CN106636197A - Method for directional knockdown of multi-copy gene in zebrafish genome - Google Patents
Method for directional knockdown of multi-copy gene in zebrafish genome Download PDFInfo
- Publication number
- CN106636197A CN106636197A CN201610843834.8A CN201610843834A CN106636197A CN 106636197 A CN106636197 A CN 106636197A CN 201610843834 A CN201610843834 A CN 201610843834A CN 106636197 A CN106636197 A CN 106636197A
- Authority
- CN
- China
- Prior art keywords
- gene
- eve
- dcas9
- embryo
- sgrna
- 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
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 53
- 241000252212 Danio rerio Species 0.000 title claims abstract description 47
- 238000000034 method Methods 0.000 title claims abstract description 38
- 108091027544 Subgenomic mRNA Proteins 0.000 claims abstract description 38
- 210000001161 mammalian embryo Anatomy 0.000 claims abstract description 38
- 238000013518 transcription Methods 0.000 claims abstract description 28
- 230000035897 transcription Effects 0.000 claims abstract description 28
- 230000014509 gene expression Effects 0.000 claims abstract description 20
- 238000000338 in vitro Methods 0.000 claims abstract description 19
- 108020004999 messenger RNA Proteins 0.000 claims abstract description 19
- 230000000694 effects Effects 0.000 claims abstract description 13
- 108091033409 CRISPR Proteins 0.000 claims abstract description 12
- 238000007901 in situ hybridization Methods 0.000 claims abstract description 11
- 102000037865 fusion proteins Human genes 0.000 claims abstract description 6
- 108020001507 fusion proteins Proteins 0.000 claims abstract description 6
- -1 dCas9-Eve Proteins 0.000 claims abstract description 5
- 239000013604 expression vector Substances 0.000 claims abstract description 5
- 238000011530 RNeasy Mini Kit Methods 0.000 claims abstract description 4
- 239000007788 liquid Substances 0.000 claims description 27
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 24
- 108020004414 DNA Proteins 0.000 claims description 19
- 239000000243 solution Substances 0.000 claims description 13
- WVDDGKGOMKODPV-UHFFFAOYSA-N Benzyl alcohol Chemical compound OCC1=CC=CC=C1 WVDDGKGOMKODPV-UHFFFAOYSA-N 0.000 claims description 12
- ZHNUHDYFZUAESO-UHFFFAOYSA-N Formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 claims description 10
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 10
- 230000013011 mating Effects 0.000 claims description 10
- 229920000136 polysorbate Polymers 0.000 claims description 10
- 108091032973 (ribonucleotides)n+m Proteins 0.000 claims description 9
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 claims description 9
- 239000000706 filtrate Substances 0.000 claims description 9
- 238000000605 extraction Methods 0.000 claims description 8
- 230000013020 embryo development Effects 0.000 claims description 7
- 210000002257 embryonic structure Anatomy 0.000 claims description 7
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims description 6
- 101710163270 Nuclease Proteins 0.000 claims description 6
- 230000018044 dehydration Effects 0.000 claims description 6
- 238000006297 dehydration reaction Methods 0.000 claims description 6
- 238000005286 illumination Methods 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 6
- 238000000246 agarose gel electrophoresis Methods 0.000 claims description 5
- 229910052751 metal Inorganic materials 0.000 claims description 5
- 239000002184 metal Substances 0.000 claims description 5
- 238000002360 preparation method Methods 0.000 claims description 5
- 210000000998 shell membrane Anatomy 0.000 claims description 5
- 239000011780 sodium chloride Substances 0.000 claims description 5
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 5
- 239000012498 ultrapure water Substances 0.000 claims description 5
- 235000019445 benzyl alcohol Nutrition 0.000 claims description 4
- 229960004217 benzyl alcohol Drugs 0.000 claims description 4
- 239000002299 complementary DNA Substances 0.000 claims description 4
- 238000001514 detection method Methods 0.000 claims description 4
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 3
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 claims description 3
- 238000012408 PCR amplification Methods 0.000 claims description 3
- 241001494479 Pecora Species 0.000 claims description 3
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 3
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 3
- 229920002334 Spandex Polymers 0.000 claims description 3
- 230000000903 blocking effect Effects 0.000 claims description 3
- 238000005119 centrifugation Methods 0.000 claims description 3
- 238000010790 dilution Methods 0.000 claims description 3
- 239000012895 dilution Substances 0.000 claims description 3
- 239000012634 fragment Substances 0.000 claims description 3
- 229960002897 heparin Drugs 0.000 claims description 3
- 229920000669 heparin Polymers 0.000 claims description 3
- 238000009396 hybridization Methods 0.000 claims description 3
- 229910001629 magnesium chloride Inorganic materials 0.000 claims description 3
- 238000000520 microinjection Methods 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 3
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims description 3
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 3
- 239000000523 sample Substances 0.000 claims description 3
- 210000002966 serum Anatomy 0.000 claims description 3
- 239000004759 spandex Substances 0.000 claims description 3
- IOVCWXUNBOPUCH-UHFFFAOYSA-M Nitrite anion Chemical compound [O-]N=O IOVCWXUNBOPUCH-UHFFFAOYSA-M 0.000 claims description 2
- 229930040373 Paraformaldehyde Natural products 0.000 claims description 2
- 235000013399 edible fruits Nutrition 0.000 claims description 2
- 229940005654 nitrite ion Drugs 0.000 claims description 2
- 229920002866 paraformaldehyde Polymers 0.000 claims description 2
- 239000003643 water by type Substances 0.000 claims description 2
- 238000004140 cleaning Methods 0.000 claims 2
- 238000002798 spectrophotometry method Methods 0.000 claims 1
- 231100000419 toxicity Toxicity 0.000 abstract description 3
- 230000001988 toxicity Effects 0.000 abstract description 3
- 230000002401 inhibitory effect Effects 0.000 abstract description 2
- 238000003197 gene knockdown Methods 0.000 abstract 3
- 210000004027 cell Anatomy 0.000 description 6
- 230000003321 amplification Effects 0.000 description 5
- 238000005516 engineering process Methods 0.000 description 5
- 235000019441 ethanol Nutrition 0.000 description 5
- 238000003199 nucleic acid amplification method Methods 0.000 description 5
- 230000015572 biosynthetic process Effects 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 239000013612 plasmid Substances 0.000 description 4
- 238000003786 synthesis reaction Methods 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 238000010354 CRISPR gene editing Methods 0.000 description 3
