CN109402170A - A kind of method for building up of fish male sterility model - Google Patents
A kind of method for building up of fish male sterility model Download PDFInfo
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- CN109402170A CN109402170A CN201811297606.0A CN201811297606A CN109402170A CN 109402170 A CN109402170 A CN 109402170A CN 201811297606 A CN201811297606 A CN 201811297606A CN 109402170 A CN109402170 A CN 109402170A
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- 208000007466 Male Infertility Diseases 0.000 title claims abstract description 27
- 206010021929 Infertility male Diseases 0.000 title claims abstract description 24
- 241000251468 Actinopterygii Species 0.000 title claims abstract description 18
- 238000000034 method Methods 0.000 title claims abstract description 14
- 101100366466 Caenorhabditis elegans spo-11 gene Proteins 0.000 claims abstract description 44
- 108091033409 CRISPR Proteins 0.000 claims abstract description 29
- 238000010354 CRISPR gene editing Methods 0.000 claims abstract description 7
- 238000005516 engineering process Methods 0.000 claims abstract description 7
- 230000021121 meiosis Effects 0.000 claims abstract description 6
- 239000013612 plasmid Substances 0.000 claims description 31
- 230000035772 mutation Effects 0.000 claims description 28
- 108020004414 DNA Proteins 0.000 claims description 22
- 241000588724 Escherichia coli Species 0.000 claims description 15
- 241000252212 Danio rerio Species 0.000 claims description 13
- 108020005004 Guide RNA Proteins 0.000 claims description 12
- 108020004999 messenger RNA Proteins 0.000 claims description 12
- 108090000790 Enzymes Proteins 0.000 claims description 11
- 102000004190 Enzymes Human genes 0.000 claims description 11
- 239000000284 extract Substances 0.000 claims description 9
- 238000000520 microinjection Methods 0.000 claims description 9
- 238000012408 PCR amplification Methods 0.000 claims description 8
- 238000001712 DNA sequencing Methods 0.000 claims description 7
- 239000002773 nucleotide Substances 0.000 claims description 7
- 125000003729 nucleotide group Chemical group 0.000 claims description 7
- 108090000623 proteins and genes Proteins 0.000 claims description 6
- 238000012216 screening Methods 0.000 claims description 6
- 238000013518 transcription Methods 0.000 claims description 6
- 230000035897 transcription Effects 0.000 claims description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- 239000000243 solution Substances 0.000 claims description 5
- 102000002322 Egg Proteins Human genes 0.000 claims description 4
- 108010000912 Egg Proteins Proteins 0.000 claims description 4
- 210000004681 ovum Anatomy 0.000 claims description 4
- BELBBZDIHDAJOR-UHFFFAOYSA-N Phenolsulfonephthalein Chemical compound C1=CC(O)=CC=C1C1(C=2C=CC(O)=CC=2)C2=CC=CC=C2S(=O)(=O)O1 BELBBZDIHDAJOR-UHFFFAOYSA-N 0.000 claims description 3
- 238000007796 conventional method Methods 0.000 claims description 3
- 238000013461 design Methods 0.000 claims description 3
- 238000001514 detection method Methods 0.000 claims description 3
- 230000004720 fertilization Effects 0.000 claims description 3
- 238000000338 in vitro Methods 0.000 claims description 3
- 238000002347 injection Methods 0.000 claims description 3
- 239000007924 injection Substances 0.000 claims description 3
- 230000009027 insemination Effects 0.000 claims description 3
- 238000004519 manufacturing process Methods 0.000 claims description 3
- 229960003531 phenolsulfonphthalein Drugs 0.000 claims description 3
- 239000011535 reaction buffer Substances 0.000 claims description 3
- 238000011084 recovery Methods 0.000 claims description 3
- 238000010839 reverse transcription Methods 0.000 claims description 3
- 238000012795 verification Methods 0.000 claims description 3
- 238000010362 genome editing Methods 0.000 claims description 2
- 238000010367 cloning Methods 0.000 claims 1
- 210000004027 cell Anatomy 0.000 description 4
- 230000002710 gonadal effect Effects 0.000 description 4
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 102100035102 E3 ubiquitin-protein ligase MYCBP2 Human genes 0.000 description 1
- 241000305071 Enterobacterales Species 0.000 description 1
- 108020005038 Terminator Codon Proteins 0.000 description 1
- 101710183280 Topoisomerase Proteins 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 210000002257 embryonic structure Anatomy 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 230000006801 homologous recombination Effects 0.000 description 1
- 238000002744 homologous recombination Methods 0.000 description 1
- 208000000509 infertility Diseases 0.000 description 1
- 230000036512 infertility Effects 0.000 description 1
- 231100000535 infertility Toxicity 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 230000000869 mutational effect Effects 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
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- C12N15/09—Recombinant DNA-technology
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/90—Stable introduction of foreign DNA into chromosome
- C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
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- A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
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- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/40—Fish
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
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Abstract
The invention discloses a kind of method for building up of fish male sterility model, meiosis related gene spo11 is knocked out using CRISPR/Cas9 technology, heterozygote is fertile, the male infertility in the homozygote group that heterozygote is selfed, to establish fish male sterility model.By means of the invention it is also possible to which specificity obtains male sterility individual, family can be knocked out by establishing, male sterility individual is continually provided, it is easy to operate, save the time.
