CN112226465A - 一段分离的核苷酸序列在无矿化肌间骨斑马鱼构建中的应用 - Google Patents
一段分离的核苷酸序列在无矿化肌间骨斑马鱼构建中的应用 Download PDFInfo
- Publication number
- CN112226465A CN112226465A CN202011115842.3A CN202011115842A CN112226465A CN 112226465 A CN112226465 A CN 112226465A CN 202011115842 A CN202011115842 A CN 202011115842A CN 112226465 A CN112226465 A CN 112226465A
- Authority
- CN
- China
- Prior art keywords
- zebra fish
- fish
- seq
- intermuscular
- bone
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 241000252212 Danio rerio Species 0.000 title claims abstract description 55
- 210000000988 bone and bone Anatomy 0.000 title claims abstract description 52
- 239000002773 nucleotide Substances 0.000 title claims abstract description 15
- 125000003729 nucleotide group Chemical group 0.000 title claims abstract description 15
- 238000010276 construction Methods 0.000 title claims abstract description 9
- 241000251468 Actinopterygii Species 0.000 claims abstract description 24
- 238000000034 method Methods 0.000 claims abstract description 16
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 12
- 238000012216 screening Methods 0.000 claims abstract description 5
- 238000012258 culturing Methods 0.000 claims abstract description 3
- 108091027544 Subgenomic mRNA Proteins 0.000 claims description 17
- 108091033409 CRISPR Proteins 0.000 claims description 14
- 210000002257 embryonic structure Anatomy 0.000 claims description 9
- 230000035772 mutation Effects 0.000 claims description 9
- 238000010354 CRISPR gene editing Methods 0.000 claims description 7
- 238000000338 in vitro Methods 0.000 claims description 7
- 238000011144 upstream manufacturing Methods 0.000 claims description 6
- 238000003205 genotyping method Methods 0.000 claims description 5
- 238000000746 purification Methods 0.000 claims description 5
- 238000012408 PCR amplification Methods 0.000 claims description 3
- 239000013612 plasmid Substances 0.000 claims description 3
- 230000008569 process Effects 0.000 claims description 3
- 238000009396 hybridization Methods 0.000 claims description 2
- 238000011084 recovery Methods 0.000 claims description 2
- 238000010453 CRISPR/Cas method Methods 0.000 claims 1
- 210000001161 mammalian embryo Anatomy 0.000 abstract description 7
- 238000011160 research Methods 0.000 abstract description 6
- 206010064571 Gene mutation Diseases 0.000 abstract description 5
- 230000015572 biosynthetic process Effects 0.000 abstract description 2
- 108020004414 DNA Proteins 0.000 description 12
- 238000000246 agarose gel electrophoresis Methods 0.000 description 7
- RGCKGOZRHPZPFP-UHFFFAOYSA-N alizarin Chemical compound C1=CC=C2C(=O)C3=C(O)C(O)=CC=C3C(=O)C2=C1 RGCKGOZRHPZPFP-UHFFFAOYSA-N 0.000 description 6
- 241000252210 Cyprinidae Species 0.000 description 5
- 238000006243 chemical reaction Methods 0.000 description 5
