessagemMachine SP6kit kit, mRNA purifying and resuspension are carried out, by reagent: 20 μ l of RNA； Licl 30μl；30 μ l of Nuclease Free water, after mixing, -20 DEG C of precipitatings are greater than 30min, 4 DEG C of 13200rpm centrifugations 15min, goes supernatant (paying attention to EP bottom of the tube RNA precipitate), the cleaning of 70% ethyl alcohol of 1ml, and 4 DEG C of centrifugation 15min of 13200rpm are gone Clearly.Nuclease Free water is added after slightly drying, RNA is resuspended, after measuring concentration, packing, -80 DEG C of preservations.
4, it injects, identification and hybridization
(1) by the mRNA mixture of the left and right arm of TALEN of final concentration of 400ng/ μ l be injected into 10 have Tg (flk1: Egfp/gata1:dsRed it) in the zebrafish embryo of transgene background, supports to adult fish and obtains F0 generation, then by F0 for adult fish and open country Raw type zebra fish mating, obtains heterozygote F1 generation, F1 is carried out genomic DNA sequencing, identification obtains+2bp mutant and -7bp Mutant heterozygote, wherein there are 6 embryos to be mutated, mutation efficiency 60%.
(2) in order to purify transgene genetic background, respectively by the heterozygote and wild type of two kinds of mutant of+2bp and -7bp Zebra fish mated for 3 generations, obtained F2.
(3)+2bp mutant is mated with+2bp mutant respectively, -7bp mutant mates with -7bp mutant, finally obtains Obtain RNF213 gene completely knockout+2bp mutant homozygote and -7bp mutant homozygote.
5, phenotype is observed
By laser lamella microscope and the micro- sem observation rnf213a- of single photon/- phenotype, find mutant (+2bp: 49.4%；- 7bp:46.4%) in rear cerebral veins, venae cerebri, there are the vascular buds of similar smoke-like to give birth to original hindbrain traffic intersection, and it should Phenotype is similar to Moyamoya Disease (such as Fig. 2A-C)；More importantly occur between rear cerebral veins, venae cerebri mulberries sample vascular bud it is raw (+2bp: 69.4%；- 7bp:71.4%), the phenotype similar to people cvernous hemangioma (such as Fig. 2 D-F), further in cvernous hemangioma Patient tests and is confirmed, it was confirmed that RNF213 gene is the tumor susceptibility gene of cvernous hemangioma.The two phenotypes are equal It is confirmed in two kinds of genotype (+2bp and -7bp), further demonstrates the specificity of RNF213 gene and phenotype.Therefore should Genotype zebra fish (rnf213a-/-) can be used as the zebra fish animal model of cvernous hemangioma and Moyamoya Disease.
The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously It cannot therefore be construed as limiting the scope of the patent.It should be pointed out that coming for those of ordinary skill in the art It says, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to protection of the invention Range.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.
SEQUENCE LISTING
<110>No.1 Hospital Affiliated to Zhongshan Univ.
<120>RNF213 gene completely knocks out the preparation method and purposes of homozygote zebra fish
<130> 7.2
<160> 13
<170> PatentIn version 3.3
<210> 1
<211> 16
<212> DNA
<213>it synthesizes
<400> 1
taaactgcaa tctcaa 16
<210> 2
<211> 19
<212> DNA
<213>it synthesizes
<400> 2
ctgcagtgag tgtggacat 19
<210> 3
<211> 18
<212> DNA
<213>it synthesizes
<400> 3
tacgagacaa cacaaggt 18
<210> 4
<211> 102
<212> DNA
<213>it synthesizes
<400> 4
ctcactccgg aacaagtggt cgcaatcgcc tccaacattg gcgggaaaca ggcactcgag 60
actgtccagc gcctgcttcc cgtgctgtgc caagcgcacg gt 102
<210> 5
<211> 34
<212> PRT
<213>it synthesizes
<400> 5
Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys
1 5 10 15
Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala
20 25 30
His Gly
<210> 6
<211> 102
<212> DNA
<213>it synthesizes
<400> 6
ctgaccccag agcaggtcgt ggccattgcc tcgaatggag ggggcaaaca ggcgttggaa 60
accgtacaac gattgctgcc ggtgctttgt caggcacacg gc 102
<210> 7
<211> 34
<212> PRT
<213>it synthesizes
<400> 7
Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys
1 5 10 15
Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala
20 25 30
His Gly
<210> 8
<211> 102
<212> DNA
<213>it synthesizes
<400> 8
ctgaccccag agcaggtcgt ggcgatcgca agccacgacg gaggaaagca agccttggaa 60
acagtacaga ggctgttgcc tgtgctttgt caggcacacg gc 102
<210> 9
<211> 34
<212> PRT
<213>it synthesizes
<400> 9
Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys
1 5 10 15
Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala
20 25 30
His Gly
<210> 10
<211> 102
<212> DNA
<213>it synthesizes
<400> 10
ctcactccgg aacaagtggt cgcaatcgcg agcaataacg gcggaaaaca ggctttggaa 60
acggtgcaga ggctccttcc agtgctgtgc caagcgcacg gt 102
<210> 11
<211> 34
<212> PRT
<213>it synthesizes
<400> 11
Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys
1 5 10 15
Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala
20 25 30
His Gly
<210> 12
<211> 19
<212> DNA
<213>it synthesizes
<400> 12
ctccccttca gctggacac 19
<210> 13
<211> 18
<212> DNA
<213>it synthesizes
<400> 13
agctgggcca cgattgac 18