Description
Technical field
The present invention relates to a kind of method for building up of fish male sterility model, belong to molecular biology field.
Background technique
Fish obtain energy from the external world, and for a part for growing, a part is used for reproduction, during reproduction, slow growth
Even stop, and the reproduction period of many fish was between growth period.Because the growth of fish all-male is very fast, individual is relatively large,
Thus become everybody and fall over each other the object of cultivation, so in recent years, it is more popular research that how fish male sterile line, which is established,
One of direction.Spo11 is the homologous protein of II type topoisomerase, participates in the formation of DNA double chain cleaved complex (DSBs),
Critical function is played in the generation of meiotic homologous recombination.In the meiosis of the biology of most of zoogamy, recombination
Vital role is play, it is the important sources of genetic diversity, is capable of the gamete of Haploid production, while sister contaminates
Chromophoric monomer cross exchanged also increases the diversity of gamete.For inventor in the experiment of many years, discovery knocks out a meiosis
Related gene spo11 leads to zebra fish male specificity infertility, and female is barely affected, and there is presently no people to report base
Because spo11 has this function.
Summary of the invention
In view of the deficiency of the prior art, building the object of the present invention is to provide a kind of fish male sterility model
Cube method.
To achieve the above object, the technical solution adopted by the present invention is that: the method for building up of fish male sterility model, it is special
Sign is, is knocked out using CRISPR/Cas9 technology to meiosis related gene spo11, and heterozygote is fertile, and heterozygote is certainly
The male infertility in obtained homozygote group is handed over, to establish fish male sterility model.
Preferably, when establishing, homozygous males and heterozygous male are distinguished using round pcr.
Preferably, the step of knocking out zebra fish spo11 gene above by CRISPR/Cas9 gene editing technology is as follows:
(1) pass through ensembl online data library lookup zebra fish spo11 genome sequence no:ENSDART00000005373.9,
Design knocks out target site on first exon of the gene, and target site sequence is SEQ ID NO:1, it may be assumed that TGGAAACGGTCGA
CAGATGCCAGG；
(2) detection target site whether there is single nucleotide polymorphism according to a conventional method；
(3) acquisition of gRNA:
Using gRNA-plasmid as template, using gRNA-F/gRNA-R as primer, the sequence of gRNA-F is SEQ ID NO:2:TAA
TACGACTCACTATAGGGTGGAAACGGTCGACAGATGCCGTTTTAGAGCTAGAAATAAG；The nucleotide sequence of gRNA-R
For SEQ ID NO:3:AGCACCGACTCGGTGCCACT, KOD Plus(TAKARA is utilized) high fidelity enzyme progress PCR amplification, expands
Volume increase object is tapped and recovered, and recovery product connects pMD18-T carrier (TAKARA) transformed competence colibacillus Escherichia coli afterwards, picking 5 big
Enterobacteria monoclonal extracts Plasmid DNA after expanding culture, and then Plasmid DNA sequencing carries out sequence verification, and sequence correctly clones expansion
The template that Plasmid DNA transcribes gRNA as next step is extracted after big culture；Recycle MEGAscript T7
Transcription Kit (InvitrogenTM) by the external reverse transcription 20ul of the plasmid containing spo11 target sequence built
Reaction system is as follows:
10x Reaction Buffer 2ul；
Plasmid DNA 4ul (about 1ug)；
T7 Enzyme 2ul；
10mmol/L NTP 1ul；
DEPC water 11ul；
It is purified after 37 DEG C of the above system one hour of reaction spare；
(4) it is transcribed in vitro and obtains Cas9 mRNA:
Cas9 plasmid comes from AddgeneTM(#63154) uses MAXIscript T7/T3 Transcription Kit
(InvitrogenTM), Cas9 mRNA is synthesized, system and reaction condition are the same as step 3；
(5) the micro- co-injection of Cas9 mRNA and gRNA: configuration microinjection system first is as follows:
300 ng/ul of Cas9 mRNA；
GRNA 30ng/ul；
Phenol-red 0.2ul；
DEPC Water up to 2ul；
After above-mentioned solution is prepared mixing, in the zebra fish fertilized egg in microinjection unicellular period, cultivation detects after 24 hours
The mutation efficiency of spo11 gene；
(6) screening of spo11 F1 generation heterozygous mutant individual and mutation type determine:
The zebra fish fertilized egg after step (5) microinjection hybridizes with wild type individual and obtains to after sexal maturity for cultivation 3 months
First familiar generation is obtained, after F1 generation individual cultivates 2 months, clip part tail fin tissue extracts genomic DNA, with F-spo11/R-
Spo11 is primer, F-spo11:5 '-ATGGCTTACCAGTTTACTG-3 '; R-spo11: 5'-
CGTAGCTCTTTCAGTAACTC-3 ' carries out PCR amplification, PCR product connection using KOD PLUS high fidelity enzyme (TAKARA)
PMD18-T carrier (TAKARA) transformed competence colibacillus Escherichia coli afterwards pick them separately after 10 Escherichia coli clones expand culture
Plasmid DNA is extracted, screening obtains 10 F1 generation idiovariation efficiency and mutation type after then Plasmid DNA sequencing compares；
(7) acquisition of the spo11 F2 for homozygous mutation individual:
After spo11 F1 generation heterozygous mutant individual sexal maturity, each progress of the consistent male and female F1 generation individual of mutation type is chosen
Artificial insemination obtains spo11 F2 for homozygous mutation individual；
(8) selection of male sterility male:
Spo11 F2 is male sterility individual for male all in homozygous mutation group, but has ovum in female individuals
Son, and being capable of normal fertilization with wild milter.
Compared with prior art, beneficial effects of the present invention: by means of the invention it is also possible to which specificity obtains male not
Individual is educated, family can be knocked out by establishing, male sterility individual is continually provided, it is easy to operate, save the time.
Detailed description of the invention
Fig. 1 be the present invention relates to related target site, ammonia corresponding to mutation type and target site mutant gene sequence
The variation of base acid, wherein A is CRISPR/Cas9 technical role target site；For target site sequence abrupt climatic change, (dotted line institute's frame is to build to B
Mutation type selected by vertical family)；For the variation of amino acid corresponding to target site mutant gene sequence, (dotted line institute's frame is to establish house to C
It is selected amino acid mutation type)
Fig. 2 is spo11 raun reproduction cell (100 times) microscope figure: wherein A is the ovum compareed in sexal maturity raun gonadal tissue
Cell, B are the egg cell in spo11 sexal maturity raun gonadal tissue；
Fig. 3 is spo11 milter reproduction cell (100 times) microscope figure: wherein A is fine in control sexal maturity hero and gonadal tissue
Born of the same parents' quantity, B are in spo11 sexal maturity milter gonadal tissue without spermatid.
Specific embodiment
Illustrate technical solution of the present invention and technical effect now in conjunction with specific embodiment, but is not to limit guarantor of the present invention
Protect the foundation of range.
Embodiment one
The method for building up of fish male sterility model, be using CRISPR/Cas9 technology to meiosis related gene spo11 into
Row knocks out, the Serial No. no:ENSDART00000005373.9 of spo11；Heterozygote is fertile, distinguishes heterozygosis using round pcr
Homozygote male and the heterozygote male that body is selfed, wherein the male in homozygote group is sterile, to establish fish
Class male sterility model, specific step is as follows for method for building up:
(1) pass through ensembl online data library lookup zebra fish spo11 genome sequence no:ENSDART00000005373.9,
Design knocks out target site on first exon of the gene, and target site sequence is sequence 1:TGGAAACGGTCGACAGATGCC
[AGG], interior bracket is PAM sequence.