- 238000011161 development Methods 0.000 description 5
- 238000002347 injection Methods 0.000 description 5
- 239000007924 injection Substances 0.000 description 5
- 238000010186 staining Methods 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 238000010362 genome editing Methods 0.000 description 4
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 230000003321 amplification Effects 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 238000000520 microinjection Methods 0.000 description 3
- 239000011259 mixed solution Substances 0.000 description 3
- 238000003199 nucleic acid amplification method Methods 0.000 description 3
- 238000012163 sequencing technique Methods 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 125000003275 alpha amino acid group Chemical group 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 238000000137 annealing Methods 0.000 description 2
- 229910021538 borax Inorganic materials 0.000 description 2
- 238000009395 breeding Methods 0.000 description 2
- 230000001488 breeding effect Effects 0.000 description 2
- 238000004925 denaturation Methods 0.000 description 2
- 230000036425 denaturation Effects 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 235000013601 eggs Nutrition 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 231100000221 frame shift mutation induction Toxicity 0.000 description 2
- 230000037433 frameshift Effects 0.000 description 2
- 230000002068 genetic effect Effects 0.000 description 2
- KWGKDLIKAYFUFQ-UHFFFAOYSA-M lithium chloride Chemical compound [Li+].[Cl-] KWGKDLIKAYFUFQ-UHFFFAOYSA-M 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 230000011164 ossification Effects 0.000 description 2
- 238000005192 partition Methods 0.000 description 2
- 238000012257 pre-denaturation Methods 0.000 description 2
- 229920006395 saturated elastomer Polymers 0.000 description 2
- 239000004328 sodium tetraborate Substances 0.000 description 2
- 235000010339 sodium tetraborate Nutrition 0.000 description 2
- 238000013518 transcription Methods 0.000 description 2
- 230000035897 transcription Effects 0.000 description 2
- JKYKXTRKURYNGW-UHFFFAOYSA-N 3,4-dihydroxy-9,10-dioxo-9,10-dihydroanthracene-2-sulfonic acid Chemical compound O=C1C2=CC=CC=C2C(=O)C2=C1C(O)=C(O)C(S(O)(=O)=O)=C2 JKYKXTRKURYNGW-UHFFFAOYSA-N 0.000 description 1
- RBTBFTRPCNLSDE-UHFFFAOYSA-N 3,7-bis(dimethylamino)phenothiazin-5-ium Chemical compound C1=CC(N(C)C)=CC2=[S+]C3=CC(N(C)C)=CC=C3N=C21 RBTBFTRPCNLSDE-UHFFFAOYSA-N 0.000 description 1
- 241001083841 Aquatica Species 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 241000144950 Colossoma Species 0.000 description 1
- 241000252228 Ctenopharyngodon Species 0.000 description 1
