CN105594664B - A kind of method of gene knockout selection and breeding stat1a Gene Deletion zebra fish - Google Patents
A kind of method of gene knockout selection and breeding stat1a Gene Deletion zebra fish Download PDFInfo
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Abstract
A kind of stat1a Gene Deletions zebra fish, sequence is made of as shown in SEQ ID No.1 following steps after experimental design area knocks out：CRISPR/Cas9 gene knockout target position point designs：Structure gRNA expression vectors and gRNA are synthesized in vitro；The microinjection of zebrafish embryo；The validity of T7E1 methods and Sanger sequencing detection target sites；After injection two months, carry out cutting tail identification, ibid authentication step；The TA of aim sequence is cloned；The Sanger of plasmid is sequenced；Obtain the F1 generation of heritable zebra fish mutant；The F2 of zebra fish mutant is obtained for homozygote, the F3 generation pure lines heredity of the Gene Deletion zebra fish is carried out according to the above method, obtains this new zebra fish strain.
Description
Technical field
The invention belongs to gene Knockout fields, are related to a kind of stat1a Gene Deletions zebra fish.
Background technology
STAT1 (signal transducer and activator of transcription 1) gene is located at the mankind
2q32.3 is the transcription factor for encoding 750 amino acid.It is generally acknowledged that the gene mediated interferon signal path, can directly adjust
The transcription of target gene is controlled, and participates in the processes such as cell Proliferation and differentiation.Pass through gene differential expression spectrum analysis and genome association
Analysis etc. finds that STAT1 genes and osteoporosis are closely related.
Zebra fish has high homology, and STAT1 genes with gene, signal path of the mankind in bone development
More conservative in evolution, two different spliceosome STAT1-alpha and STAT1-beta of mankind's STAT1 genes correspond to spot
Horse fish two different genes stat1a and stat1b, research find that stat1a is especially high in zebrafish embryo early expression amount.And
And compared with other animal models, zebra fish individual is small, juvenile fish entire body is transparent, is conducive to the observation of skeleton development.
By CRISPR/Cas9 gene targetings, suitable target practice site is designed on zebra fish stat1a genes, it will
Specific sgRNA (final concentration 20ng/ μ L) and Cas9-mRNA (300 ng/ μ L of final concentration) the micro- co-injection synthesized in vitro into
Zebra fish one is intracellular, and the validity in selected site is confirmed by Activity determination.
Gene targeting originates from late 1980s, is that one kind is studied by carrying out pointed decoration to genome
The important Method means of gene function, it can also be used to treat the various genetic diseases of the mankind.The technology is mainly using scarce
The modes such as mutation, inactivation of gene, the deletion of chromosome large fragment and foreign gene importing are lost to change the hereditary information of biology, and
And mutant character is expressed after in system genitale stablizing heredity, to study in organism specific gene in growth and development process
Effect, so this kind of technological means has become modern molecular biology research hotspot.Traditional gene targeting is built upon
On embryonic stem cell (ESC) and the basis of homologous recombination technique, therefore Knockout technology is extremely inefficient.It is a kind of completely new at the beginning of 2013
Artificial endonucleases clustered regularly interspaced short palindromic repeats
(CRISPR)/CRISPR-associated (Cas) 9, can the more efficient and more accurately specific base of silence in organism genome
Cause, and making is simple, at low cost, and multiple sites on target gene can be sheared simultaneously, any number of single base of silence
Cause, but for the technology there are certain defect, miss rate is relatively high simultaneously.
Based on a kind of this stat1a Gene Deletions zebra fish of offer, improves the research to statla genes and increase spot simultaneously
The commercial value of horse fish just seems particularly necessary.
Invention content
The object of the present invention is to provide a kind of stat1a Gene Deletions zebra fish.In the method for the present invention, stat1 genes
It is related to the growth of cell, differentiation, apoptosis and immune gene expression.Research finds that stat1a genes are related to the development of bone,
The method of the present invention, can be used for carry out stat1a genes and skeleton development correlative study, also can income other aspect exploratory developments,
Whether the missing for detecting stat1a genes and the development of other organs there is correlation, such as heart etc. to have good medicine
Researching value, while knocking out the zebra fish of stat1a genes its growth cycle and being obviously shortened, it may have good commercial value.
In order to achieve the above objectives, the technical solution adopted in the present invention is：
A kind of stat1a Gene Deletions zebra fish, zebra fish stat1a gene delection experimental designs area knock out postorder
Row are as shown in SEQ ID No.1.