(2) detection target site whether there is single nucleotide polymorphism (SNP) according to a conventional method:
The genomic DNA that 6 zebra fish extract tail fin tissue respectively is randomly selected, as pcr template, with F-spo11/R-
Spo11 is primer, F-spo11:5 '-ATGGCTTACCAGTTTACTG-3 '; R-spo11: 5'-
CGTAGCTCTTTCAGTAACTC-3 ' carries out PCR amplification, PCR product connection using KOD PLUS high fidelity enzyme (TAKARA)
PMD18-T carrier (TAKARA) transformed competence colibacillus Escherichia coli afterwards pick them separately after 5 Escherichia coli clones expand culture and mention
Plasmid DNA is taken, then Plasmid DNA sequencing is found in selected target site after comparing without SNP site.
(3) acquisition of gRNA:
Using gRNA-plasmid as template, using gRNA-F/gRNA-R as primer, the nucleotide sequence of gRNA-F such as sequence 2:TAA
TACGACTCACTATAGGGTGGAAACGGTCGACAGATGCCGTTTTAGAGCTAGAAATAAG；The nucleotide sequence of gRNA-R
Such as sequence 3:AGCACCGACTCGGTGCCACT, KOD Plus(TAKARA is utilized) high fidelity enzyme progress PCR amplification, amplified production
It is tapped and recovered, recovery product connects pMD18-T carrier (TAKARA) transformed competence colibacillus Escherichia coli afterwards, 5 Escherichia coli of picking
Monoclonal extracts Plasmid DNA after expanding culture, and then Plasmid DNA sequencing carries out sequence verification, and sequence correctly clones expansion culture
The template that Plasmid DNA transcribes gRNA as next step is extracted afterwards；Recycle MEGAscript T7 Transcription Kit
(InvitrogenTM) the external reverse transcription 20ul reaction system of the plasmid containing spo11 target sequence built is as follows:
10x Reaction Buffer 2ul；
Plasmid DNA 4ul (about 1ug)；
T7 Enzyme 2ul；
10mmol/L NTP 1ul；
DEPC water 11ul；
It is purified after 37 DEG C of the above system one hour of reaction spare.
(4) it is transcribed in vitro and obtains Cas9 mRNA:
Cas9 plasmid comes from AddgeneTM(#63154) uses MAXIscript T7/T3 Transcription Kit
(InvitrogenTM), Cas9 mRNA is synthesized, system and reaction condition are the same as step 3.
(5) the micro- co-injection of Cas9 mRNA and gRNA: configuration microinjection system first is as follows:
300 ng/ul of Cas9 mRNA；
GRNA 30ng/ul；
Phenol-red 0.2ul；
DEPC Water up to 2ul；
After above-mentioned solution is prepared mixing, in the zebra fish fertilized egg in microinjection unicellular period, cultivation detects after 24 hours
The mutation efficiency of spo11 gene.Method is as follows: randomly selecting 8 embryos and extracts genomic DNA, is with F-spo11/R-spo11
Primer, F-spo11:5 '-ATGGCTTACCAGTTTACTG-3 ';R-spo11:5 '-CGTAGCTCTTTCAGTAACTC-3 ',
PCR amplification is carried out using KOD PLUS high fidelity enzyme (TAKARA), PCR product connection pMD18-T carrier (TAKARA) converts afterwards
Competent E.coli picks them separately after 20 Escherichia coli clones expand culture and extracts Plasmid DNA, and then Plasmid DNA is surveyed
Sequence obtains mutation efficiency after comparing.
(6) screening of spo11 F1 generation heterozygous mutant individual and mutation type determine:
The zebra fish fertilized egg after step (5) microinjection hybridizes with wild type individual and obtains to after sexal maturity for cultivation 3 months
First familiar generation is obtained, after F1 generation individual cultivates 2 months, clip part tail fin tissue extracts genomic DNA, with F-spo11/R-
Spo11 is primer, F-spo11:5 '-ATGGCTTACCAGTTTACTG-3 '; R-spo11: 5'-
CGTAGCTCTTTCAGTAACTC-3 ' carries out PCR amplification, PCR product connection using KOD PLUS high fidelity enzyme (TAKARA)
PMD18-T carrier (TAKARA) transformed competence colibacillus Escherichia coli afterwards pick them separately after 10 Escherichia coli clones expand culture
Plasmid DNA is extracted, screening obtains 10 F1 generation idiovariation efficiency and mutation type after then Plasmid DNA sequencing compares.