- 241000252230 Ctenopharyngodon idella Species 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 241000255601 Drosophila melanogaster Species 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 241000282414 Homo sapiens Species 0.000 description 1
- 241000252232 Hypophthalmichthys Species 0.000 description 1
- 241000720946 Hypophthalmichthys molitrix Species 0.000 description 1
- 241000252234 Hypophthalmichthys nobilis Species 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 241001275890 Megalobrama amblycephala Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000699660 Mus musculus Species 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 241000277334 Oncorhynchus Species 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 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 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 description 1
- 241000269319 Squalius cephalus Species 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 238000004061 bleaching Methods 0.000 description 1
- 244000309464 bull Species 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 210000002808 connective tissue Anatomy 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 230000035558 fertility Effects 0.000 description 1
- 239000013505 freshwater Substances 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000012139 lysis buffer Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 229960000907 methylthioninium chloride Drugs 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 230000003387 muscular Effects 0.000 description 1
- 230000017448 oviposition Effects 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 229960003531 phenolsulfonphthalein Drugs 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 108010008359 protein kinase C lambda Proteins 0.000 description 1
- 239000001044 red dye Substances 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 230000035938 sexual maturation Effects 0.000 description 1
- 229910052709 silver Inorganic materials 0.000 description 1
- 239000004332 silver Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000007447 staining method Methods 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000002054 transplantation Methods 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/89—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microinjection
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/22—Ribonucleases RNAses, DNAses
-
- 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
- A01K67/027—New or modified breeds of vertebrates
- A01K67/0275—Genetically modified vertebrates, e.g. transgenic
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/461—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from fish
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/102—Mutagenizing nucleic acids
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
-