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 3
- 210000001647 gastrula Anatomy 0.000 description 3
- 238000012163 sequencing technique Methods 0.000 description 3
- 102100035102 E3 ubiquitin-protein ligase MYCBP2 Human genes 0.000 description 2
- 108020005004 Guide RNA Proteins 0.000 description 2
- 101000911390 Homo sapiens Coagulation factor VIII Proteins 0.000 description 2
- YNAVUWVOSKDBBP-UHFFFAOYSA-N Morpholine Chemical compound C1COCCN1 YNAVUWVOSKDBBP-UHFFFAOYSA-N 0.000 description 2
- 239000000969 carrier Substances 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 102000057593 human F8 Human genes 0.000 description 2
- KWGKDLIKAYFUFQ-UHFFFAOYSA-M lithium chloride Chemical compound [Li+].[Cl-] KWGKDLIKAYFUFQ-UHFFFAOYSA-M 0.000 description 2
- 238000005192 partition Methods 0.000 description 2
- 229940047431 recombinate Drugs 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 230000008685 targeting Effects 0.000 description 2
- QRXMUCSWCMTJGU-UHFFFAOYSA-N 5-bromo-4-chloro-3-indolyl phosphate Chemical compound C1=C(Br)C(Cl)=C2C(OP(O)(=O)O)=CNC2=C1 QRXMUCSWCMTJGU-UHFFFAOYSA-N 0.000 description 1
- 241000251468 Actinopterygii Species 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 description 1
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 description 1
- 108010053770 Deoxyribonucleases Proteins 0.000 description 1
- 102000016911 Deoxyribonucleases Human genes 0.000 description 1
- SHIBSTMRCDJXLN-UHFFFAOYSA-N Digoxigenin Natural products C1CC(C2C(C3(C)CCC(O)CC3CC2)CC2O)(O)C2(C)C1C1=CC(=O)OC1 SHIBSTMRCDJXLN-UHFFFAOYSA-N 0.000 description 1
- LTMHDMANZUZIPE-AMTYYWEZSA-N Digoxin Natural products O([C@H]1[C@H](C)O[C@H](O[C@@H]2C[C@@H]3[C@@](C)([C@@H]4[C@H]([C@]5(O)[C@](C)([C@H](O)C4)[C@H](C4=CC(=O)OC4)CC5)CC3)CC2)C[C@@H]1O)[C@H]1O[C@H](C)[C@@H](O[C@H]2O[C@@H](C)[C@H](O)[C@@H](O)C2)[C@@H](O)C1 LTMHDMANZUZIPE-AMTYYWEZSA-N 0.000 description 1
- 108010042407 Endonucleases Proteins 0.000 description 1
- 102000004533 Endonucleases Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 241000283070 Equus zebra Species 0.000 description 1
- 102000001706 Immunoglobulin Fab Fragments Human genes 0.000 description 1
- 108010054477 Immunoglobulin Fab Fragments Proteins 0.000 description 1
- 241000244206 Nematoda Species 0.000 description 1
- 108091034057 RNA (poly(A)) Proteins 0.000 description 1
- 238000012228 RNA interference-mediated gene silencing Methods 0.000 description 1
- 229920002684 Sepharose Polymers 0.000 description 1
- 108091023040 Transcription factor Proteins 0.000 description 1
- 102000040945 Transcription factor Human genes 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 1
- 230000004721 adaptive immunity Effects 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- QONQRTHLHBTMGP-UHFFFAOYSA-N digitoxigenin Natural products CC12CCC(C3(CCC(O)CC3CC3)C)C3C11OC1CC2C1=CC(=O)OC1 QONQRTHLHBTMGP-UHFFFAOYSA-N 0.000 description 1
- SHIBSTMRCDJXLN-KCZCNTNESA-N digoxigenin Chemical compound C1([C@@H]2[C@@]3([C@@](CC2)(O)[C@H]2[C@@H]([C@@]4(C)CC[C@H](O)C[C@H]4CC2)C[C@H]3O)C)=CC(=O)OC1 SHIBSTMRCDJXLN-KCZCNTNESA-N 0.000 description 1
- LTMHDMANZUZIPE-PUGKRICDSA-N digoxin Chemical compound C1[C@H](O)[C@H](O)[C@@H](C)O[C@H]1O[C@@H]1[C@@H](C)O[C@@H](O[C@@H]2[C@H](O[C@@H](O[C@@H]3C[C@@H]4[C@]([C@@H]5[C@H]([C@]6(CC[C@@H]([C@@]6(C)[C@H](O)C5)C=5COC(=O)C=5)O)CC4)(C)CC3)C[C@@H]2O)C)C[C@@H]1O LTMHDMANZUZIPE-PUGKRICDSA-N 0.000 description 1
- 229960005156 digoxin Drugs 0.000 description 1
- LTMHDMANZUZIPE-UHFFFAOYSA-N digoxine Natural products C1C(O)C(O)C(C)OC1OC1C(C)OC(OC2C(OC(OC3CC4C(C5C(C6(CCC(C6(C)C(O)C5)C=5COC(=O)C=5)O)CC4)(C)CC3)CC2O)C)CC1O LTMHDMANZUZIPE-UHFFFAOYSA-N 0.000 description 1
- 229910000397 disodium phosphate Inorganic materials 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 125000002485 formyl group Chemical group [H]C(*)=O 0.000 description 1
- 230000030279 gene silencing Effects 0.000 description 1
- 230000009368 gene silencing by RNA Effects 0.000 description 1
- 238000012226 gene silencing method Methods 0.000 description 1
- 238000010362 genome editing Methods 0.000 description 1
- 238000003205 genotyping method Methods 0.000 description 1
- 230000005484 gravity Effects 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- 239000010977 jade Substances 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 150000002689 maleic acids Chemical class 0.000 description 1
- 125000004573 morpholin-4-yl group Chemical group N1(CCOCC1)* 0.000 description 1
- 238000002703 mutagenesis Methods 0.000 description 1
- 231100000350 mutagenesis Toxicity 0.000 description 1
- 230000030648 nucleus localization Effects 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 1
- 229910000162 sodium phosphate Inorganic materials 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 230000014621 translational initiation Effects 0.000 description 1
- 238000000870 ultraviolet spectroscopy Methods 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 229910052725 zinc Inorganic materials 0.000 description 1
- 239000011701 zinc Substances 0.000 description 1
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
- 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
- C12N2800/00—Nucleic acids vectors
- C12N2800/80—Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Wood Science & Technology (AREA)
- Molecular Biology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Gastroenterology & Hepatology (AREA)
- Physics & Mathematics (AREA)
- Toxicology (AREA)
- Veterinary Medicine (AREA)
- Plant Pathology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a method for directional knockdown of a multi-copy gene in zebrafish genome. The method comprises the following steps: I, preparing a zebrafish embryo; II, constructing an expression vector of dCas9-Eve fusion protein; III, conducting in-vitro transcription of sgRNA, dCas9-Eve, Cas9 and gene, and purifying the sgRNA, dCas9-Eve, Cas9mRNA and gene mRNA, which are synthesized through the in-vitro transcription, by virtue of RNeasy Mini Kit; IV, identifying activity of the sgRNA; and V, knocking out expression of the multi-copy gene znfl1s by virtue of a dCas9-Eve method, and judging the situation that the gene expression is knocked out by detecting the expression of the gene by virtue of an embryo whole-mount in situ hybridization method. The method provided by the invention, by virtue of the dCas9-Eve, can specifically knock down the transcription of the gene under the circumstance of implementing the effective targeted identification of sgRNA existence of a gene promoter, so that the researched gene is prevented from being affected by MO toxicity or non-specific phenotype caused by target missing; and moreover, since an Eve inhibiting transcription structural domain is derived from zebrafish, the dCas9-Eve is applicable to knockdown of any zebrafish genes at a transcription level.