(7) acquisition of the spo11 F2 for homozygous mutation individual:
After spo11 F1 generation heterozygous mutant individual sexal maturity, each progress of the consistent male and female F1 generation individual of mutation type is chosen
Artificial insemination obtains spo11 F2 for homozygous mutation individual.The mutation type marked in selection attached drawing 1B, the mutation type is prominent
Become location proximate and produce termination codon, i.e., can only generate the corresponding amino of presequence in the mutational site spo11 in mutated individual
Acid.
(8) selection of male sterility male:
Spo11 F2 is male sterility individual for male all in homozygous mutation group, such as attached drawing 2, spo11 F2 generation
Without sperm in homozygous mutation male spermary, but there is ovum in female individuals, and being capable of normal fertilization with wild milter.
Sequence table
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<120>a kind of method for building up of fish male sterility model
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tggaaacggt cgacagatgc cagg 24
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Claims (3)
1. a kind of method for building up of fish male sterility model, which is characterized in that using CRISPR/Cas9 technology to meiosis
Related gene spo11 is knocked out, and heterozygote is fertile, the male infertility in the homozygote group that heterozygote is selfed,
To establish fish male sterility model.
2. the method for building up of fish male sterility model according to claim 1, which is characterized in that when establishing, utilize PCR
Technology distinguishes homozygous males and heterozygous male.
3. the method for building up of fish male sterility model according to claim 2, which is characterized in that above by
It is as follows that CRISPR/Cas9 gene editing technology knocks out the step of zebra fish spo11 gene:
(1) pass through ensembl online data library lookup zebra fish spo11 genome sequence no:ENSDART00000005373.9,
Design knocks out target site on first exon of the gene, and target site sequence is sequence 1, it may be assumed that TGGAAACGGTCGACAGATG
CCAGG；
(2) detection target site whether there is single nucleotide polymorphism according to a conventional method；
(3) acquisition of gRNA:
Using gRNA-plasmid as template, using gRNA-F/gRNA-R as primer, the sequence of gRNA-F such as sequence 2:TAATACGAC
TCACTATAGGGTGGAAACGGTCGACAGATGCCGTTTTAGAGCTAGAAATAAG；The nucleotide sequence of gRNA-R such as sequence
3:AGCACCGACTCGGTGCCACT utilizes KOD Plus(TAKARA) high fidelity enzyme carries out PCR amplification, and amplified production taps rubber back
It receives, recovery product connects pMD18-T carrier (TAKARA) transformed competence colibacillus Escherichia coli afterwards, 5 Escherichia coli clones of picking
Plasmid DNA is extracted after expanding culture, then Plasmid DNA sequencing carries out sequence verification, and sequence is extracted after correctly cloning expansion culture
Plasmid DNA transcribes the template of gRNA as next step；Recycle MEGAscript T7 Transcription Kit
(InvitrogenTM) the external reverse transcription 20ul reaction system of the plasmid containing spo11 target sequence built is as follows:
10x Reaction Buffer 2ul；
Plasmid DNA 4ul (about 1ug)；
T7 Enzyme 2ul；
10mmol/L NTP 1ul；
DEPC water 11ul；
It is purified after 37 DEG C of the above system one hour of reaction spare；
(4) it is transcribed in vitro and obtains Cas9 mRNA:
Cas9 plasmid comes from AddgeneTM(#63154) uses MAXIscript T7/T3 Transcription Kit
(InvitrogenTM), Cas9 mRNA is synthesized, system and reaction condition are the same as step 3；
(5) the micro- co-injection of Cas9 mRNA and gRNA: configuration microinjection system first is as follows:
300 ng/ul of Cas9 mRNA；
GRNA 30ng/ul；
Phenol-red 0.2ul；
DEPC Water up to 2ul；
After above-mentioned solution is prepared mixing, in the zebra fish fertilized egg in microinjection unicellular period, cultivation detects after 24 hours