Further, a kind of stat1a Gene Deletions zebra fish is made of following steps：
Step 1: CRISPR/Cas9 gene knockout target position point designs：
The genomic dna sequence and its functional domain that zebra fish stat1a genes are inquired on NCBI, according to CRISPR/
Cas knocks out principle, designs the target site of a pair of stat1a genes, and the selection of target spot follows this standard：5’-GG-(N)18-NGG-
3’；The GG dinucleotides at wherein 5 ' ends is a part for T7 promoters, and 3 ' ends of target site are NGG；
Step 2: structure gRNA expression vectors and gRNA are synthesized in vitro
Step 3: the microinjection of zebrafish embryo
Within after fertilization 30min, embryo transfer is drawn to in the microinjection special culture dish of agarose production with suction pipe；
Before carrying out microinjection, Cas9mRNA and gRNA are made into mixed liquor, mixed well, makes Cas9mRNA's
The final concentration of 20ng/ μ L of final concentration of 300ng/ μ L, gRNA；It is thin in one to inject 1.8nL Cas9 mRNA and gRNA mixed liquors
In the fertilized eggs of born of the same parents' phase；The fertilized eggs injected are positioned over 5mmol/L NaCl, 0.33mmol/L CaCl2,0.33mmol/L
In the E3 water of MgSO4,0.17mmol/L KCl, 28 DEG C of hatchings；Embryonic phenotypes are observed under Stereo microscope, screen normal hair
The embryo educated is used for target position point mutation analysis；
Step 4: the validity of T7E1 methods and Sanger sequencing detection target sites
After carrying out microinjection to zebrafish embryo, the normal body early embryo of individual developmental is selected, its stat1a is detected
Gene with the presence or absence of mutation, in advance confirm this time selection target site it is whether effective, microinjection operation whether specification；
Step 5: after injecting two months, carry out cutting tail identification, ibid authentication step；
Step 6: the TA clones of aim sequence；
T7E1 digestion Preliminary Identifications have the possible aim sequence of mutation to carry out Sanger sequencings again；If sequencing peak figure has double
Peak, and sequencing result shows the aim sequence of insertion or deficient phenomena, picking monoclonal after being cloned followed by TA
Make further detection；
Step 7: the Sanger sequencings of plasmid
The plasmid that double digestion testing result display stripe size is met to expected results is sent to sequencing, is given later according to sequencing
The peak figure and sequence gone out, is compared on NCBI with standard aim sequence, according to comparison result, analyzes each monoclonal
Mutation type；
Step 8: obtaining the F1 generation of heritable zebra fish mutant
By front it is a series of screening zebra fish mutant F0 generations are determined, and then by F0 for mutant respectively with it is wild
Type zebra fish hybridizes to obtain F1 generation embryo, is placed in 28 DEG C of cultures, observes the survival rate of F1 generation in the early stage；It is fertilized two days later, each
Mutant F1 generation takes 10 embryos to carry out mutation heredity identification respectively；Each embryo is individually extracted into genome, then PCR expands
Increase the target site near zone for 700bp, then carry out T7E1 restriction analysis and part is sent to go to be sequenced, determines that this mutation whether may be used
To be genetic to F1 generation；
If detecting the presence of mutation from F1 generation embryo, the F1 generation of zebra fish mutant was brought up to 2-3 months；
Every F1 generation zebra fish adult fish is carried out respectively again to cut tail, screens F1 generation mutant；
Step 9: obtaining the F2 of zebra fish mutant for homozygote
The raun and milter of identical mutation are selected from F1 generation mutant, hybridization obtains F2 generations, is positioned over 28 DEG C of cultures, by
Essence takes partial embryonic to be identified after four days.Each embryo is individually extracted into genome, PCR amplification goes out near 700bp target sites
Region by I restricted enzyme cutting analysis of Bal and is sequenced, and whether preliminary test can obtain stat1a mutant homozygotes.Such as inspection
Testing result proves that there are homozygotes, then single cuts tail identification again after bringing up.
Step 10: carrying out the F3 generation pure lines heredity of the Gene Deletion zebra fish according to step 9 method, obtain this new
Zebra fish strain.