Description
Technical field
The present invention relates to biological technical field, more particularly to a kind of orientation strikes multi-copy gene in drop zebra fish genome
Method.
Background technology
When function of the gene in early embryo development is studied, we are generally in certain region design morpholine of gene
The expression of gene drops to strike in (morpholino, MO), but because multi-copy gene has the inconsistency of montage, it is impossible to design pin
Verify the function of the gene to the MO of same splice site, and MO toxicity or miss the target and often result in nonspecific phenotype.
CRISPR/Cas9 is a kind of adaptive immunity defence that bacterium and archeobacteria are formed during long-term evolution, can be used to resist
The virus of invasion and foreign DNA, it has also become a kind of base suitable for realizing directed mutagenesis cultured cells and whole organism
Because of a group edit tool.In recent years, researcher to have gone out some based on tradition CRISPR/Cas9 gene editing system constructings new
Gene regulation instrument：CRISPR for down-regulated gene disturbs (CRISPRi) technology.
CRISPRi to be referred to and be oriented to RNA coexpressions with wall scroll using the Cas9 (dCas9) of absence of endonuclease activity, comes
Produce a kind of DNA recognition complexes, using this compound specificity interference transcription extend, RNA polymerase combine, or transcription because
Son is combined.The technology can pass through the importing of two sgRNA and realize very strong Gene silencing efficacy.It is dry different from traditional RNA
Technology is disturbed, CRISPRi can be while any number of individual gene of silence.Also, there is very little risk to close non-targeted gene.This
Outward, prevent protein generation different from RNA interference, CRISPRi is played a role by preventing DNA information from being read and write as RNA.
Existing researcher confirms to merge Kr ü ppel associated box (KRAB) domains and dCas9, in nematode and zebra
Target gene (Lijiang Long et al., 2015, Cell Research, 25 can effectively be suppressed in fish:638-641).
But KRAB domains do not find homologous gene in zebra fish, a kind of dCas9 emerging systems are needed badly, it suppresses to turn
The domain of record exists in zebra fish, is changed on or near endogenous gene expression sites by the sgRNAs of targeting specific
Gene expression.
The content of the invention
The invention aims to shortcoming present in prior art is solved, the Eve domains originated with zebra fish,
And the method that a kind of orientation for proposing strikes multi-copy gene in drop zebra fish genome, comprise the following steps：
Step one：The preparation of zebrafish embryo；
Step 2：The expression vector establishment of dCas9-Eve fusion proteins, by the cDNA sequence of coding dCas9 and coding Eve
CDNA sequence recombinate when, a linker sequence is inserted between；
Step 3：Carry out the in-vitro transcription of sgRNA, dCas9-Eve, Cas9 and gene, then in-vitro transcription is synthesized
SgRNA, dCas9-Eve, Cas9mRNA and gene mRNA are purified with RNeasy Mini Kit；
Step 4：The activity identification of sgRNA, 1- cell stage zebrafish embryos are injected into by sgRNA and Cas9mRNA simultaneously,
Treat that embryonic development, to 24hpf, collects 5 embryos, shell membrane is torn off with metal tweezers, in being put into PCR pipe, with zebra fish genotype
Identification kit rapid extraction genomic DNA, then with it is amplifiable go out the gene promoter forward primer containing sgRNA recognition sequences and
Reverse primer is amplimer, enters performing PCR amplification by template of the genomic DNA of said extracted, and afterwards checking PCR primer is big
It is little whether consistent with purpose fragment, and PCR primer is sequenced, judge whether sgRNA is active；
Step 5：DCas9-Eve methods strike the expression of drop znfl1s, will identify activated sgRNA with final concentration 50ng/ μ l
Mixed with 250ng/ μ l dCas9-Eve, then microinjection enters the zebrafish embryo of 1- cells, treats embryonic development extremely
8hpf, the expression for passing through detection gene with embryo's whole mount in situ hybridization method judges that the situation of drop is struck in gene expression.
Preferably, the preparation method of the zebrafish embryo is comprised the following steps：Zebra fish is raised in circulation
In, 28.5 DEG C of room temperature, daily fixed-illumination 14 hours, dark 10 hours, upper and lower noon each feeding once, when collecting embryo,
Carry the previous day by the raun and milter of mating be respectively placed in mating box two ends, centre separated with dividing plate, treat the next morning
During illumination, by drawing out partition plate, embryo is collected in male and female mating, and embryo is placed in 28.5 DEG C of constant incubator cultivates standby.