- 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
- A01K2207/00—Modified animals
- A01K2207/05—Animals modified by non-integrating nucleic acids, e.g. antisense, RNAi, morpholino, episomal vector, for non-therapeutic purpose
-
- 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
- A01K2217/00—Genetically modified animals
- A01K2217/07—Animals genetically altered by homologous recombination
- A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
-
- 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
- A01K2217/00—Genetically modified animals
- A01K2217/07—Animals genetically altered by homologous recombination
- A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
- A01K2217/077—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out heterozygous knock out animals displaying phenotype
-
- 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
- A01K2227/00—Animals characterised by species
- A01K2227/40—Fish
-
- 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
- A01K2267/00—Animals characterised by purpose
- A01K2267/02—Animal zootechnically ameliorated
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2800/00—Nucleic acids vectors
- C12N2800/80—Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
- Y02A40/81—Aquaculture, e.g. of fish
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Environmental Sciences (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Veterinary Medicine (AREA)
- Medicinal Chemistry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Animal Husbandry (AREA)
- Animal Behavior & Ethology (AREA)
- Toxicology (AREA)
- Gastroenterology & Hepatology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Crystallography & Structural Chemistry (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明属于分子生物学领域，涉及了一段分离的核苷酸序列在无肌间骨斑马鱼构建中的应用，所述的核苷酸序列为SEQ ID NO.1所示。以SEQ ID NO.1为靶基因进行基因突变，挑选有突变的F0的胚胎培养成亲本鱼，通过F0突变体亲本与野生鱼杂交产生F1胚胎，筛选出有义的突变杂合子F1培养至成鱼，然后F1杂合子自交能产生纯合子，杂合子，野生型的三种基因类型的F2后代。利用基因突变的方法获得无矿化肌间骨斑马鱼品系，为后续研究鱼类肌间骨的分子形成机制和培育无肌间骨经济鱼类提供基础，具有科学研究的基础价值和应用价值。
Description
技术领域
本发明属于分子生物学领域，具体涉及一段分离的核苷酸序列在无矿化肌间骨斑马鱼构建中的应用，通过对该核苷酸序列进行突变，可获得无矿化肌间骨的斑马鱼。
背景技术
斑马鱼(Danio rerio)是一种热带观赏鱼，属于辐鳍亚纲(Actinopterygii)鲤科(Cypri nidae)鱼丹属(Danio)。因其体侧具有像斑马样纵向暗蓝色与银色相间的条纹而得名。斑马鱼具有体型小，易饲养发育快速，性成熟周期短，繁殖力强，雌雄易分辨，胚胎体外发育，易于观察和操作等优点，是一种用于研究脊椎动物胚胎学和发育遗传学的经典模式动物(Br ooks et al.,2014)。斑马鱼的基因组及转录组数据已较完善。斑马鱼产卵量大，成活率高，体外受精，胚胎在母体外发育并且透明。受精卵的直径约1mm，易于进行显微注射和细胞移植等操作。
鱼类肌间骨(intermuscular bone)是由肌膈结缔组织连续同源骨化而来的膜骨，根据其附着位置不同，分为髓弓小骨、脉弓小骨和椎体小骨三种类型。肌间骨的存在与鱼类的分类地位有极大的关系,其仅存在低等真骨鱼类中,系统演化过程表现为数目由少到多，多到少，再到无的过程。我国的大宗淡水鱼类都含有一定数量的肌间骨，如青鱼(Mylopharyngodonpi ceus)、草鱼(Ctenopharyngodonidellus)、鲢(Hypophthalmichthysmolitrix)、鳙(H.nobilis)、团头鲂(Megalobramaamblycephala)等鲤科鱼类，对鲜鱼的食用和鱼糜制品的加工带来极大不便，这对我国鲤科鱼类养殖业的发展和产值产生了直接的影响。研究表明斑马鱼肌间骨的骨化顺序与类型与鲤科鱼类相似(Nie et al.,2018)；巴西无肌间骨的大盖巨脂鲤(Colossoma macropomum)个体的发现证明肌间骨对鱼类本身的生命活动无显著影响(Perazza et al.,2017)；研究表明由雌核发育筛选获得的无肌间骨的草鱼的生长发育正常(徐晓峰等，2015)。因此，在一定程度上，去除或减少肌间骨的数量是可行的。已有研究也对肌间骨发生发育及数目相关的分子遗传信息进行了发掘(Wan etal.,2016；Nieet al.,2017；Wan et al.,2017；Wan et al.,2019)，提供了肌间骨研究的遗传数据基础，但之前未筛选出与肌间骨数目直接相关的基因信息。