Further, in the step 1, primer sequence is：
F1- target site a forward primers：
tgtaatacgactcactataggagaacgagtctctggccagttttagagctagaaatagc
F2- target site b forward primers：
tgtaatacgactcactatagacctctgacattccagcaagttttagagctagaaatagc
Reverse primer shared R-：
Aagcaccgactcggtgccact。
Further, the step 2 specifically includes：
(1) gRNA skeletons are cloned on p42250 carriers first, 1-2 μ L plasmids is taken to be detected into row agarose gel electrophoresis；
(2) specificity gRNA is synthesized in vitro
This plasmid is linearized with BsaI restriction enzymes；Endonuclease reaction total volume is 20 μ L, and system is as follows：
In 37 DEG C of water-baths, digestion 2h or more after centrifugation mixing；
(3) using the p42250 carriers of linearisation as template, PCR is carried out by following specific primer, is amplified for spy
The double-stranded DNA of anisotropic gRNA synthesis；
PCR primer upstream and downstream primer is located on No. 1 and No. 3 intrones：
F：5’-CAGAAATCGGGGGAAAAATATAC-3’
R：5’-TGCTGTTGTACCATGGCTATACTT-3’
Forward primer F：T7 promoters _ 20bp target sequences _ the upstreams 20bp gRNA skeleton
Reverse primer R：The downstreams 20bp gRNA skeleton
PCR reaction systems are as follows：
After shaking mixing, 4 DEG C of centrifugations, in being carried out amplification reaction in PCR instrument；Reaction condition is：95 DEG C of 8min of pre-degeneration,
95 DEG C of 30s are wherein denaturalized, anneal 64 DEG C of 30s, extends 72 DEG C of 20s and carries out 30 cycles, then 72 DEG C of 8min；It waits for after reaction,
PCR product is centrifuged, takes 1 μ L samples point sample in carrying out electrophoresis, gel imaging system shooting result on 2.0% Ago-Gel；
(4) it detects after determining that band is correct, carries out Ago-Gel DNA recycling, purifying recycling PCR products；
(5) DNA concentration after purifying is measured, then using this DNA as template, in-vitro transcription is carried out with 20 μ L systems, is synthesized
Specific gRNA；Tip heads used in transcription experiment, EP pipes are the processed RNase-Free products of DEPC, and concrete operations are as follows：
In-vitro transcription reaction system：
Reactant is all added in the EP pipes of 1.5mL RNase-Free, after mixing, in 37 DEG C of 1.5 h of water-bath；
After water-bath, 1 μ L samples are taken, electrophoresis is carried out with prepared 2.0% Ago-Gel, with detection transcription knot
Fruit illustrates to transcribe successfully if transcription product size is consistent with expected；
1 μ L DNA enzymatics are added into transcription system, is positioned in 37 DEG C of water-baths and reacts 20min, to digest DNA moulds
Plate, then take 1 μ L transcription final products, i.e. gRNA is into row agarose gel electrophoresis, to detect transcriptional efficiency；
(6) purifying of specificity gRNA
Successful gRNA is transcribed with RNeasy Mini kit kits, is stored in -20 DEG C；Draw 1 μ L solution into
Row agarose gel electrophoresis to examine purified product, and measures the gRNA concentration after purifying.
Further, in the step 3, microinjection system is as follows：
Further, the concrete operations of the step 4 are：
(1) zebra fish genome is extracted
After zebrafish embryo 50hpf is fertilized 50 hours, embryo is in 1.5mL after collection wild type is compareed and injected respectively
In Ep pipes, often 5 embryos of pipe, extract genomic DNA, are as follows by the following method：
400 μ L cell pyrolysis liquids are added into the Ep pipes equipped with embryo, 2 μ L Proteinase Ks are positioned in 55 DEG C of water-baths and split
Solve 2 hours or more, during which every half an hour, mixing is gently overturned, to ensure that embryo is complete by abundant cracking；
After the completion of cracking, fully shaking on the oscillator is put, the isopropanol pre-cooled in equal volume is added in Ep pipes,
Fully reverse mixing, under the conditions of 4 DEG C, 12000 × g centrifuges 10min, outwells supernatant；
75% ethyl alcohol, 500 μ L are added, under the conditions of 4 DEG C, 12000 × g centrifuges 5min, abandons supernatant, room temperature air-dries 20min；
60-100 μ L deionized waters are added, fully blow and beat mixing, agarose gel electrophoresis Detection and Extraction efficiency
(2) PCR amplification aim sequence
After extracting genomic DNA, according to the genome area of CRISPR target sites upstream and downstream about 150-200bp, utilize
5.0 software Design primers sequences of Primer Premier are to amplify target DNA fragment.
PCR reaction systems are as follows：
After shaking mixing, 4 DEG C of centrifugations, in being carried out amplification reaction in PCR instrument, reaction condition is：94 DEG C of 2min of pre-degeneration,
94 DEG C of 30s are wherein denaturalized, anneal 55 DEG C of 30s, extends 72 DEG C of 23s totally 30 cycles, then 72 DEG C of 2min；Wait for after reaction, from
Heart PCR product takes 1 μ L samples point sample in carrying out electrophoresis on 2.0% Ago-Gel, and whether detection PCR product size is correct；
(3) if PCR product is correct, PCR product is detached with 2.0% agarose gel electrophoresis, under ultraviolet
Purpose band is cut, purifying recycling is carried out；
(4) T7 endonucleases I methods
Detect whether there is mutation with T7E1 analyses, first, the DNA15 μ L after taking purifying to recycle are loaded on the Ep of 150 μ L
Guan Zhong is placed in 95 DEG C of hot water and is denaturalized, then cooled to room temperature, then the DNA after denaturation is taken to carry out T7E1 digestions,
System is as follows：
After mixing system, the endonuclease reaction 30min in 37 DEG C of water-baths, then electrophoresis is carried out with 2% gel, to detect mesh
DNA fragmentation whether be cut open.If there is the band being cut open below target DNA fragment, then ImageJ softwares are used, enzyme is passed through
The brightness of rear band is cut to estimate the frequency of non-homologous end joining；
In addition, the target DNA fragment after sending partial purification carries out Sanger sequencings, tentatively obtained by the peak figure being sequenced
The information be inserted into or lacked.
Further, application of the stat1a Gene Deletions zebra fish in terms of zebra fish economy cultivation.