The mutation efficiency of spo11 gene；
(6) screening of spo11 F1 generation heterozygous mutant individual and mutation type determine:
The zebra fish fertilized egg after step (5) microinjection hybridizes with wild type individual and obtains to after sexal maturity for cultivation 3 months
First familiar generation is obtained, after F1 generation individual cultivates 2 months, clip part tail fin tissue extracts genomic DNA, with F-spo11/R-
Spo11 is primer, F-spo11:5 '-ATGGCTTACCAGTTTACTG-3 '; R-spo11: 5'-CGTAGCTCTTTCAGTAAC
TC-3 ' carries out PCR amplification using KOD PLUS high fidelity enzyme (TAKARA), and PCR product connects pMD18-T carrier (TAKARA)
Transformed competence colibacillus Escherichia coli afterwards pick them separately after 10 Escherichia coli clones expand culture and extract Plasmid DNA, then plasmid
Screening obtains 10 F1 generation idiovariation efficiency and mutation type after DNA sequencing compares；
(7) acquisition of the spo11 F2 for homozygous mutation individual:
After spo11 F1 generation heterozygous mutant individual sexal maturity, each progress of the consistent male and female F1 generation individual of mutation type is chosen
Artificial insemination obtains spo11 F2 for homozygous mutation individual；
(8) selection of male sterility male:
Spo11 F2 is male sterility individual for male all in homozygous mutation group, but has ovum in female individuals
Son, and being capable of normal fertilization with wild milter.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113789352A (en) * | 2021-03-15 | 2021-12-14 | 中国科学院水生生物研究所 | Method for realizing sex control breeding of XX/XY sex genetic determination type fish and application |
| CN113817779A (en) * | 2021-03-15 | 2021-12-21 | 中国科学院水生生物研究所 | Breeding method for obtaining pseudo male fish parent determined by XX/XY sex in large scale and application thereof |
| CN114686524A (en) * | 2022-06-01 | 2022-07-01 | 中山大学 | Method for producing 1-year-old female yellow fin sea bream by using gene editing |
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| CN102392073A (en) * | 2011-11-03 | 2012-03-28 | 温州医学院 | Analysis method for activity of mismatch repair of zebra fish embryo DNA and application thereof |
| CN103957698A (en) * | 2011-09-29 | 2014-07-30 | 瑞克斯旺种苗集团公司 | Quartet breeding |
| CN105647969A (en) * | 2016-02-16 | 2016-06-08 | 湖南师范大学 | Method for breeding stat1a (signal transducer and activator of transcription 1) gene-deleted zebra fish through gene knockout |
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| CN103957698A (en) * | 2011-09-29 | 2014-07-30 | 瑞克斯旺种苗集团公司 | Quartet breeding |
| CN102392073A (en) * | 2011-11-03 | 2012-03-28 | 温州医学院 | Analysis method for activity of mismatch repair of zebra fish embryo DNA and application thereof |
| CN105647969A (en) * | 2016-02-16 | 2016-06-08 | 湖南师范大学 | Method for breeding stat1a (signal transducer and activator of transcription 1) gene-deleted zebra fish through gene knockout |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113789352A (en) * | 2021-03-15 | 2021-12-14 | 中国科学院水生生物研究所 | Method for realizing sex control breeding of XX/XY sex genetic determination type fish and application |
| CN113817779A (en) * | 2021-03-15 | 2021-12-21 | 中国科学院水生生物研究所 | Breeding method for obtaining pseudo male fish parent determined by XX/XY sex in large scale and application thereof |
| CN113789352B (en) * | 2021-03-15 | 2023-03-28 | 中国科学院水生生物研究所 | Method for realizing sex control breeding of XX/XY sex genetic determination type fish and application |
| CN113817779B (en) * | 2021-03-15 | 2023-05-19 | 中国科学院水生生物研究所 | Breeding method for obtaining XX/XY sex determined pseudo-male parent on large scale and application thereof |
| CN114686524A (en) * | 2022-06-01 | 2022-07-01 | 中山大学 | Method for producing 1-year-old female yellow fin sea bream by using gene editing |
| CN114686524B (en) * | 2022-06-01 | 2022-09-30 | 中山大学 | Method for producing 1-year-old female yellow-fin sparus by gene editing |
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