Clustered Regularly Interspaced Short Palindromic Repeats(CRISPR)/CRISPR-associated(C as9)是2013年初出现的新一代基因编辑技术。它主要是基于细菌的一种获得性免疫系统人工改造而成，具有制作简单、使用方便、成本低、作用效率高等特点(Mussolino&Cathomen,2013)。CRISPR/Cas9技术自发现至今，已被证实能在不同物种广泛使用，包括哺乳动物、微生物和植物，如人(Homo sapiens)(Liang et al.,2015)、小鼠(Musmusculus)(Coppola et al.,2015)、果蝇(Drosophila melanogaster)(Port&Bullock,2016)、斑马鱼(Xie etal.,2016)等。CRISP R/Cas9基因编辑技术为筛选鱼类肌间骨发生发育关键调控基因以及培育无肌间骨的鱼类新品系提供了高效的途径。
发明内容
本发明的目的在于提供一段分离的核苷酸序列在无矿化肌间骨斑马鱼构建中的应用，所述的核苷酸序列为SEQ ID NO.1所示。以SEQ ID NO.1序列为靶序列，进行基因突变，可获得无矿化肌间骨的斑马鱼，方法易行，操作简单。
本发明的另一个目的在于提供了一种无矿化肌间骨的斑马鱼品系的包含的突变基因序列，所述的基因序列为SEQ ID NO.2所示。
为了达到上述目的，本发明采用以下技术措施：
一段分离的核苷酸序列在无矿化肌间骨斑马鱼构建中的应用，所述的核苷酸序列为SEQ ID NO.1所示。
以上所述的应用中，优选的，所述的核苷酸序列为SEQ ID NO.3所示。
以上所述的应用中，是将SEQ ID NO.1所示序列进行突变，突变后的序列为SEQ IDNO.2所示。
以上所述的应用中，是将SEQ ID NO.1所示序列突变后，筛选出有突变的F0突变体，将该突变体与野生斑马鱼杂交产生F1代，培养至成鱼，筛选出F1代有突变体的杂合子，自交产生F2代，F2代中的纯合子即为无矿化肌间骨斑马鱼。
以上所述的应用中，优选的，是采取CRISPR/Cas9的方式，以SEQ ID NO.1所示序列为靶位点进行基因编辑。
以上所述的应用中，优选的，在无矿化肌间骨品系中，包含SEQ ID NO.2所示的基因序列。
以上所述的应用中，优选的，在进行CRISPR/Cas9编辑时，包括下述步骤：sgRNA以保守的下游引物Scaffold(GATCCGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC)与带有T7启动子的特异性包含靶点序列的上游引物(AA TTAATACGACTCACTATAGGGGAACATCGGTGAGTCTGGTTTTAGAGCTAGAAATAGC)进行overlap PCR扩增，纯化回收sgRNA；线性化pT3TS-nCas9n质粒，纯化回收后体外转录zCas9mRNA；sgRNA和zCas9mRNA注射到1细胞期的斑马鱼胚胎，筛选突变的F0，对F0与野生型杂交产生的F1进行基因分型，筛选出突变基因型相同的F1并自交，对产生的F2基因分型后获得的纯合子即为无矿化肌间骨斑马鱼品系。
本发明的保护内容还包括：通过在其它有肌间骨鱼类中，筛选与本发明SEQ IDNO.1同源的片段，通过突变该同源片段，也能获得对应的无矿化肌间骨的鱼类。
与现有技术相比，本发明具有以下优点：
本发明的方法可用于获得无矿化肌间骨斑马鱼品系。该方法简单易行，操作简单。利用基因突变的方法获得无矿化肌间骨的斑马鱼品系，具有研究鱼类肌间骨功能的科学研究的基础价值和应用价值。该方法可以成为一种获得无矿化肌间骨的斑马鱼品系。
附图说明
图1斑马鱼靶基因序列和突变后的序列示意图。
图2野生型斑马鱼的茜素红整体骨骼染色示意图。
图3野生型斑马鱼尾部和背部茜素红骨骼染色示意图；其中箭头所示为矿化的肌间骨。
图4纯合子成鱼斑马鱼的茜素红整体骨骼染色示意图。
图5纯合子斑马鱼尾部和背部茜素红骨骼染色示意图。
具体实施方式
本发明所述技术方案，如未特别说明，均为本领域的常规方式；所用试剂或材料，如未特别说明，均来源于商业渠道。本发明实施例以CRISPR/Cas9的方法对靶序列进行突变来制备无矿化肌间骨斑马鱼品系，本领域的其他基因编辑方式，只要是针对SEQ ID NO.1做的突变，本发明所述的突变包括错义突变、移码突变等，只要突变后的序列编辑的氨基酸序列与SEQ ID NO.1编辑的氨基酸序列不同，都能成功制备无肌间骨斑马鱼品系。
实施例1：
一段分离的核苷酸序列在无矿化肌间骨斑马鱼构建中的应用：
1.1实验材料
野生型荧光斑马鱼(eGFP:sp7)(Liang et al.,2019)饲养于华中农业大学水产学院鱼房，28℃水温，14:10光照周期条件下。斑马鱼显微注射所用的胚胎由雌雄斑马鱼自然产卵获得。
1.2实验方法
1.2.1确定sgRNA靶位点
确定sgRNA靶位点为：5‘AGCTCAGGAATGCCTCAG’3(SEQ ID NO.3)。
1.2.2体外合成sgRNA
sgRNA以保守的下游引物Scaffold(GATCCGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC)与带有T7启动子的特异性包含靶点序列的上游引物(AATTAATACGACTCACTATAGGAGCTCAGGAATGCCTCAGGTTTTAGAGCTAGAAATAGC)进行overlap PCR扩增。PCR体系为：Primer STAR Max DNA Polymerase 10.5μL,Scaffold 5μL,sgRNA 5μL,ddH2O 4.5μL。PCR反应条件98℃预变性30s，98℃变性10s，60℃退火10s，72℃延伸15s，45个循环，72℃再延伸5min。取5μL PCR产物进行2％琼脂糖凝胶电泳，条带大小验证正确后纯化回收PCR产物，并用Nanodrop 2000测浓度(Thermo Scientific，美国)。根据(TranscriptAID T7 High Yield Tr anscription Kit(Thermo scientific，美国)试剂盒转录RNA，然后使用RNA纯化试剂盒(ZYM O,美国)纯化回收sgRNA。取出1μL测RNA浓度，并通过2％琼脂糖凝胶电泳检测RNA的质量后装置于-80℃保存待用，即为sgRNA。