Compared with the existing technology, beneficial effects of the present invention are：It is dry thin that traditional gene targeting is built upon embryo
On born of the same parents (ESC) and the basis of homologous recombination technique, therefore Knockout technology is extremely inefficient.This method can be more efficient and more accurately exists
Silence specific gene in organism genome, and making is simple, at low cost, and multiple sites on target gene can be cut simultaneously
It cuts, any number of individual gene of silence.
Stat1 genes are related to the growth of cell, differentiation, apoptosis and immune gene expression.Research finds stat1a genes
Related to the development of bone, the method for the present invention can be used for carrying out the correlative study of stat1a genes and skeleton development, also can income
Whether other aspect exploratory developments, the missing of detection stat1a genes have correlation, such as heart with the development of other organs
Deng there is good medical research value, while knocking out the zebra fish of stat1a genes its growth cycle to be obviously shortened, it may have
Good commercial value.
Description of the drawings
Fig. 1 is the schematic diagram of CRISPR/Cas9 targeting systems；
Fig. 2 is the structure chart of target site on stat1a genes.
Fig. 3 deletion forms and wildtype gene sequence reverse contrast figure, the figure illustrate the position of deletion segment and the alkali of missing
Radix mesh.
Fig. 4 target sites nearby lack comparison diagram, and display knocks out successfully, there is the appearance of saltant type fish.
Fig. 5 is the prognostic chart of No. 17 zebra fish F1 generation mutant secondary structural changes.
Specific implementation mode
The following describes the present invention in detail with reference to the accompanying drawings and specific embodiments.
As shown in Figs. 1-3, a kind of stat1a Gene Deletions zebra fish of the present invention is made of following steps：
A, CRISPR/Cas9 gene knockouts target position point design
Zebra fish is inquired on National Center for Biotechnology Information (NCBI)
The genomic dna sequence and its functional domain of stat1a genes knock out principle, in website The according to CRISPR/Cas
ZiFiT Targeter(http://zifit.partners.org/ZiFiT_Cas9) on design a pair of stat1a genes target
Site.The selection of target spot must comply with this standard：5’-GG-(N)18-NGG-3’.The GG dinucleotides at wherein 5 ' ends is that T7 starts
A part for son, can be not limited when designing target site, but it must be ensured that 3 ' ends of target site are NGG.The choosing of target spot
The total domain of stat1a genes can be influenced by selecting the insertion that must assure that target position base or missing, to change gene
Expression.It is the schematic diagram of CRISPR/Cas9 targeting systems as shown in Figure 1, Fig. 2 is the structure chart of target site on stat1a genes.
Two pairs of Specific PCR primers are as follows：
F1 (target site a forward primers)：
tgTAATACGACTCACTATAggagaacgagtctctggccaGTTTTAGAGCTAGAAATAGC
F2 (target site b forward primers)：
tgTAATACGACTCACTATAgacctctgacattccagcaaGTTTTAGAGCTAGAAATAGC
R (shared reverse primer)：AAGCACCGACTCGGTGCCACT
B, gRNA expression vectors are built and gRNA is synthesized in vitro
(2) gRNA skeletons are cloned on p42250 carriers first, 1-2 μ L plasmids is taken to be detected into row agarose gel electrophoresis.
(2) specificity gRNA is synthesized in vitro
This plasmid is linearized with BsaI restriction enzymes.In general, endonuclease reaction total volume is 20 μ L, and system is as follows：
In 37 DEG C of water-baths, digestion 2h or more after centrifugation mixing.
(3) using the p42250 carriers of linearisation as template, PCR is carried out by following specific primer, is amplified for spy
The double-stranded DNA of anisotropic gRNA synthesis.
>PCR primer
PCR primer upstream and downstream primer is located on No. 1 and No. 3 intrones：
F(5’-CAGAAATCGGGGGAAAAATATAC-3’)
R(5’-TGCTGTTGTACCATGGCTATACTT-3’)
Forward primer F：T7 promoters _ 20bp target sequences _ the upstreams 20bp gRNA skeleton
Reverse primer R：The downstreams 20bp gRNA skeleton
PCR reaction systems (25 μ L) are as follows：
After shaking mixing, 4 DEG C of centrifugations, in being carried out amplification reaction in PCR instrument.Reaction condition is：95 DEG C of 8min of pre-degeneration,
(95 DEG C of 30s of denaturation, anneal 64 DEG C of 30s, extends 72 DEG C of 20s) 30 cycles, then 72 DEG C of 8min.It waits for after reaction, centrifuging PCR
Product takes 1 μ L samples point sample in carrying out electrophoresis, gel imaging system shooting result on 2.0% Ago-Gel
(4) it detects after determining that band is correct, carries out Ago-Gel DNA recycling, purifying recycling PCR products.
(5) DNA concentration (reaching 1 μ g as possible) after purifying is measured, then using this DNA as template, is carried out with 20 μ L systems
In-vitro transcription, synthesis specificity gRNA.Tip heads used in transcription experiment, EP pipes are the processed RNase-Free productions of DEPC
Product, concrete operations are as follows.
In-vitro transcription reaction system (20 μ L)：
Reactant is all added in the EP pipes of 1.5mL RNase-Free, after mixing, in 37 DEG C of 1.5 h of water-bath；
After water-bath, 1 μ L samples are taken, electrophoresis is carried out with prepared 2.0% Ago-Gel, with detection transcription knot
Fruit illustrates to transcribe successfully if transcription product size is consistent with expected；
1 μ L DNA enzymatics are added into transcription system, is positioned in 37 DEG C of water-baths and reacts 20min, to digest DNA moulds
Plate, then take 1 μ L transcription final products, i.e. gRNA is into row agarose gel electrophoresis, to detect transcriptional efficiency.
(6) purifying of specificity gRNA
Successful gRNA is transcribed with RNeasy Mini kit kits, is stored in -20 DEG C.Draw 1 μ L solution into
Row agarose gel electrophoresis to examine purified product, and measures the gRNA concentration after purifying.
C, the microinjection of zebrafish embryo
As possible within after fertilization 30min, draws embryo transfer with suction pipe and extremely use the microinjection of agarose production special
In culture dish.
Before carrying out microinjection, Cas9mRNA and gRNA are made into mixed liquor, mixed well, makes Cas9mRNA's
The final concentration of 20ng/ μ L of final concentration of 300ng/ μ L, gRNA.It is thin in one to inject about 1.8nL Cas9mRNA and gRNA mixed liquors
In the fertilized eggs of born of the same parents' phase.The fertilized eggs injected be positioned over E3 water (5mmol/L NaCl, 0.33mmol/L CaCl2,
0.33mmol/L MgSO4,0.17mmol/L KCl) in, 28 DEG C of hatchings.Embryonic phenotypes are observed under Stereo microscope, are screened
The embryo of normal development is used for target position point mutation analysis.
Microinjection system is as follows：
D, the validity of T7E1 methods and Sanger sequencing detection target sites
After carrying out microinjection to zebrafish embryo, the normal body early embryo of individual developmental is selected, its stat1a is detected
Gene with the presence or absence of mutation, can confirm in advance this time selection target site it is whether effective, microinjection operation whether specification.
(5) zebra fish genome is extracted
Zebrafish embryo be fertilized 50 hours after (50hpf), respectively collect wild type (control) and inject after embryo in
In 1.5mL Ep pipes (often 5 embryos of pipe), genomic DNA is extracted by the following method, is as follows：
400 μ L cell pyrolysis liquids are added into the Ep pipes equipped with embryo, 2 μ L Proteinase Ks are positioned in 55 DEG C of water-baths and split
Solution 2 hours above (period every half an hour, gently overturns mixing, to ensure embryo by abundant cracking completely)；
After the completion of cracking, fully shaking on the oscillator is put, isometric (400 μ L) isopropanol (pre-cooling) is added in Ep
Guan Zhong, fully reverse mixing, under the conditions of 4 DEG C, 12000 × g centrifuges 10min, outwells supernatant；
75% ethyl alcohol, 500 μ L are added, under the conditions of 4 DEG C, 12000 × g centrifuges 5min, abandons supernatant, and room temperature air-dries
20min；
60-100 μ L deionized waters are added, fully blow and beat mixing, agarose gel electrophoresis Detection and Extraction efficiency
(6) PCR amplification aim sequence
After extracting genomic DNA, according to the genome area of CRISPR target sites upstream and downstream about 150-200bp, utilize
5.0 software Design primers sequences of Primer Premier are to amplify target DNA fragment.
PCR reaction systems (50 μ L) are as follows：
After shaking mixing, 4 DEG C of centrifugations, in being carried out amplification reaction in PCR instrument.Reaction condition is：94 DEG C of 2min of pre-degeneration,
(94 DEG C of 30s of denaturation, anneal 55 DEG C of 30s, extends 72 DEG C of 23s) 30 cycles, then 72 DEG C of 2min.It waits for after reaction, centrifuging PCR
Product takes 1 μ L samples point sample in carrying out electrophoresis on 2.0% Ago-Gel, and whether detection PCR product size is correct.
(7) if PCR product is correct, PCR product is detached with 2.0% agarose gel electrophoresis, purpose is cut under ultraviolet
Band carries out purifying recycling.
(8) T7 endonucleases I (T7E1) method
Detect whether there is mutation with T7E1 analyses.First, the DNA15 μ L after taking purifying to recycle are loaded on the Ep of 150 μ L
Guan Zhong is placed in 95 DEG C of hot water and is denaturalized, then cooled to room temperature (at least 30 min).The DNA after denaturation is taken again
T7E1 digestions are carried out, system is as follows：
After mixing system, the endonuclease reaction 30min in 37 DEG C of water-baths, then electrophoresis is carried out with 2% gel, to detect mesh
DNA fragmentation whether be cut open.If there is the band being cut open below target DNA fragment, then ImageJ softwares are used, enzyme is passed through
The brightness of rear band is cut to estimate the frequency of non-homologous end joining.
(9) in addition, target DNA fragment after sending partial purification carries out Sanger sequencings, come tentatively by the peak figure being sequenced
The information be inserted into or lacked.
D, it after injecting two months, carries out cutting tail identification, ibid authentication step.
E, the TA clones of aim sequence
T7E1 digestion Preliminary Identifications have the possible aim sequence of mutation to carry out Sanger sequencings again.If sequencing peak figure has double
Peak, and sequencing result shows the aim sequence of insertion or deficient phenomena, picking monoclonal after being cloned followed by TA
Make further detection.
F, the Sanger sequencings of plasmid
The plasmid that double digestion testing result display stripe size is met to expected results is sent to sequencing, is given later according to sequencing
The peak figure and sequence gone out, is compared on NCBI with standard aim sequence, according to comparison result, analyzes each monoclonal
Mutation type.
G, the F1 generation of heritable zebra fish mutant is obtained
By front it is a series of screening zebra fish mutant F0 generations are determined, and then by F0 for mutant respectively with it is wild
Type zebra fish hybridizes to obtain F1 generation embryo, is placed in 28 DEG C of cultures, observes the survival rate of F1 generation in the early stage.It is fertilized two days later, each
Mutant F1 generation takes 10 embryos to carry out mutation heredity identification respectively.Each embryo is individually extracted into genome, then PCR expands
Increase the target site near zone for 700bp, then carry out T7E1 restriction analysis and part is sent to go to be sequenced, determines that this mutation whether may be used
To be genetic to F1 generation.
If detecting the presence of mutation from F1 generation embryo, the F1 generation of zebra fish mutant was brought up to 2-3 months.
Every F1 generation zebra fish adult fish is carried out respectively again to cut tail, screening F1 generation mutant (specific method is as previously described).
H, the F2 of zebra fish mutant is obtained for homozygote
The raun and milter of identical mutation are selected from F1 generation mutant, hybridization obtains F2 generations, is positioned over 28 DEG C of cultures, by
Essence takes partial embryonic to be identified after four days.Each embryo is individually extracted into genome, PCR amplification goes out near 700bp target sites
Region by I restricted enzyme cutting analysis of Bal and is sequenced, and whether preliminary test can obtain stat1a mutant homozygotes.Such as inspection
Testing result proves that there are homozygotes, then single cuts tail identification again after bringing up.Fig. 3 is that deletion form and wildtype gene sequence are reversely right
Than figure, which illustrates the position of deletion segment and the base number of missing.
Fig. 4 target sites nearby lack comparison diagram, and display knocks out successfully, there is the appearance of saltant type fish.The zebra fish stat1a
Sequence is as shown in SEQ ID No.1 after gene delection experimental design area knocks out.
Sequencing result and wild-type sequence (700bp) are compared, find (runic table at two target sites of stat1a
Show, underscore) there is base deletion, there is the missing (2 amino acid of missing) of 6 bases at target site a, has 18 at target site b
The missing (6 amino acid of missing) of a base.
I, the F3 generation pure lines heredity that can ibid carry out the Gene Deletion zebra fish, obtains this new zebra fish strain.
Such as the prediction that Fig. 5 is No. 17 zebra fish F1 generation mutant secondary structural changes
Since the stat1a Gene Partial base deletions of the F1 generation of No. 17 mutant screened do not cause whole gene
Frameshift mutation, the expression of zebra fish stat1a genes cannot be changed completely.Next, soft using Protein Conformation Prediction
Part Phyre predicts the structure of the F1 generation mutant STAT1 screened, is wild-type zebrafish as a result as shown above
With the comparing result of the secondary structure of the STAT1 albumen of No. 17 zebra fish F1 generation mutant, certain conserved structures of STAT1 are shown
There is change (being identified with circle in figure) at domain.It is homologous dimerization body interface at the V638 and S639 of STAT1 albumen, corresponds to 17
The STAT1 albumen of number zebra fish F1 generation, the two amino acid sites, V630 and S631 are but no longer homologous dimerization contact levels
.Although as a result illustrating change of the F1 generation without entire STAT1 albumen of No. 17 screened zebra fish, due to certain guarantors
The variation for keeping structural domain site influences the combination of STAT1 protein dimers, can influence the bone growth mechanism of zebra fish, add
The speed of growth of fast zebra fish.
Test example：
In 3 different phases (development 20 days, 30 days and 47 days) of embryonic development, 10 zebra fish of every group of random picking,
The length of every zebra fish is measured respectively, and surveyed length mean value is reported in Table 1 below.
The measurement length of the F1 generation body early embryo of 1 mutant zebra fish
Every group of data are analyzed, are as a result reported in Table 2 below.The results show that under same culture conditions, 3 differences
The F1 generation of No. 17 zebra fish in period all obviously grows fast than other two groups.At 20 days, No. 17 groups are compared with wild type group, as a result
Highly significant (P=0.002), at 30 days, No. 17 groups are compared with wild type group, as a result reach significance (P=0.015)；
As a result it may imply, the mutation of zebra fish stat1a genes can accelerate the speed of growth of zebra fish.
Significance analysis of the table 2F1 for the measurement length of body early embryo
* is 0.05 for standard with significance, and the P values for reaching significance are marked with runic.
Claims (2)
1. a kind of method of gene knockout selection and breeding stat1a Gene Deletion zebra fish, which is characterized in that the zebra fish
As shown in SEQIDNo.1, which is obtained sequence by following steps after stat1a gene delection experimental designs area knocks out：
Step 1: CRISPR/Cas9 gene knockout target position point designs：
The genomic dna sequence and its functional domain that zebra fish stat1a genes are inquired on NCBI, according to CRISPR/Cas
Principle is knocked out, the target site of a pair of stat1a genes is designed, the selection of target spot follows this standard：5’-GG-(N)18-NGG-3’；
The GG dinucleotides at wherein 5 ' ends is a part for T7 promoters, and 3 ' ends of target site are NGG；
Step 2: structure gRNA expression vectors and gRNA are synthesized in vitro：
(1) gRNA skeletons are cloned on p42250 carriers first, 1-2 μ L plasmids is taken to be detected into row agarose gel electrophoresis；
(2) specificity gRNA is synthesized in vitro
This plasmid is linearized with BsaI restriction enzymes；Endonuclease reaction total volume is 20 μ L, and system is as follows：
In 37 DEG C of water-baths, digestion 2h or more after centrifugation mixing；
(3) using the p42250 carriers of linearisation as template, PCR is carried out by following specific primer, is amplified for specificity
The double-stranded DNA of gRNA synthesis；
PCR primer upstream and downstream primer is located on No. 1 and No. 3 intrones：
F：5’-CAGAAATCGGGGGAAAAATATAC-3’
R：5’-TGCTGTTGTACCATGGCTATACTT-3’
Forward primer F：T7 promoters _ 20bp target sequences _ the upstreams 20bpgRNA skeleton
Reverse primer R：The downstreams 20bpgRNA skeleton
PCR reaction systems are as follows：
After shaking mixing, 4 DEG C of centrifugations, in being carried out amplification reaction in PCR instrument；Reaction condition is：95 DEG C of 8min of pre-degeneration, wherein
95 DEG C of 30s are denaturalized, anneal 64 DEG C of 30s, extends 72 DEG C of 20s and carries out 30 cycles, then 72 DEG C of 8min；It waits for after reaction, centrifugation
PCR product takes 1 μ L samples point sample in carrying out electrophoresis, gel imaging system shooting result on 2.0% Ago-Gel；
(4) it detects after determining that band is correct, carries out Ago-Gel DNA recycling, purifying recycling PCR product；
(5) DNA concentration after purifying is measured, then using this DNA as template, carries out in-vitro transcription with 20 μ L systems, synthesis is special
Property gRNA；Tip heads used in transcription experiment, EP pipes are the processed RNase-Free products of DEPC, and concrete operations are as follows：
In-vitro transcription reaction system：
Reactant is all added in the EP pipes of 1.5mLRNase-Free, after mixing, in 37 DEG C of water-bath 1.5h；
After water-bath, 1 μ L samples are taken, electrophoresis is carried out with prepared 2.0% Ago-Gel, to detect transcription result,
If transcription product size is consistent with expected, illustrate to transcribe successfully；
1 μ LDNA enzymes are added into transcription system, is positioned in 37 DEG C of water-baths and reacts 20min, to digest DNA profiling, then take
1 μ L transcribe final product, i.e. gRNA is into row agarose gel electrophoresis, to detect transcriptional efficiency；
(6) purifying of specificity gRNA
Successful gRNA is transcribed with RNeasyMinikit kits, is stored in -20 DEG C；It draws 1 μ L solution and carries out agarose
Gel electrophoresis to examine purified product, and measures the gRNA concentration after purifying；
Step 3: the microinjection of zebrafish embryo, microinjection system is as follows：
Within after fertilization 30min, embryo transfer is drawn to in the microinjection special culture dish of agarose production with suction pipe；
Before carrying out microinjection, Cas9mRNA and gRNA are made into mixed liquor, mixed well, makes the final concentration of Cas9mRNA
For the final concentration of 20ng/ μ L of 300ng/ μ L, gRNA；Inject 1.8nLCas9mRNA and gRNA mixed liquors in one cell stage by
In smart ovum；The fertilized eggs injected are positioned over 5mmol/LNaCl, 0.33mmol/LCaCl2,0.33mmol/LMgSO4,
In the E3 water of 0.17mmol/LKCl, 28 DEG C of hatchings；Embryonic phenotypes are observed under Stereo microscope, screen the embryo of normal development
For target position point mutation analysis；
Step 4: the validity of T7E1 methods and Sanger sequencing detection target sites：
After carrying out microinjection to zebrafish embryo, the normal body early embryo of individual developmental is selected, its stat1a gene is detected
With the presence or absence of mutation, confirm whether the target site of this time selection effective in advance, microinjection operation whether specification；
Step 5: after injecting two months, carry out cutting tail identification, ibid authentication step；
Step 6: the TA clones of aim sequence：
T7E1 digestion Preliminary Identifications have the possible aim sequence of mutation to carry out Sanger sequencings again；If sequencing peak figure have it is bimodal, and
And sequencing result shows the aim sequence of insertion or deficient phenomena, picking monoclonal is made into one after being cloned followed by TA
Step detection；
Step 7: the Sanger sequencings of plasmid：