Preferably, purify in the step 3 and comprise the steps：First to Buffer RPE 4 times of volumes of addition
Nuclease free absolute ethyl alcohol, the ultra-pure water for then adding DEPC to process toward the mRNA samples of in-vitro transcription so that volume expands
It is big to add the RLT of 350 μ l to 100 μ l, mix, then mixed liquor is added to into RNeasy Mini and prepare in pipe, pipe will be prepared and put
In entering 2ml collecting pipes, 12000g is centrifuged 1 minute, loses filtrate, adds 500 μ l Buffer RPE, 12000g to be centrifuged 1 minute,
Filtrate is lost, adds 500 μ l Buffer RPE, 12000g to be centrifuged again 1 minute, lose filtrate, again 12000g is centrifuged 1 point
Zhong Hou, will prepare pipe and is put in the EP of nuclease free pipes, and the ultra-pure water that 30-50 μ l DEPC were processed is added dropwise to pipe central authorities are prepared,
12000g is centrifuged 1 minute, finally determines mRNA concentration, the agarose gel electrophoresis of Jing 1% identification mRNA with ultraviolet specrophotometer
After quality, -80 DEG C are dispensed and are stored in.
Preferably, the process of rapid extraction genomic DNA is in step 4：10 μ l A liquid are added in PCR pipe, then will
PCR pipe is placed in PCR instrument device, and Jing 65 DEG C completes extracting genome DNA, the base of extraction for 1 minute in 5 minutes, 16 DEG C in 15 minutes, 95 DEG C
Save backup because a group DNA is placed in -20 DEG C.
Preferably, the concrete operation step of embryo's whole mount in situ hybridization method is that embryo is used into 4% poly in the step 5
Formaldehyde is fixed, and 4 DEG C are overnight, after second day tears shell membrane off with tip metal tweezer under anatomical lens, with 25%, 50%, absolute ethyl alcohol
Serial dehydration, each room temperature 5 minutes, the embryo after dehydration is placed in -20 DEG C 1 hour, and 25%PBST is used afterwards：75% ethanol,
50%PBST：50 ethanol, 1 × PBST gradient rehydrations, the embryo after rehydration adds prehybridization solution, and in 65 DEG C of hybrid heaters 5 are placed
Minute, with 65 DEG C of hybridization solution for adding yeast rna and heparin and rna probe overnight afterwards, the 3rd day, in 65 DEG C, with containing
The solution of 50% formamide, 10%20 × SSC and 0.1%Tween is washed twice, 30 minutes every time, is washed once with 2 × SSCT afterwards
15 minutes, then washed twice with 0.2 × SSCT, 30 minutes every time, then wash 3 times with the MABT room temperatures of pH7.5,5 minutes every time, afterwards
The confining liquid of the DigiTAb containing 1/2000-1/5000 dilutions is changed to after being closed 1 hour with confining liquid room temperature, 4 DEG C are overnight,
4th day, washed eight times with MABT, room temperature, 30 minutes every time, developed the color afterwards, and be initially charged equilibrium liquid, room temperature place 10 minutes it
Afterwards, equilibrium liquid is suctioned out, changes to nitrite ion, lucifuge, room temperature is placed 6-8 hours, after colour developing completely, cleaned with PBST one time, room temperature 5
Minute, with twice of washes of absolute alcohol, room temperature 10 minutes, add embedding liquid (2:1 mixing Bezylbenzoate and
Benzylalcohol), in situ hybridization result Lycra MZ16F stereoscope Taking Pictures recordings, image Adobe
Photoshop CS5Extended softwares carry out contrast and luminous intensity is processed.
Preferably, the prehybridization solution is the mixing of 50% deionized formamide, 25%20 × SSC and 0.1%Tween
Liquid.
Preferably, the confining liquid be 2%Blocking regent, 10% sheep serum, 70%MAB and 0.1%
The mixed liquor of Tween.
Preferably, the equilibrium liquid is 0.1M NaCl, 0.1M Tris-HCl (PH=9.5), 0.05M MgCl2, 0.1%
The mixed liquor of Tween-20 and 0.5mg/ml Lavamisol.
Preferably, the embedding liquid is 2:The Bezylbenzoate and Benzylalcohol of 1 mixing.
The method that a kind of orientation that the present invention is provided strikes multi-copy gene in drop zebra fish genome, using dCas9-Eve
Method specifically strike the transcription of drop gene, Eve in fruit bat suppresses functional domain, finds homologous zebra fish Eve functional domains,
Suppress functional domain to do fusion protein the Eve of dCas9 and zebra fish, can recognize that the sgRNA of gene promoter is deposited with efficient targeting
Situation, while the dCas9-Eve after Eve and dCas9 are combined together can significantly inhibit the expression of any zebra fish gene,
It is applied widely, and avoid research gene in early embryo development by MO toxicity or caused non-specificity of missing the target
The impact of phenotype.
Description of the drawings
Fig. 1 is that the method for striking multi-copy gene in drop zebra fish genome according to a kind of orientation proposed by the present invention is implemented
Experiment shows figure.
Wherein：A is the structural representation of dCas9 and the Eve fusion protein constructions in zebra fish source, and NLS refers to nuclear location
Sequence (Nuclear localization sequence), B is to inject sgRNA1, sgRNA2, sgRNA3 and Cas9mRNA
Embryo afterwards, extracts the result collection of illustrative plates that is sequenced of genomic DNA, the sequence in square frame for sgRNA3 binding site, arrow
The initiation site that effect occurs for sgRNAs where head indication, C is the znfl1s for not adding dCas9-Eve in the table of primitive gut mid-term
Up to situation, D is the znfl1s for adding dCas9-Eve in the expression of primitive gut mid-term.
Specific embodiment
The present invention is made with reference to specific embodiment further explain.