1.2.3体外转录zCas9mRNA
使用XbaI(NEB，美国)限制性内切酶线性化pT3TS-nCas9n质粒，经过1％琼脂糖凝胶电泳确认线性化完全后用Gel Extraction Kit(Omega，美国)液体纯化回收。根据mMESSAG E mMACHINE T3 Transcription Kit(Invitrogen，美国)说明书体外转录zCas9mRNA，使用氯化锂沉淀法纯化回收zCas9mRNA，加入无酶水溶解并用Nanodrop 2000(Thermo Scientific，美国)测浓度，通过2％琼脂糖凝胶电泳检测转录的mRNA质量后分装置于-80℃保存待用。
1.2.4显微注射
注射前晚上将雌雄斑马鱼按2:3比例配对并用隔板隔开，次日注射前30min抽出隔板使其自然产卵，20min后收集胚胎并按顺序排列在胚胎模具中。使用sgRNA和zCas9mRNA配制体积为5μL注射样品，其中sgRNA的终浓度为80ng/μL，zCas9mRNA的终浓度为500ng/μL，并加入终浓度为0.2％的酚红做指示剂。利用PicoliterMicroinjector注射仪(Warner，美国)将实验样品注射到1细胞期的斑马鱼胚胎中。注射完成后用亚甲基蓝培养液培养斑马鱼胚胎，并置于28℃光照恒温培养箱中。
1.2.5检测靶点突变率
选取30颗注射后48h的胚胎，用裂解法快速提取斑马鱼胚胎基因组DNA。将待裂解的胚胎置于96孔200μL的孔板中，加入50μL Lysis buffer(10mmol/L Tris+50mmol/LKCl+1.5mmol/L MgCl2+0.3％Tween-20+0.3％Nonident P-40)94℃PCR反应20min，55℃结束反应；置于冰上加入5μL Pk酶(10mg/ml)涡旋混匀,55℃PCR反应60min，94℃PCR反应20min，16℃结束反应。用靶点扩增检测引物(check F：5’TGTATCTTGTTCTCTCCACAGG；check R：5’TGTACTGACCTCTTCCGCTTC)扩增出靶点附近187bp序列。PCR反应体系为：
Master Mix(翊圣，上海)10μL，上下游引物各0.5μL，基因组DNA模板2μL，无菌水7μL。PCR反应条件为：94℃预变性5min，94℃变性30s，54℃退火30s，72℃延伸30s，35个循环，72℃再延伸5min。取10μL PCR产物进行3％琼脂糖凝胶电泳，检测后在PCR产物附近有双条带的杂合个体F0挑选出来培养至成鱼。30颗胚胎的突变率是30％。
1.2.6获得杂合子F1
用F0与正常野生型繁殖F1代个体，按照上述提取F1代胚胎DNA，利用靶点检测引物进行PCR产物扩增，并3％琼脂糖凝胶电泳检测筛选出有突变杂合子F1。运用靶点扩增测序引物(seq F：5’GACCAAACCCCCTCTAAA；seq R：5’CGAGTACTTGATGAACGCT)扩增出靶点附近417bp的序列，取2μLPCR产物进行1％的琼脂糖凝胶电泳，检测单一条带目的片段大小正确的产物送到公司测序(擎科生物技术有限公司，武汉)。分析每个个体的序列，与野生正常的基因序列相比较，分析突变靶序列。
1.2.7获得纯合子F2
将获得的同种突变类型的F1个体培养至成鱼，杂合子F1自交产生有纯合子，杂合子，野生型三种基因类型的F2代。基因分型确定F2代纯合子的基因突变序列，即获得无矿化肌间骨存在的突变体斑马鱼品系。
1.2.8无矿化肌间骨斑马鱼品系基因型与表型分析
野生型斑马鱼与无矿化肌间骨突变体斑马鱼的靶位点基因序列比较结果如图1所示。SEQ ID NO.1为野生型斑马鱼靶位点序列，SEQ ID NO.2为无矿化肌间骨突变体斑马鱼靶位点序列。
通过茜素红整体骨骼染色法(成鱼在4％多聚甲醛中固定48h后，在ddH2O水中漂洗30min；随后在3％H2O2与1％KOH等体积混合溶液中漂白4h；在ddH2O中漂洗30min；在饱和硼砂溶液中处理12h；ddH2O漂洗30min；在1％茜素红S(Sigma)与1％KOH混合溶液中染色24h；在ddH2O中漂洗1h(直至洗净茜素红染液)；在1％胰蛋白酶(Solarbio)和2％饱和硼砂混合溶液中漂洗24-48h，去除杂质；在50％，100％甘油中梯度透明，储存)对斑马鱼肌间骨表型进行观察。
野生型斑马鱼整体肌间骨表型如图2所示，其背部与尾部分布有数目显著的肌间骨，单侧肌间骨总数目约为35-40根(图2、图3)；纯合子突变体斑马鱼整体肌间骨表型如图4所示，其矿化的肌间骨已全部消失，只有印迹存在(图4、图5)。测序分析表明该稳定遗传的无肌间骨品系来自于一种类型的突变体，与野生型SEQ ID NO.1相比，靶序列附近的序列突变为SEQ ID NO.2所示序列，导致移码突变。
序列表
<110> 华中农业大学
<120> 一段分离的核苷酸序列在无矿化肌间骨斑马鱼构建中的应用
<160> 9
<170> SIPOSequenceListing 1.0
<210> 1
<211> 47
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
tcagcggagc tcaggaatgc ctcaggggtt atgaagaacc aggtggc 47
<210> 2
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
tcaggggtta tgaagaacca ggtggc 26
<210> 3
<211> 18
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
agctcaggaa tgcctcag 18
<210> 4
<211> 81
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
gatccgcacc gactcggtgc cactttttca agttgataac ggactagcct tattttaact 60
tgctatttct agctctaaaa c 81
<210> 5
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