The plasmid that double digestion testing result display stripe size is met to expected results is sent to sequencing, is provided later according to sequencing
Peak figure and sequence are compared on NCBI with standard aim sequence, according to comparison result, analyze the mutation of each monoclonal
Type；
Step 8: obtaining the F1 generation of heritable zebra fish mutant：
By front it is a series of screening zebra fish mutant F0 generations are determined, and then by F0 for mutant respectively with wild type spot
Horse fish hybridizes to obtain F1 generation embryo, is placed in 28 DEG C of cultures, observes the survival rate of F1 generation in the early stage；Fertilization is two days later, each to be mutated
Body F1 generation takes 10 embryos to carry out mutation heredity identification respectively；Each embryo is individually extracted into genome, then PCR amplification goes out
The target site near zone of 700bp, then carry out T7E1 restriction analysis and part is given to go to be sequenced, determine whether this mutation can heredity
To F1 generation；
If detecting the presence of mutation from F1 generation embryo, the F1 generation of zebra fish mutant was brought up to 2-3 months；Distinguish again
Every F1 generation zebra fish adult fish is carried out to cut tail, screens F1 generation mutant；
Step 9: obtaining the F2 of zebra fish mutant for homozygote：
The raun and milter of identical mutation are selected from F1 generation mutant, hybridization obtains F2 generations, is positioned over 28 DEG C of cultures, fertilization four
It taking partial embryonic to be identified after it, each embryo is individually extracted into genome, PCR amplification goes out 700bp target site near zones,
It by I restricted enzyme cutting analysis of Bal and is sequenced, whether preliminary test can obtain stat1a mutant homozygotes；Such as examine knot
Fruit proves that there are homozygotes, then single cuts tail identification again after bringing up；
Step 10: carrying out the F3 generation pure lines heredity of the Gene Deletion zebra fish according to step 9 method, this new spot is obtained
Horse fish products system.
2. a kind of method of gene knockout selection and breeding stat1a Gene Deletion zebra fish according to claim 1, feature
It is, the concrete operations of the step 4 are：
(1) zebra fish genome is extracted
After zebrafish embryo 50hpf is fertilized 50 hours, embryo after wild type is compareed and injected is collected respectively and is managed in 1.5mLEp
In, often 5 embryos of pipe, extract genomic DNA, are as follows by the following method：
400 μ L cell pyrolysis liquids are added into the Ep pipes equipped with embryo, 2 μ L Proteinase Ks are positioned in 55 DEG C of water-baths and crack 2
Hour or more, during which every half an hour, mixing is gently overturned, to ensure that embryo is complete by abundant cracking；
After the completion of cracking, fully shaking on the oscillator is put, the isopropanol pre-cooled in equal volume is added in Ep pipes, fully runs
Mixing, under the conditions of 4 DEG C, 12000 × g centrifuges 10min, outwells supernatant；
75% ethyl alcohol, 500 μ L are added, under the conditions of 4 DEG C, 12000 × g centrifuges 5min, abandons supernatant, room temperature air-dries 20min；
60-100 μ L deionized waters are added, fully blow and beat mixing, agarose gel electrophoresis Detection and Extraction efficiency
(2) PCR amplification aim sequence
After extracting genomic DNA, according to the genome area of CRISPR target sites upstream and downstream about 150-200bp, utilize
PrimerPremier5.0 software Design primers sequence is to amplify target DNA fragment；
After shaking mixing, 4 DEG C of centrifugations, in being carried out amplification reaction in PCR instrument, reaction condition is：94 DEG C of 2min of pre-degeneration, wherein
94 DEG C of 30s are denaturalized, anneal 55 DEG C of 30s, extends 72 DEG C of 23s totally 30 cycles, then 72 DEG C of 2min；It waits for after reaction, centrifuging PCR
Product takes 1 μ L samples point sample in carrying out electrophoresis on 2.0% Ago-Gel, and whether detection PCR product size is correct；
(3) if PCR product is correct, PCR product is detached with 2.0% agarose gel electrophoresis, purpose band is cut under ultraviolet,
Carry out purifying recycling；
(4) T7 endonucleases I methods
Detect whether there is mutation with T7E1 analyses, first, in Ep pipes of the DNA15 μ L loaded on 150 μ L after taking purifying to recycle,
It is placed in 95 DEG C of hot water and is denaturalized, then cooled to room temperature, then the DNA after denaturation is taken to carry out T7E1 digestions, system
It is as follows：
After mixing system, the endonuclease reaction 30min in 37 DEG C of water-baths, then electrophoresis is carried out with 2% gel, with testing goal
Whether DNA fragmentation is cut open, if there is the band being cut open below target DNA fragment, then uses ImageJ softwares, passes through digestion
The frequency of non-homologous end joining is estimated in the brightness of band afterwards；
In addition, the target DNA fragment after sending partial purification carries out Sanger sequencings, tentatively it is inserted by the peak figure being sequenced
Or the information of missing.
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