Claims (9)

1. the preparation method that a kind of rnf213a gene completely knocks out homozygote zebra fish, which comprises the following steps:
S1, design are directed to the TALEN sequence of 2 exon target sequence of zebra fish rnf213a gene；
S2, the building left and right arm plasmid of TALEN, and the left and right arm mRNA of TALEN is transcribed in vitro out respectively；
S3, the left and right arm mRNA of TALEN is mixed and is injected into transgenic zebrafish embryo, extract the discovery of embryo's DNA sequencing The mRNA of TALEN can act on 2 exon of rnf213a gene；
S4, it mates by the Embryo Culture after injection to F0 generation, and with wild type adult fish, obtains F1 generation adult fish；
S5, identification F1 whether there is rnf213a gene mutation, and identify rnf213a gene mutation type through DNA sequencing, will contain There are heterozygote and wild type diplomatic 3 generations of rnf213a gene mutation, purifies its genetic background；
S6, the heterozygote crosses of syngeneic type are obtained to two kinds of genotype of homozygote :+2bp and -7bp.
2. preparation method as described in claim 1, which is characterized in that the zebra fish rnf213a gene 2 in the step S1 Exon target sequence is shown in SEQ ID NO.1.
3. preparation method as described in claim 1, which is characterized in that the left arm sequence of the TALEN sequence in the step S1 As shown in SEQ ID NO.2；Right arm sequence is as shown in SEQ ID NO.3.
4. preparation method as described in claim 1, which is characterized in that the TALEN sequence in the step S1 further includes that can know The nucleotide sequence of other different bases and its amino acid sequence of coding, wherein the nucleotide sequence such as SEQ ID of identification base A Shown in NO.4, the amino acid sequence of coding is as shown in SEQ ID NO.5；Identify the nucleotide sequence such as SEQ ID of base T Shown in NO.6, the amino acid sequence of coding is as shown in SEQ ID NO.7；Identify the nucleotide sequence such as SEQ ID of base C Shown in NO.8, the amino acid sequence of coding is as shown in SEQ ID NO.9；Identify the nucleotide sequence such as SEQ ID of bases G Shown in NO.10, the amino acid sequence of coding is as shown in SEQ ID NO.11.
5. preparation method as described in claim 1, which is characterized in that further include the plasmid conversion that will be constructed in the step S2 The competent cell of low-copy after being expanded culture, extracts plasmid, identifies through restricted interior digestion DNA sequencing, determines plasmid DNA sequence dna.
6. preparation method as claimed in claim 5, which is characterized in that the forward primer for plasmid order-checking design is such as Shown in SEQ ID NO.12；Reverse primer is as shown in SEQ ID NO.13.
7. preparation method as described in claim 1, which is characterized in that identified in the step S5 to contain rnf213a gene The adult fish F1 heterozygote of mutation is+2bp mutant and -7bp mutant.
8. preparation method as described in claim 1, which is characterized in that homozygote is respectively+2bp mutant in the step S6 And -7bp mutant.
9. a kind of rnf213a gene knockout zebra fish mutant homozygosis submodule that preparation method as described in claim 1 is established Type is as the purposes in the medicaments sifting model for Moyamoya Disease or/and cvernous hemangioma.
CN201910592731.2A 2019-07-03 2019-07-03 RNF213 gene completely knocks out the preparation method and purposes of homozygote zebra fish Pending CN110396525A (en)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2010259390A (en) * 2009-05-08 2010-11-18 Tohoku Univ Method for detecting or diagnosing moyamoya disease onset risk by detecting gene mutation
WO2011049207A1 (en) * 2009-10-23 2011-04-28 国立大学法人京都大学 Moyamoya disease-related gene and utilization of same
CN108018316A (en) * 2017-12-20 2018-05-11 湖南师范大学 A kind of method of gene knockout selection and breeding rmnd5b Gene Deletion zebra fish
CN108103108A (en) * 2018-01-30 2018-06-01 上海交通大学医学院附属瑞金医院 Preparation and application of Cebpa gene-deleted zebra fish mutant

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2010259390A (en) * 2009-05-08 2010-11-18 Tohoku Univ Method for detecting or diagnosing moyamoya disease onset risk by detecting gene mutation
WO2011049207A1 (en) * 2009-10-23 2011-04-28 国立大学法人京都大学 Moyamoya disease-related gene and utilization of same
CN108018316A (en) * 2017-12-20 2018-05-11 湖南师范大学 A kind of method of gene knockout selection and breeding rmnd5b Gene Deletion zebra fish
CN108103108A (en) * 2018-01-30 2018-06-01 上海交通大学医学院附属瑞金医院 Preparation and application of Cebpa gene-deleted zebra fish mutant

Non-Patent Citations (3)

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Title
JUN WEN等: "Mutation of rnf213a by TALEN causes abnormal angiogenesis and circulation defects in zebrafish", 《BRAIN RESEARCH》 *
YONG-GANG MA等: "Role of Ring Finger Protein 213 in Moyamoya Disease", 《CHIN MED J (ENGL)》 *
沈延等: "类转录激活因子效应物核酸酶(TALEN)介导的基因组定点修饰技术", 《遗传》 *

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Application publication date: 20191101