With reference to Fig. 1, the method that a kind of orientation proposed by the present invention strikes multi-copy gene in drop zebra fish genome, including with
Lower step：
Step one：The preparation of zebrafish embryo, zebra fish is raised in circulation, 28.5 DEG C of room temperature, daily
Fixed-illumination 14 hours, dark 10 hours, upper and lower noon each feeding once, when collecting embryo, carried the previous day by the raun of mating
With the two ends that milter is respectively placed in mating box, centre is separated with dividing plate, when the next morning illumination, by drawing out partition plate, male and female
Mating, produces embryo because gravity can sink to the cassette bottom portion of mating, collection embryo, and embryo is placed in into 28.5 DEG C incubated
Cultivate in case standby；
Step 2：The expression vector establishment of dCas9-Eve fusion proteins, by the cDNA sequence of coding dCas9 and coding Eve
CDNA sequence recombinate when, a linker sequence is inserted between, wherein：DCas9 expression vectors are by Nanjing Yao along Yu
Bio tech ltd provides, and the sequence for encoding the albumen is humanized sequence；
Zebra fish Eve sequences are：
ATGGAAACCACAATAAAGGTTTGGTTTCAGAACCGTCGCATGAAGGACAAGAGACAGCGTCTGGCTATGACCTGGCC
GCATCCTGCCGACCCCGCCTTTTACACCTATATGATGAGCCATGCTGCAGCAACGGGCAGTCTGCCCTATCCATTTC
AATCTCATCTTCCCCTTCC TTACTATTCTCCACTAAGCAGTGTGACTGCAGGTTCAGCCACTGCCACTGCGGGTCC
ATTCTCAAATCCCCTGCGCTCGCTGGATAGTTTTCGGGTGCTTTCGCATCCATACCCGCGACCTGAACTGCTGTGCG
CCTTCAGACACCCATCACTGTACCCCAGCCCGGGTCATGGGCTTGGTCCCGGTGGAAGTCCATGCTCCTGCCTTGCT
TGCCACGCTTCCAGTCAAACAAACGGGCTCCAACATAGATCCAATAATGCAGAATTCTCGTGTTCGCCCACGACCAG
GACTGAGGCCTTCCTCACTTTCTCGCCAGCAGTCATCAGCAAATCATCTTCGGTGTCTTTGGACCAGAGGGAGGAAG
TGCCACTAACTAGA；
Linker sequences are：
AGCCCCAAGAAGAAGAGAAAGGTGGAGGCCAGCGGAGGTACGCGTTCTAGAACC；
Step 3：The in-vitro transcription of sgRNA, dCas9-Eve, Cas9 and znfl1s is carried out, 3 identification znfl1s is designed and is opened
The sequence of the gRNA of sub-area is respectively：
gRNA1(GCACTGTCCTGTACTTCCCATGG)、
gRNA2(GTTAGTAGCCAAATAAGATTTGG)、
gRNA3(ACAACATCAGAAAAACATGG), wherein underlined sequences are PAM sequences, and corresponding sgRNA adopts body
The method synthesis of outer transcription, synthesizes in vitro template used amplification by PCR and obtains, and the forward primer of PCR reactions is respectively：
gRNA1F(TAATACGACTCACTATAggcactgtcctgtacttcccaGTTTTAGAGCTAGAAATAGC)、
gRNA2F(TAATACGACTCACTATAggttagtagccaaataagattGTTTTA GAGCTAGAAATAGC)、
GRNA3F (TAATACGACTCACTATAggacaacatcagaaaaacaGTTTTAGAGCTAGAAATAGC -3 '),
The big write sequence in front end of the primer is T7 promoter sequences, and middle little write sequence is former interval, and PCR reverse primer sequences are
CTATTTCTAGCTCTAAAAC；The template that amplification is used is pMD 19-gRNA carriers；The plasmid is respected dimension laboratory and is given by bear；
PCR reacted constituents are：10 μ l2MasterMix, 7 μ l ultra-pure waters, 5 μM it is positive/negative to each 1 μ l and pMD 19-gRNA carriers of primer
1 μ l, reaction condition is：94 DEG C 2 minutes, 35 circulation (94 DEG C 15 seconds, 58 DEG C 30 seconds and 72 DEG C 20 seconds), 72 DEG C 10 minutes；PCR
Product Jing PCR Clean Up kits after amplification；During in-vitro transcription sgRNA, to purify the PCR primer for obtaining as mould
Plate, using MAXIscript In Vitro Transcription Kit in-vitro transcriptions sgRNA, and transcription synthesis in vitro
During gRNA, the UTP of digoxigenin labeled is not added with；The kit of in-vitro transcription mRNA is mMESSAGE mMACHINE T7, and mRNA adds
The kit of tail is Poly (A) Tailing Kit；
When in-vitro transcription Cas9 and dCas9-Eve synthesis Cas9 and dCas9-Eve mRNA, distinguished using XbaI and XhoI
Digested plasmid PXT7-cas9 and pKS-dCas9-dmEVE linearize it, are detected with 1% agarose gel electrophoresis, will be linear
Change completely plasmid PCR Clean Up kits and reclaim template, use DEPC water dissolves, add in the EP pipes containing DNA profiling
Enter 10 μ l 2NTP/CAP, 2 μ l 10Reaction Buffer and 2 μ l T7enzyme Mix, flick the mixing of EP pipes, and after being centrifuged
It is put in 37 DEG C to react 2 hours, adds 1 μ l TURBO DNase to react 15 minutes after 37 DEG C, when carrying out tailings reactions, to 20 μ l
36 μ l DEPC water, 20 μ l 5E-PAP Buffer, 10 μ l 25mM MnCl are added in above-mentioned reaction system2, 10 μ l ATP
Solution, adds 4 μ l E-PAP, soft to mix, and reacts 45 minutes in 37 DEG C, and 50 μ l LiCl are added in reactant liquor, mixes
- 80 DEG C are put in after even 1 hour, 4 DEG C of centrifugations, 12000g removes supernatant after 10 minutes, after adding 75% without enzyme ethanol, 12000g 4
DEG C centrifugation 10 minutes, removes supernatant, collects mRNA, is dissolved in DEPC water, Jing after 1% agarose gel electrophoresis identification mRNA mass, point
Fill and be stored in -80 DEG C；
During in-vitro transcription znfl1s mRNA, using SmaI digested plasmid pXT7-znfl1s it is linearized, with 1% fine jade
After sepharose electrophoresis detection confirms that linearisation is complete, linearisation template is reclaimed with PCR Clean Up kits, be dissolved in DEPC
Water, the in-vitro transcription of in-vitro transcription step same sgRNA, dCas9-Eve and Cas9mRNA；
SgRNA, dCas9-Eve, Cas9mRNA and znfl1s mRNA RNeasy Mini Kit of in-vitro transcription synthesis
Purified, purification step is as follows：The nuclease free absolute ethyl alcohol of 4 times of volumes is added to Buffer RPE first, then toward body
The ultra-pure water for adding DEPC to process in the mRNA samples of outer transcription so that volume is extended to 100l, adds the RLT of 350l, mixes
It is even, then mixed liquor is added to into RNeasy Mini prepares in pipe, pipe being prepared and be put in 2ml collecting pipes, 12000g is centrifuged 1 point
Clock, loses filtrate, adds 500l Buffer RPE, 12000g to be centrifuged 1 minute, loses filtrate, and 500l Buffer are added again
RPE, 12000g be centrifuged 1 minute, lose filtrate, again 12000g be centrifuged 1 minute after, by prepare pipe be put into nuclease free EP pipe
It is interior, the ultra-pure water that 30-50l DEPC were processed is added dropwise to pipe central authorities are prepared, 12000g is centrifuged 1 minute, finally uses ultraviolet spectrometry light