aattaatacg actcactata ggagctcagg aatgcctcag gttttagagc tagaaatagc 60
<210> 6
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
tgtatcttgt tctctccaca gg 22
<210> 7
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
tgtactgacc tcttccgctt c 21
<210> 8
<211> 18
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
gaccaaaccc cctctaaa 18
<210> 9
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
cgagtacttg atgaacgct 19
Claims (7)
1.一段分离的核苷酸序列在无矿化肌间骨斑马鱼构建中的应用，所述的核苷酸序列为SEQ ID NO.1。
2.根据权利要求1所述的应用，所述的应用的过程包括将SEQ ID NO.1所示序列进行突变，突变后的序列为SEQ ID NO.2所示。
3.根据权利要求2所述的应用，所述的应用的过程是将SEQ ID NO.1所示序列突变后，筛选出有突变的F0突变体，将该突变体与野生斑马鱼杂交产生F1代，培养至成鱼，筛选出F1代有突变体的杂合子，F1代杂合子自交产生F2代，F2代中的纯合子即为无矿化肌间骨斑马鱼。
4.根据权利要求3所述的应用，所述突变是采取CRISPR/Cas9的方式。
5.根据权利要求1所述的应用，是制备的无矿化肌间骨斑马鱼中，包含SEQ ID NO.2所示的基因序列。
6.根据权利要求4所述的应用，在进行CRISPR/Cas9编辑时，包括下述步骤：sgRNA以保守的下游引物Scaffold：gatccgcaccgactcggtgccactttttcaagttgataacggactagccttattttaacttgctatttctagctctaaaac)与带有T7启动子的特异性包含靶点序列的上游引物：AATTAATACGACTCACTATAGGGGAACATCGGTGAGTCTGGTTTTAGAGCTAGAAATAGC)进行overlap pcr扩增，纯化回收sgRNA；线性化pT3TS-nCas9n质粒，纯化回收后体外转录zCas9mRNA；sgRNA和zCas9mRNA注射到1细胞期的斑马鱼胚胎，筛选突变的F0，对F0与野生型杂交产生的F1进行基因分型，筛选出突变基因型相同的F1并自交，对产生的F2基因分型后获得的纯合子即为无矿化肌间骨斑马鱼品系。
7.根据权利要求1所述的应用，所述的核苷酸序列为SEQ ID NO.3所示。
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011115842.3A CN112226465B (zh) | 2020-10-19 | 2020-10-19 | 一段分离的核苷酸序列在无矿化肌间骨斑马鱼构建中的应用 |
| US17/554,881 US11685931B2 (en) | 2020-10-19 | 2021-12-17 | Application of a fragment of an isolated nucleotide sequence in construction of non-mineralized intermuscular bone of Danio rerio |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011115842.3A CN112226465B (zh) | 2020-10-19 | 2020-10-19 | 一段分离的核苷酸序列在无矿化肌间骨斑马鱼构建中的应用 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112226465A true CN112226465A (zh) | 2021-01-15 |
| CN112226465B CN112226465B (zh) | 2021-09-24 |
Family
ID=74118895
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011115842.3A Active CN112226465B (zh) | 2020-10-19 | 2020-10-19 | 一段分离的核苷酸序列在无矿化肌间骨斑马鱼构建中的应用 |
Country Status (2)
| Country | Link |
|---|---|
| US (1) | US11685931B2 (zh) |
| CN (1) | CN112226465B (zh) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110684777A (zh) * | 2019-11-13 | 2020-01-14 | 华中农业大学 | 一段分离的核苷酸序列在肌间刺减少的斑马鱼构建中的应用 |
| CN115943930A (zh) * | 2022-12-30 | 2023-04-11 | 中国科学院水生生物研究所 | 一种无肌间刺银鲫的创制方法 |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106978392A (zh) * | 2017-05-19 | 2017-07-25 | 华中农业大学 | 一种团头鲂骨组织细胞培养的方法 |
| CN110684777A (zh) * | 2019-11-13 | 2020-01-14 | 华中农业大学 | 一段分离的核苷酸序列在肌间刺减少的斑马鱼构建中的应用 |
-
2020
- 2020-10-19 CN CN202011115842.3A patent/CN112226465B/zh active Active
-
2021
- 2021-12-17 US US17/554,881 patent/US11685931B2/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106978392A (zh) * | 2017-05-19 | 2017-07-25 | 华中农业大学 | 一种团头鲂骨组织细胞培养的方法 |