Degree meter determines mRNA concentration, Jing after 1% agarose gel electrophoresis identification mRNA mass, dispenses and be stored in -80 DEG C；
Step 4：The activity identification of sgRNA, comprises the following steps：First by 50pg sgRNA and 250pg Cas9mRNA
Simultaneously 1- cell stage zebrafish embryos are injected into, treat that embryonic development, to 24hpf, collects 5 embryos, with metal tweezers shell is torn off
Film, in being put into PCR pipe, with zebra fish genotyping kit rapid extraction genomic DNA, its process is：To in PCR pipe
10 μ l A liquid are added, then PCR pipe is placed in PCR instrument device, Jing 65 DEG C completes base in 1 minute in 5 minutes, 16 DEG C in 15 minutes, 95 DEG C
Because group DNA is extracted, the genomic DNA of extraction is placed in -20 DEG C and saves backup, then with it is amplifiable go out containing sgRNA recognition sequences
Znfl1s promoter forward primers (GGCTTGATTCTTGCTTTG) and reverse primer (GTGAGTGCTTGATGCTGT) are amplification
Primer, with the genomic DNA of said extracted as template, enter performing PCR amplification, amplification condition is：95 DEG C 3 minutes, 35 circulation (95
DEG C 30 seconds, 58 DEG C 30 seconds and 72 DEG C 30 seconds 1 minute), 72 DEG C 10 minutes, 16 DEG C 10 minutes, it is big with electrophoresis checking PCR primer afterwards
It is little whether consistent with purpose fragment 1004bp, PCR primer is directly sent sequencing company be sequenced, sequencing primer is reversely to draw
Whether thing, occur substantially covering peak according to starting in PAM sequence annexes of showing of sequencing result, judges whether sgRNA is active；
Step 5：DCas9-Eve methods strike the expression of drop znfl1s, will identify activated sgRNA with final concentration 50ng/ μ l
Mixed with 250ng/ μ l dCas9-Eve, then microinjection enters the zebrafish embryo of 1- cells, treats embryonic development extremely
8hpf, the expression for passing through detection zfnl1s with embryo's whole mount in situ hybridization method judges that the situation of drop is struck in zfnl1s gene expressions；
In the present invention, Znfl1s is the transcription factor containing a Zinc finger domain, and znfl1s genes contain in zebra fish
There are 13 copies, the genome sequence that this 13 are copied is (to translation termination sequence from the about 2kbp of translation initiation site upstream
Downstream 1kbp stops) sequence of about 7k compares, it is found that these genes are mutually unison very high on genome sequence.
In the present invention, the concrete operation step of embryo's whole mount in situ hybridization method is to fix embryo with 4% paraformaldehyde, 4
DEG C overnight；After second day tears shell membrane off with tip metal tweezer under anatomical lens, with 25% (1 time), 50% (1 time), absolute ethyl alcohol
(2 times) serial dehydration, each room temperature 5 minutes, the embryo after dehydration is placed in -20 DEG C 1 hour, 25%PBST is used afterwards：75% second
Alcohol (1 time), 50%PBST：50 ethanol (1 time), 1 × PBST (130mM NaCl, 7mM Na2HPO4With 7mM NaH2PO4) gradient
Rehydration, wherein T refer to 0.1%Tween；Embryo after rehydration add prehybridization solution (50% deionized formamide, 25%20 ×
SSC and 0.1%Tween), place in 65 DEG C of hybrid heaters 5 minutes, afterwards with addition yeast rna (500 μ g/ml) and heparin (50
μ g/ml) and final concentration of 1ng/ μ l rna probe 65 DEG C of hybridization solution overnight；3rd day, in 65 DEG C, with containing 50% formyl
The solution of amine, 10%20 × SSC and 0.1%Tween is washed twice, 30 minutes every time, is washed one time 15 minutes with 2 × SSCT afterwards,
Washed twice with 0.2 × SSCT again, 30 minutes every time, then with MABT (100mM maleic acids, 150mM sodium chloride, the pH=of pH7.5
7.5,0.1%Tween-20) room temperature washes 3 times, 5 minutes every time, (2%Blocking regent, 10% continuous with confining liquid afterwards
Sheep blood serum, 70%MAB and 0.1%Tween) room temperature close 1 hour after change to containing 1/2000-1/5000 dilution digoxin resist
The confining liquid of body (Anti-Digoxigenin-AP Fab fragment), 4 DEG C overnight；4th day, eight times are washed with MABT, room
Temperature, 30 minutes every time, develops the color afterwards, and is initially charged equilibrium liquid (0.1M NaCl, 0.1M Tris-HCl (PH=9.5), 0.05M
MgCl2, 0.1%Tween-20,0.5mg/ml Lavamisol), room temperature place 10 minutes afterwards, suction out equilibrium liquid, change to aobvious
Color liquid (adds 1.2mg/ml Lavamisol, the NBT/BCIP storing solutions of 20 μ l) in equilibrium liquid, lucifuge, and it is little that room temperature places 6-8
When, after colour developing completely, clean with PBST one time, room temperature 5 minutes, with twice of washes of absolute alcohol, room temperature 10 minutes is added and embedded
Liquid (2:The Bezylbenzoate and Benzylalcohol of 1 mixing), in situ hybridization result is clapped with Lycra MZ16F stereoscopes
According to record, image Adobe Photoshop CS5Extended softwares carry out contrast and luminous intensity is processed.
The present embodiment passes through to combine in target gene promoter sequence using dCas9-Eve under sgRNA guiding, affects to turn
Functional transcription of the record complex to target gene, and for having synthesized recognizable different sequences on the promoter sequence of znfl1s altogether
3 sgRNA of row, activity checking shows the activity of sgRNA1 substantially, but sgRNA3 is relatively low without activity or activity, and sgRNA2
Activity due to sgRNA1 activity the bad judgement of impact, be the activity for accurately judging sgRNA, we strike drop in dCas9-Eve
In experiment, after being mixed using these three sgRNA with dCas9-EvemRNA co-injections in 1-2 cell stage zebrafish embryos, so
Afterwards expressions of the znfl1s in primitive gut mid-term is observed by embryo's whole mount in situ hybridization.As a result show, the expression of znfl1s is received
To significantly suppression, the expression for inhibiting znfl1s to a great extent using dCas9-Eve and sgRNA is illustrated, so as to realize
Drop is struck to znfl1s functions.
The above, the only present invention preferably specific embodiment, but protection scope of the present invention is not limited thereto,
Any those familiar with the art the invention discloses technical scope in, technology according to the present invention scheme and its
Inventive concept equivalent or change in addition, all should be included within the scope of the present invention.
Claims (9)
1. a kind of method that orientation strikes multi-copy gene in drop zebra fish genome, it is characterised in that comprise the following steps：
Step one：The preparation of zebrafish embryo；
Step 2：The expression vector establishment of dCas9-Eve fusion proteins, by the cDNA sequence of coding dCas9 with coding Eve's
When cDNA sequence is recombinated, a linker sequence is inserted between；
Step 3：Carry out the in-vitro transcription of sgRNA, dCas9-Eve, Cas9 and gene, then in-vitro transcription is synthesized sgRNA,
DCas9-Eve, Cas9mRNA and gene mRNA are purified with RNeasy Mini Kit；
Step 4：The activity identification of sgRNA, 1- cell stage zebrafish embryos are injected into by sgRNA and Cas9mRNA simultaneously, treat embryo
Tire is developed to 24hpf, collects 5 embryos, shell membrane is torn off with metal tweezers, in being put into PCR pipe, with zebra fish genotype identification
Kit rapid extraction genomic DNA, then with it is amplifiable go out the gene promoter forward primer containing sgRNA recognition sequences and reversely
Primer is amplimer, enters performing PCR amplification by template of the genomic DNA of said extracted, and afterwards whether checking PCR primer size
It is consistent with purpose fragment, and PCR primer is sequenced, judge whether sgRNA is active；
Step 5：DCas9-Eve methods strike drop znfl1s expression, will identify activated sgRNA with final concentration 50ng/ μ l with
250ng/ μ l dCas9-Eve are mixed, and then microinjection enters the zebrafish embryo of 1- cells, treats embryonic development to 8hpf,
The expression for passing through detection gene with embryo's whole mount in situ hybridization method, judges that the situation of drop is struck in gene expression.
2. the method that a kind of orientation according to claim 1 strikes multi-copy gene in drop zebra fish genome, its feature exists
In the preparation method of the zebrafish embryo is comprised the following steps：Zebra fish is raised in circulation, room temperature
28.5 DEG C, daily fixed-illumination 14 hours, dark 10 hours, once, when collecting embryo, carrying the previous day will for upper and lower noon each feeding
The raun and milter of mating is respectively placed in the two ends of mating box, and centre is separated with dividing plate, when the next morning illumination, will be every
Plate is extracted out, male and female mating, collects embryo, and embryo is placed in 28.5 DEG C of constant incubator cultivates standby.
3. the method that a kind of orientation according to claim 1 strikes multi-copy gene in drop zebra fish genome, its feature exists
In purifying comprises the steps in the step 3：First nuclease free absolute ethyl alcohol is added to Buffer RPE, then toward body
The ultra-pure water for adding DEPC to process in the mRNA samples of outer transcription so that volume is extended to 100 μ l, adds the RLT of 350 μ l,
Mix, then mixed liquor be added to into RNeasy Mini and prepare in pipe, pipe will be prepared and be put in collecting pipe, be centrifuged and lose filtrate,
Buffer RPE are added, filtrate is centrifuged and loses, Buffer RPE are added again, be centrifuged and lose filtrate, after being centrifuged again, will
Prepare pipe to be put in the EP of nuclease free pipes, to pipe central authorities are prepared the ultra-pure waters that processed of DEPC are added dropwise, centrifugation, finally with ultraviolet
Spectrophotometric determination mRNA concentration, Jing after 1% agarose gel electrophoresis identification mRNA mass, dispenses and is stored in -80 DEG C.
4. the method that a kind of orientation according to claim 1 strikes multi-copy gene in drop zebra fish genome, its feature exists
In the process of rapid extraction genomic DNA is in the step 4：10 μ l A liquid are added in PCR pipe, then puts PCR pipe
In PCR instrument device, Jing 65 DEG C completes extracting genome DNA, the genomic DNA of extraction for 1 minute in 5 minutes, 16 DEG C in 15 minutes, 95 DEG C
It is placed in -20 DEG C to save backup.
5. the method that a kind of orientation according to claim 1 strikes multi-copy gene in drop zebra fish genome, its feature exists
In the concrete operation step of embryo's whole mount in situ hybridization method is to fix embryo with 4% paraformaldehyde in the step 5,4 DEG C
Overnight, tear off after shell membrane within second day, carry out serial dehydration, the embryo after dehydration is placed in -20 DEG C 1 hour, then carry out gradient rehydration,
Embryo after rehydration adds prehybridization solution, places 5 minutes in 65 DEG C of hybrid heaters, afterwards with add yeast rna and heparin and
65 DEG C of the hybridization solution of rna probe overnight, the 3rd day, in 65 DEG C, is used contain 50% formamide, 10%20 × SSC and 0.1% successively
The solution of Tween, 2 × SSCT, 0.2 × SSCT cleaning, then cleaned with the MABT room temperatures of pH7.5, sealed with confining liquid room temperature afterwards
The confining liquid of the DigiTAb containing 1/2000-1/5000 dilutions is changed to after closing 1 hour, 4 DEG C overnight, the 4th day, uses MABT
Develop the color after room temperature cleaning, and be initially charged equilibrium liquid, room temperature places 10 minutes afterwards, suctions out equilibrium liquid, changes to nitrite ion, keeps away
Light, room temperature places 6-8 hours, after colour developing completely, successively with PBST and washes of absolute alcohol, adds embedding liquid, in situ hybridization knot
Fruit Lycra MZ16F stereoscope Taking Pictures recordings, contrast is carried out to image and luminous intensity is processed.
6. the method that a kind of orientation according to claim 5 strikes multi-copy gene in drop zebra fish genome, its feature exists
In the prehybridization solution is the mixed liquor of 50% deionized formamide, 25%20 × SSC and 0.1%Tween.
7. the method that a kind of orientation according to claim 5 strikes multi-copy gene in drop zebra fish genome, its feature exists
In the confining liquid is 2%Blocking regent, the mixed liquor of 10% sheep serum, 70%MAB and 0.1%Tween.
8. the method that a kind of orientation according to claim 5 strikes multi-copy gene in drop zebra fish genome, its feature exists
In the equilibrium liquid is 0.1M NaCl, 0.1M Tris-HCl, 0.05M MgCl2, 0.1%Tween-20 and 0.5mg/ml
The mixed liquor of Lavamisol.
9. the method that a kind of orientation according to claim 5 strikes multi-copy gene in drop zebra fish genome, its feature exists
In the embedding liquid is 2:The Bezylbenzoate and Benzylalcohol of 1 mixing.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610843834.8A CN106636197B (en) | 2016-09-22 | 2016-09-22 | A method of orientation strikes multi-copy gene in drop zebra fish genome |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610843834.8A CN106636197B (en) | 2016-09-22 | 2016-09-22 | A method of orientation strikes multi-copy gene in drop zebra fish genome |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106636197A true CN106636197A (en) | 2017-05-10 |
| CN106636197B CN106636197B (en) | 2019-09-03 |
Family
ID=58853153
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610843834.8A Active CN106636197B (en) | 2016-09-22 | 2016-09-22 | A method of orientation strikes multi-copy gene in drop zebra fish genome |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106636197B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109207524A (en) * | 2017-06-29 | 2019-01-15 | 南京尧顺禹生物科技有限公司 | The foundation and application of human obesity's zebra fish model based on FTO gene |
| CN110669795A (en) * | 2019-10-18 | 2020-01-10 | 福州大学 | Technology for realizing precise fixed-point RNA shearing in fish embryo |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102653756A (en) * | 2011-03-04 | 2012-09-05 | 南京大学 | Directional modification method of specific gene of animal genome and application thereof |
| CN105142669A (en) * | 2012-12-06 | 2015-12-09 | 西格马-奥尔德里奇有限责任公司 | Crispr-based genome modification and regulation |
| CN105594664A (en) * | 2016-02-16 | 2016-05-25 | 湖南师范大学 | Statla gene deletion type zebra fish |
-
2016
- 2016-09-22 CN CN201610843834.8A patent/CN106636197B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102653756A (en) * | 2011-03-04 | 2012-09-05 | 南京大学 | Directional modification method of specific gene of animal genome and application thereof |
| CN105142669A (en) * | 2012-12-06 | 2015-12-09 | 西格马-奥尔德里奇有限责任公司 | Crispr-based genome modification and regulation |
| CN105594664A (en) * | 2016-02-16 | 2016-05-25 | 湖南师范大学 | Statla gene deletion type zebra fish |
Non-Patent Citations (4)
| Title |
|---|
| DONG X 等: "Zebrafish Znfl1 proteins control the expression of hoxb1b gene in the posterior neuroectoderm by acting upstream of pou5f3 and sall4 genes", 《J BIOL CHEM》 * |
| HE L 等: "Danio rerio even-skipped homeobox 1 (evx1), mRNA", 《GENBANK DATABASE》 * |
| JOLY JS 等: "The ventral and posterior expression of the zebrafish homeobox gene eve1 is perturbed in dorsalized and mutant embryos", 《DEVELOPMENT》 * |
| LIJIANG LONG 等: "Regulation of transcriptionally active genes via the catalytically inactive Cas9 in C. elegans and D. rerio", 《CELL RES》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109207524A (en) * | 2017-06-29 | 2019-01-15 | 南京尧顺禹生物科技有限公司 | The foundation and application of human obesity's zebra fish model based on FTO gene |
| CN110669795A (en) * | 2019-10-18 | 2020-01-10 | 福州大学 | Technology for realizing precise fixed-point RNA shearing in fish embryo |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106636197B (en) | 2019-09-03 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102884191B (en) | For parallel separation and/or the method for purifying RNA and DNA | |
| Watkins et al. | Assembly and maturation of the U3 snoRNP in the nucleoplasm in a large dynamic multiprotein complex | |
| CN106636471B (en) | Multiplex PCR detection kit for simultaneously detecting WSSV, AHPND, EHP and IHHNV of prawns | |
| US20050069910A1 (en) | Nucleic acid ligands to complex targets | |
| CN110343785B (en) | Kit for detecting hepatitis B virus covalent closed circular DNA based on PCR-CRISPR-cas13a | |
| CN104450920A (en) | MicroRNA trace detection method based on exponential order non-enzymatic amplification and electrochemical luminescence principle | |
| Tang et al. | Transcriptionally inactive hepatitis B virus episome DNA preferentially resides in the vicinity of chromosome 19 in 3D host genome upon infection | |
| CN111073891B (en) | Aptamer for detecting grouper iridovirus as well as construction method and application thereof | |
| CN103205431A (en) | Nucleic acid aptamer and derivatives thereof, screening method of nucleic acid aptamer, application of nucleic acid aptamer and derivatives in detecting human biliary duct carcinoma cell line | |
| Wang et al. | Genome oligopaint via local denaturation fluorescence in situ hybridization | |
| CN105120986B (en) | A step program for nucleic acid purification | |
| CN106801096A (en) | A kind of chromatin immune is co-precipitated sample fast preparation method | |
| CN108841934A (en) | A kind of method and kit of the N7- methylguanosine modification of the full inside transcript profile range single base resolution ratio detection RNA | |
| CN106636197A (en) | Method for directional knockdown of multi-copy gene in zebrafish genome | |
| CN102037140A (en) | Method for determination of DNA methylation | |
| CN103911379B (en) | Nucleic acid aptamer, derivatives, screening method and application thereof | |
| CN109929911A (en) | A kind of novel translation group Ribosome-seq banking process | |
| CN109022419A (en) | A kind of preparation method of fragmentation DNA | |
| CN103160514A (en) | cDNA overall length nucleotide sequence of human EBLN-1 gene and cloning method thereof | |
| EP2143806A1 (en) | Method of measuring dna methylation | |
| KR20240051337A (en) | Improved CRISPR-CAS technology | |
| JP5303981B2 (en) | DNA methylation measurement method | |
| CN111118014B (en) | Anti-iridovirus aptamer and construction method and application thereof | |
| CN111363749A (en) | Aptamer for detecting Chinese softshell turtle iridovirus and construction method and application thereof | |
| CN106337053B (en) | Nucleic acid aptamer for detecting spring viremia of carp virus and application of nucleic acid aptamer |
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 |