| CN110684777A (zh) * | 2019-11-13 | 2020-01-14 | 华中农业大学 | 一段分离的核苷酸序列在肌间刺减少的斑马鱼构建中的应用 |
Non-Patent Citations (3)
| Title |
|---|
| CHUNHONG NIE等: "Loss of scleraxis leads to distinct reduction of mineralized intermuscular bone in zebrafish", 《AQUACULTURE AND FISHERIES》 * |
| NCBI: "PREDICTED: Danio rerio runt-related transcription factor 2b (runx2b), transcript variant X1, mRNA", 《GENBANK：XM_021468385.1》 * |
| T.VAN DER MEULEN等: "Identification and characterisation of two runx2 homologues in zebrafish with different expression patterns", 《BIOCHIMICA ET BIOPHYSICA ACTA (BBA) - GENE STRUCTURE AND EXPRESSION》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110684777A (zh) * | 2019-11-13 | 2020-01-14 | 华中农业大学 | 一段分离的核苷酸序列在肌间刺减少的斑马鱼构建中的应用 |
| CN110684777B (zh) * | 2019-11-13 | 2021-05-07 | 华中农业大学 | 一段分离的核苷酸序列在肌间刺减少的斑马鱼构建中的应用 |
| CN115943930A (zh) * | 2022-12-30 | 2023-04-11 | 中国科学院水生生物研究所 | 一种无肌间刺银鲫的创制方法 |
| CN115943930B (zh) * | 2022-12-30 | 2024-03-19 | 中国科学院水生生物研究所 | 一种无肌间刺银鲫的创制方法 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN112226465B (zh) | 2021-09-24 |
| US20220275394A1 (en) | 2022-09-01 |
| US11685931B2 (en) | 2023-06-27 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105647969B (zh) | 一种基因敲除选育stat1a基因缺失型斑马鱼的方法 | |
| CN110684777B (zh) | 一段分离的核苷酸序列在肌间刺减少的斑马鱼构建中的应用 | |
| CN106282231B (zh) | 粘多糖贮积症ii型动物模型的构建方法及应用 | |
| CN112226465B (zh) | 一段分离的核苷酸序列在无矿化肌间骨斑马鱼构建中的应用 | |
| CN110129455B (zh) | 一种生长相关的分子标记在凡纳滨对虾遗传育种中的应用 | |
| CN111387105B (zh) | 一种全雄罗氏沼虾制种的方法 | |
| WO2012083681A1 (zh) | 半滑舌鳎性别特异微卫星标记及其在超雌鱼鉴定中的应用 | |
| US20240008461A1 (en) | Crucian carp (carassius auratus) strain without intermuscular bones and breeding method thereof | |
| CN111154758A (zh) | 敲除斑马鱼slc26a4基因的方法 | |
| CN112048014B (zh) | 一种斑节对虾Pm GLUT2基因及其应用 | |
| CN111394445A (zh) | 用于斑鳢性别鉴定的Indel标记及应用 | |
| CN111549031A (zh) | 一种草鱼和青鱼肌间刺变粗的分子育种方法 | |
| Sasado et al. | The National BioResource Project Medaka (NBRP Medaka): an integrated bioresource for biological and biomedical sciences | |
| CN110894510A (zh) | 一种基因敲除选育Lgr6基因缺失型斑马鱼的方法 | |
| US20030162292A1 (en) | Method for producing heart-specific fluorescence of non-human eukaryotic animals | |
| Takasu et al. | Targeted mutagenesis in Bombyx mori using TALENs | |
| CN112779258A (zh) | 一种大黄鱼CRISPR/Cas9基因编辑方法 | |
| CN115720874A (zh) | 养殖经济鱼类无肌间刺种质创制方法及应用 | |
| Yan et al. | Disruption of mstn Gene by CRISPR/Cas9 in large yellow croaker (Larimichthys crocea) | |
| CN108251456B (zh) | 一种nod遗传背景的动脉粥样硬化小鼠模型的制备方法 | |
| CN114480497B (zh) | 一种ep400基因敲除斑马鱼心力衰竭模型的构建及其应用的方法 | |
| CN113846172B (zh) | 凡纳滨对虾耐亚硝酸盐性状相关的snp分子标记及其应用 | |
| CN114058618B (zh) | 谷氨酸脱氢酶作为靶点在防治害虫中的应用 | |
| CN113249442B (zh) | 一种筛查牡蛎不饱和脂肪酸含量相关甲基化修饰基因的方法 | |
| CN115029352A (zh) | 一种基因敲除选育adgrg1基因缺失型斑马鱼的方法 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |