CN111793653B - Construction method of dpy19l1l gene deletion type zebra fish - Google Patents
Construction method of dpy19l1l gene deletion type zebra fish Download PDFInfo
- Publication number
- CN111793653B CN111793653B CN202010656897.9A CN202010656897A CN111793653B CN 111793653 B CN111793653 B CN 111793653B CN 202010656897 A CN202010656897 A CN 202010656897A CN 111793653 B CN111793653 B CN 111793653B
- Authority
- CN
- China
- Prior art keywords
- dpy19l1l
- zebra fish
- gene
- primer
- generation
- 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.)
- Active
Links
- 241000252212 Danio rerio Species 0.000 title claims abstract description 49
- 238000010276 construction Methods 0.000 title claims abstract description 9
- 238000012224 gene deletion Methods 0.000 title abstract description 16
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 29
- 108020005004 Guide RNA Proteins 0.000 claims abstract description 18
- 238000000338 in vitro Methods 0.000 claims abstract description 13
- 108091033409 CRISPR Proteins 0.000 claims abstract description 12
- 208000000875 Spinal Curvatures Diseases 0.000 claims abstract description 9
- 238000003209 gene knockout Methods 0.000 claims abstract description 8
- 238000012258 culturing Methods 0.000 claims abstract description 7
- 206010010356 Congenital anomaly Diseases 0.000 claims abstract description 6
- 230000002068 genetic effect Effects 0.000 claims abstract 2
- 238000013518 transcription Methods 0.000 claims description 15
- 230000035897 transcription Effects 0.000 claims description 15
- 108020004414 DNA Proteins 0.000 claims description 13
- 108091092584 GDNA Proteins 0.000 claims description 13
- 238000006243 chemical reaction Methods 0.000 claims description 10
- 238000000034 method Methods 0.000 claims description 9
- 241000251468 Actinopterygii Species 0.000 claims description 7
- 235000013601 eggs Nutrition 0.000 claims description 7
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 6
- 230000035772 mutation Effects 0.000 claims description 6
- 206010064571 Gene mutation Diseases 0.000 claims description 5
- 102000006382 Ribonucleases Human genes 0.000 claims description 5
- 108010083644 Ribonucleases Proteins 0.000 claims description 5
- 238000000520 microinjection Methods 0.000 claims description 5
- 238000012216 screening Methods 0.000 claims description 5
- 230000003321 amplification Effects 0.000 claims description 4
- 238000010367 cloning Methods 0.000 claims description 4
- 108010045673 endodeoxyribonuclease XBAI Proteins 0.000 claims description 4
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 4
- 238000007480 sanger sequencing Methods 0.000 claims description 4
- 238000010354 CRISPR gene editing Methods 0.000 claims description 3
- 238000009395 breeding Methods 0.000 claims description 3
- 230000001488 breeding effect Effects 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 3
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 claims description 2
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 claims description 2
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 claims description 2
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 claims description 2
- 108091028733 RNTP Proteins 0.000 claims description 2
- 239000000872 buffer Substances 0.000 claims description 2
- 230000012447 hatching Effects 0.000 claims description 2
- 101710137500 T7 RNA polymerase Proteins 0.000 claims 1
- 239000011535 reaction buffer Substances 0.000 claims 1
- 239000003161 ribonuclease inhibitor Substances 0.000 claims 1
- 210000002257 embryonic structure Anatomy 0.000 abstract description 8
- 201000010099 disease Diseases 0.000 abstract description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 4
- 210000000988 bone and bone Anatomy 0.000 abstract description 3
- 238000011161 development Methods 0.000 abstract description 3
- 210000001161 mammalian embryo Anatomy 0.000 abstract description 3
- 238000004519 manufacturing process Methods 0.000 abstract description 2
- 238000004458 analytical method Methods 0.000 abstract 1
- 230000008685 targeting Effects 0.000 abstract 1
- 101150081199 dpy-19 gene Proteins 0.000 description 4
- 239000012634 fragment Substances 0.000 description 4
- 238000012986 modification Methods 0.000 description 4
- 230000004048 modification Effects 0.000 description 4
- 102100028693 Probable C-mannosyltransferase DPY19L1 Human genes 0.000 description 3
- 108050000654 Probable C-mannosyltransferase DPY19L1 Proteins 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 238000012163 sequencing technique Methods 0.000 description 3
- 208000020084 Bone disease Diseases 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 101150012230 DPY19L2 gene Proteins 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- 230000004075 alteration Effects 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 230000014461 bone development Effects 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 238000012217 deletion Methods 0.000 description 2
- 230000037430 deletion Effects 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 230000004720 fertilization Effects 0.000 description 2
- 238000013508 migration Methods 0.000 description 2
- 230000005012 migration Effects 0.000 description 2
- 239000011259 mixed solution Substances 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 230000021595 spermatogenesis Effects 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- 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 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- 108010008532 Deoxyribonuclease I Proteins 0.000 description 1
- 102000007260 Deoxyribonuclease I Human genes 0.000 description 1
- 101000915000 Homo sapiens Probable C-mannosyltransferase DPY19L2 Proteins 0.000 description 1
- 101000915008 Homo sapiens Probable C-mannosyltransferase DPY19L3 Proteins 0.000 description 1
- 101000914997 Homo sapiens Probable C-mannosyltransferase DPY19L4 Proteins 0.000 description 1
- 108020004485 Nonsense Codon Proteins 0.000 description 1
- 102100028694 Probable C-mannosyltransferase DPY19L2 Human genes 0.000 description 1
- 102100028677 Probable C-mannosyltransferase DPY19L3 Human genes 0.000 description 1
- 102100028695 Probable C-mannosyltransferase DPY19L4 Human genes 0.000 description 1
- 102000004357 Transferases Human genes 0.000 description 1
- 108090000992 Transferases Proteins 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000028600 axonogenesis Effects 0.000 description 1
- 206010003883 azoospermia Diseases 0.000 description 1
- 238000005452 bending Methods 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000006239 cerebral cortex development Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000002759 chromosomal effect Effects 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 230000008303 genetic mechanism Effects 0.000 description 1
- 238000003205 genotyping method Methods 0.000 description 1
- 210000001362 glutamatergic neuron Anatomy 0.000 description 1
- 208000000509 infertility Diseases 0.000 description 1
- 230000036512 infertility Effects 0.000 description 1
- 208000021267 infertility disease Diseases 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 230000036244 malformation Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 230000009456 molecular mechanism Effects 0.000 description 1
- 230000008271 nervous system development Effects 0.000 description 1
- 230000003988 neural development Effects 0.000 description 1
- 238000006386 neutralization reaction Methods 0.000 description 1
- 230000037434 nonsense mutation Effects 0.000 description 1
- 208000008634 oligospermia Diseases 0.000 description 1
- 230000036616 oligospermia Effects 0.000 description 1
- 231100000528 oligospermia Toxicity 0.000 description 1
- 238000003752 polymerase chain reaction Methods 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 108010008359 protein kinase C lambda Proteins 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 229920002477 rna polymer Polymers 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 230000012488 skeletal system development Effects 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 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
- C12N15/87—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
-
- 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
- A01K61/00—Culture of aquatic animals
- A01K61/10—Culture of aquatic animals of 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
- 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
- A01K67/0276—Knock-out vertebrates
-
- 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
- C12N15/90—Stable introduction of foreign DNA into chromosome
- C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
-
- 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
- C12N9/10—Transferases (2.)
- C12N9/1048—Glycosyltransferases (2.4)
- C12N9/1051—Hexosyltransferases (2.4.1)
-
- 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
- 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
- A01K2267/03—Animal model, e.g. for test or diseases
-
- 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)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Chemical & Material Sciences (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Environmental Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Biophysics (AREA)
- Biodiversity & Conservation Biology (AREA)
- Physics & Mathematics (AREA)
- Animal Husbandry (AREA)
- Mycology (AREA)
- Veterinary Medicine (AREA)
- Animal Behavior & Ethology (AREA)
- Marine Sciences & Fisheries (AREA)
- Medicinal Chemistry (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to the technical field of gene knockout and zebra fish models, in particular to a construction method of a dpy19l1l gene deletion type zebra fish. Designing a proper targeting site on a dpy19l1l gene of the zebra fish, microinjecting the specific gRNA and Cas9 protein synthesized in vitro into a zebra fish cell, culturing the embryo for 48 hours, and then carrying out genotype analysis by selecting the embryo, thereby confirming the effectiveness of the selected site, and obtaining the stable genetic dpy19l1l homozygous mutant zebra fish strain through culture. The invention can silence specific genes in the genome of an organism more efficiently and accurately, has simple manufacture and low cost, can simultaneously cut a plurality of sites on a target gene, silence any number of single genes, knock out the development deformity of dpy19l1l gene zebra fish embryos, construct successful dpy19l1l mutant congenital spinal curvature, provide a good zebra fish model for a spinal curvature model, and is helpful for researching bone related diseases.
Description
Technical Field
The invention relates to the technical field of gene knockout and zebra fish models, in particular to a construction method of a dpy19l1l gene deletion type zebra fish.
Background
The DPY19L1L gene is located on chromosome 16 of zebra fish, and is a member of the DPY19 gene family, which encodes the DPY19 (dumpy-19, DPY-19) protein family, including DPY19L1 (DPY-19-like 1), DPY19L2, DPY19L3 and DPY19L4, which has mannose transferase activity as a classical function. DPY19L1 protein is multi-transmembrane protein, mainly participates in nervous system development, regulates radial migration of glutamatergic neurons in the process of cerebral cortex development, and is simultaneously required for axon growth. The DPY19L2 gene plays an important role in the spermatogenesis process, and the gene deletion is an important causative factor of the oligospermia. Studies have indicated that the sterility of a round-headed sperm is related to a balance between migration of the DPY19L2 gene and gene deletion at chromosomal breakpoints. In conclusion, the DPY19 gene mainly plays a role in the processes of neural development and spermatogenesis, and is not reported in the fields related to skeletal development and diseases, so that the research on the DPY19L1L gene is deficient.
Based on the above, it is important to provide a DPY19L1L gene deletion type zebra fish and to excavate the new function and genetic mechanism of the DPY19L1L gene in the development of the zebra fish.
Disclosure of Invention
The invention aims to provide a construction method of a dpy19l1l gene deletion type zebra fish, wherein in the invention, the dpy19l1l gene is related to zebra fish bone development, and can be used for researching a deep molecular mechanism of zebra fish bone development.
In order to achieve the above purpose, the invention adopts the following technical scheme:
a construction method of dpy19l1l gene deletion type zebra fish comprises the following steps:
s1, inquiring a genomic DNA sequence of a dpy19l1l gene of the zebra fish on NCBI, analyzing a functional domain of the genomic DNA sequence, finding a target site of the dpy19l1l gene from an exon 1 of the dpy19l1l gene according to a CRISPR/Cas9 knockout principle, designing a target site long primer and a gDNA joint primer, wherein the sequence of the target site long primer of the dpy19l1l is 5'-Taatacgactcactatag GGAATAACAGTGCTGATTCT gttttagagctagaaatagc-3', gDNA joint primer sequence 5'-AGCACCGACTCGGTGCCACTT-3';
s2, obtaining an in vitro transcription template of the gRNA by primer star PCR;
s3, in-vitro transcription is carried out according to the gRNA in-vitro transcription template obtained in the S2, and the gRNA is extracted and purified to obtain purified gRNA;
s4, microinjecting the purified gRNA and Cas9 protein to fertilized eggs of the zebra fish in a cell stage, and culturing to obtain a stable inheritance dpy-19l1l homozygous mutant zebra fish strain, namely dpy19l1l gene deletion type zebra fish.
Further, the S2 reaction system is as follows: gDNA Vector Template 1. Mu.l, 1. Mu.l of target site long primer, gDNA adapter primer, 1. Mu.l of dNTP Mix, 5X Primer star buffer. Mu.l, primer star DNApolymerase. Mu.l, ddH 2 O32 μl; the gDNA Vector Template is obtained by cloning Cas9cDNAs with double NLS into a pXT7 vector and linearizing with XbaI endonuclease.
Further, the amplification procedure of S2 is: 98 ℃ for 2min; the following steps were repeated for 35 cycles: 98℃15s,58℃15s,72℃20s;72 ℃ for 5min; 30min at 72 ℃.
Further, the transcription system of S3 is: template DNA10ul, 10X RNA polymerase Reaction buffer2ul,25mM rNTP Mix0.8ul,RNase Inhibitor0.5ul,T7 RNA polymerase2ul,RNase free H 2 O4.7ul。
Further, the transcription system is: 37℃for 3h.
Further, the culturing of S4 specifically comprises the following steps:
s1, culturing and hatching microinjected fertilized eggs, detecting the effectiveness of target sites by Sanger sequencing, screening out gene mutation, marking as F0 generation, and breeding to adult fish;
s2, laterally crossing the F0 adult fish with the WT to obtain an F1 generation;
s3, determining F1 generation of dpy19l1l gene knockout by using a genotype identification method;
s4, selecting F1 generations with the same mutation type from the F1 generation mutants to mate to obtain F2 generation;
s5, genotype identification is carried out on the homozygous of the dpy19l1l gene knockout in the F2 generation, namely the stable inheritance dpy-19l1l homozygous mutant zebra fish strain, namely the dpy19l1l gene deletion type zebra fish.
Compared with the prior art, the invention has the following beneficial effects:
(1) The invention can silence specific genes in the genome of an organism more efficiently and accurately, has simple manufacture and low cost, can cut a plurality of sites on a target gene at the same time, silence any number of single genes, and knock out the development malformation of dpy19l1l gene zebra fish embryos.
(2) The DPY19l1 gene deletion type zebra fish congenital spinal curvature successfully constructed by the invention provides a good zebra fish model for a spinal curvature model, and is beneficial to researching bone related diseases and screening medicines for treating or relieving bone diseases. .
Drawings
FIG. 1 is a peak plot of wild type and Dpy19l1l heterozygotes py19l1l gene sequences of example 1, wherein A is wild type, B is one heterozygote and C is another heterozygote.
FIG. 2 is an alignment of wild type and Dpy19l1l heterozygotes py19l1l gene sequences and base mutation of example 1, wherein A is wild type, B is one heterozygote and C is another heterozygote.
FIG. 3 is a comparison of wild type and dpy19l1 gene deleted zebra fish phenotypes of example 1, wherein A is wild type and B is dpy19l1 gene deleted zebra fish.
Detailed Description
The present invention will now be described in detail with reference to the drawings and specific examples, which should not be construed as limiting the invention. Unless otherwise indicated, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples are commercially available unless otherwise indicated.
Example 1
Construction method of dpy19l1l gene deletion type zebra fish
S1, inquiring a genomic DNA sequence of a dpy19l1l gene of the zebra fish on NCBI, analyzing a functional domain of the genomic DNA sequence, finding a target site of the dpy19l1l gene from an exon 1 of the dpy19l1l gene according to a CRISPR/Cas9 knockout principle, and designing a target site long primer, wherein the sequence is 5'-GGAATAACAGTGCTGATTCT-3', as shown in SEQ ID NO.1, and the sequence of the dpy19l1l target site long primer is 5'-Taatacgactcactatag GGAATAACAGTGCTGATTCT gttttagagctagaaatagc-3', as shown in SEQ ID NO. 2;
s2, obtaining an in vitro transcription template of gRNA by primer star PCR, wherein the total reaction volume is 50 μl, and the system is shown in Table 1:
TABLE 1 primer star PCR System
The gDNA Vector Template is obtained by cloning Cas9cDNAs with double NLS into a pXT7 vector and linearizing with XbaI endonuclease;
the sequence of gDNA adaptor Primer is 5'-AGCACCGACTCGGTGCCACTT-3'; as shown in SEQ ID NO. 3.
The T7 promoter and target site carried on the long primer are connected to gDNA Vector through PCR mode in vitro amplification, then DNA fragment is recovered through DNA purification technology mode, and the concentration of the DNA fragment is determined, and the fragment is the in vitro transcription template.
After the reaction system was mixed uniformly, primer star PCR was performed on a PCR apparatus for amplification reaction, and the reaction procedure was as shown in Table 2:
TABLE 2 primer star PCR amplification procedure
The final PCR product concentration was 31.8ng/ul and OD was 1.85.
S3, carrying out in-vitro transcription according to the in-vitro transcription template obtained in the step S2, wherein the total reaction volume is 10ul, and the system is as follows:
TABLE 3 in vitro transcription reaction System
After the system is added, the electrophoresis running gel is detected immediately after the synthesis in an oven at 37 ℃ for 3 hours, and the result is seen. After 1ul DNase I was added, gRNA extraction was performed for 1 h.
Extracting and purifying the successfully transcribed gRNA of S3 by a trizol method, operating according to a kit protocol, and finally adding 10ul of RNase free H 2 O。
The protocols were as follows:
a. adding 500ul of trizol and 100ul of chloroform, shaking vigorously, mixing, standing and layering for 5min.
b. 15000xg maximum rotation speed at 4 ℃ for 15min.
c. Taking the supernatant into a new EP tube, adding an equal volume of pre-cooled isopropanol at-20 ℃, vibrating for 15s, and precipitating at-20 ℃ for half an hour.
d. 15000xg maximum rotation speed at 4 ℃ for 10min.
e. Removing the supernatant, taking 1ml of 75% ethanol, and washing for 2 times to precipitate; shaking for 15s, and carrying out maximum rotation speed of 15000xg at 4 ℃ for 5min.
f. Air-dried on ice, and dissolved by adding 10ul RNase free H2O.
The purified gRNA had a concentration of 583.1ng/ul and an OD of 2.08, and was stored at-80 ℃.
S5, sucking embryos by using a suction tube within 30min after fertilization of the zebra fish, transferring the embryos to a special microinjection culture dish made of agarose, preparing a mixed solution of Cas9 protein and gRNA (ribonucleic acid) before microinjection, enabling the final concentration of the Cas9 protein to be 5 mu g/ul, enabling the final concentration of the gRNA to be 100ng/ul, and injecting 3 mu L of the mixed solution of the Cas9 protein and the gRNA into fertilized eggs in a cell stage; placing the injected fertilized eggs in fish liquid water, and incubating at 28.5 ℃;
the microinjection system is as follows:
table 4 microinjection system
The culture of the step specifically comprises the following steps:
(1) Culturing and incubating microinjected fertilized eggs, detecting the effectiveness of target sites by Sanger sequencing, screening out the gene mutation, marking the gene mutation as F0 generation, and breeding the gene mutation to adult fish;
the Sanger sequencing assay specifically comprises the following steps:
a, extracting genome of zebra fish
After 48 hours of fertilization of zebra fish embryos (48 hpf), wild-type embryos (as control) and post-injection embryos were collected separately in 1.5ml Ep tubes, 10 embryos per tube, and genomic DNA was extracted as follows: 20 μl of 50mm NaOH solution was added to the embryo-filled Ep tube, and the mixture was placed in a metal bath for cleavage at 95deg.C for 15min; after cooling at 4℃2ul Tris-HCl (ph 8.0) was added for neutralization to obtain a crude genomic DNA.
b, PCR amplifying the target sequence
After extracting the genome DNA, designing a primer sequence to amplify a target DNA fragment;
the PCR reaction system is as follows: dpy19l1l sequencing primer: an upper primer (forward primer 5'-GTCTGCGAGAGCCAAACTAC-3' shown as SEQ ID NO. 4) and a lower primer (reverse primer 5'-TTGTTTACAATCCGAGCTCC-3' shown as SEQ ID NO. 5).
c, the PCR product is sent to a Yingjun biological company for sequencing to analyze the insertion and deletion of mutation site bases, the peak diagram and the sequence given after sequencing are compared with a standard target sequence on NCBI, the mutation type of each monoclonal is analyzed according to the comparison result, the result is shown in fig. 1 and 2, the dpy19l1l heterozygote mutant sequence is two, and nonsense mutation is generated by frame shifting.
(2) Laterally crossing the F0 adult fish with the WT to obtain an F1 generation;
(3) Determining F1 generation of dpy19l1l gene knockout by using a genotyping method;
(4) F1 generation of the same mutation type is selected from the F1 generation mutants to mate to obtain F2 generation;
(5) The homozygous of the dpy19l1l gene knockout in the F2 generation is the stable inheritance dpy-19l1l homozygous mutant zebra fish strain, namely the dpy19l1l gene deletion type zebra fish, and the dpy19l1l deletion type zebra fish has a spinal curvature phenotype after screening as shown in figure 3.
Example 2
Application of dpy19l1l gene deletion type zebra fish constructed in example 1
In this example, a study of bone-related diseases was conducted on dpy19l1l gene-deleted zebra fish constructed in example 1, and a drug for treating or alleviating bone diseases was selected to alleviate the bending degree of zebra fish ridges, which was the objective drug.
While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. It is therefore intended that the following claims be interpreted as including the preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.
Sequence listing
<110> university of Jinggang mountain
<120> construction method of dpy19l1l gene deletion type zebra fish
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> zebra fish
<400> 1
ggaataacag tgctgattct 20
<210> 2
<211> 58
<212> DNA
<213> artificial sequence
<400> 2
taatacgact cactataggg aataacagtg ctgattctgt tttagagcta gaaatagc 58
<210> 3
<211> 21
<212> DNA
<213> artificial sequence
<400> 3
agcaccgact cggtgccact t 21
<210> 4
<211> 20
<212> DNA
<213> artificial sequence
<400> 4
gtctgcgaga gccaaactac 20
<210> 5
<211> 20
<212> DNA
<213> artificial sequence
<400> 5
ttgtttacaa tccgagctcc 20
Claims (6)
- The application of the dpy19l1l gene in constructing a congenital spinal curvature zebra fish model is characterized in that the construction method of the congenital spinal curvature zebra fish model comprises the following steps:s1, inquiring a genomic DNA sequence of a dpy19l1l gene of the zebra fish, analyzing a functional domain of the genomic DNA sequence, finding a target site of the dpy19l1l gene from an exon 1 of the dpy19l1l gene according to a CRISPR/Cas9 knockout principle, designing a target site long primer and a gDNA joint primer, wherein the sequence of the dpy19l1l target site long primer is 5'-Taatacgactcactatag GGAATAACAGTGCTGATTCT gttttagagctagaaatagc-3', gDNA joint primer sequence 5'-AGCACCGACTCGGTGCCACTT-3';s2, obtaining an in vitro transcription template of the gRNA by primer star PCR;the gDNAVector Template is obtained by cloning Cas9cDNAs with double NLS into a pXT7 vector and linearizing with XbaI endonuclease;the sequence of gDNAadaptor Primer is 5'-AGCACCGACTCGGTGCCACTT-3';s3, in-vitro transcription is carried out according to the gRNA in-vitro transcription template obtained in the S2, and the gRNA is extracted and purified to obtain purified gRNA;s4, microinjection of purified gRNA and Cas9 proteins into fertilized eggs of the zebra fish in a cell stage, and culturing to obtain a stable genetic dpy-19l1l homozygous mutant zebra fish strain, namely a congenital spinal curvature zebra fish model.
- 2. The use according to claim 1, wherein the S2 reaction system is: gDNA Vector Template 1. Mu.l, 1. Mu.l of target site long primer, gDNA adapter primer, 1. Mu.l of dNTP Mix, 5X Primer star buffer. Mu.l, primer star DNA polymerase. Mu.l, ddH 2 O32 μl; the gDNA Vector Template is obtained by cloning Cas9cDNAs with double NLS into a pXT7 vector and linearizing with XbaI endonuclease.
- 3. The use according to claim 2, wherein the amplification procedure of S2 is: 98 ℃ for 2min; the following steps were repeated for 35 cycles: 98℃15s,58℃15s,72℃20s; 5min at 72 ℃; 30min at 72 ℃.
- 4. The use according to claim 3, wherein the transcription system of S3 is: template DNA 100-200ng, 10X RNA polymerase Reaction buffer 1ul,25mM rNTP Mi x 1ul,RNase Inhibitor 1ul,T7 RNApolymerase 0.5ul,RNase free H 2 O was fixed to a volume of 10 ul.
- 5. The use according to claim 4, wherein the transcription conditions are: 37℃for 3h.
- 6. The use according to claim 5, wherein the cultivation of S4 comprises the steps of:s1, culturing and hatching microinjected fertilized eggs, detecting the effectiveness of target sites by Sanger sequencing, screening out gene mutation, marking as F0 generation, and breeding to adult fish;s2, laterally crossing the F0 adult fish with the WT to obtain an F1 generation;s3, determining F1 generation of dpy19l1l gene knockout by using a genotype identification method;s4, selecting F1 generations with the same mutation type from the F1 generation mutants to mate to obtain F2 generation;s5, genotype identification is carried out on the homozygote of the dpy19l1l gene knockout in the F2 generation, namely the stable inheritance dpy-19l1l homozygote mutant zebra fish strain, namely the congenital spinal curvature zebra fish model.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202010656897.9A CN111793653B (en) | 2020-07-09 | 2020-07-09 | Construction method of dpy19l1l gene deletion type zebra fish |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202010656897.9A CN111793653B (en) | 2020-07-09 | 2020-07-09 | Construction method of dpy19l1l gene deletion type zebra fish |
Publications (2)
Publication Number | Publication Date |
---|---|
CN111793653A CN111793653A (en) | 2020-10-20 |
CN111793653B true CN111793653B (en) | 2023-06-09 |
Family
ID=72810579
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202010656897.9A Active CN111793653B (en) | 2020-07-09 | 2020-07-09 | Construction method of dpy19l1l gene deletion type zebra fish |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN111793653B (en) |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN107988268A (en) * | 2017-12-18 | 2018-05-04 | 湖南师范大学 | A kind of method of gene knockout selection and breeding tcf25 Gene Deletion zebra fish |
CN109825569A (en) * | 2019-01-14 | 2019-05-31 | 阅尔基因技术(苏州)有限公司 | PCR primer group, kit, amplification system and the detection method of gene DPY19L2 exon |
CN110066805A (en) * | 2019-04-26 | 2019-07-30 | 湖南师范大学 | The method of gene knockout breeding adgrf3b Gene Deletion zebra fish |
WO2019210268A2 (en) * | 2018-04-27 | 2019-10-31 | The Broad Institute, Inc. | Sequencing-based proteomics |
-
2020
- 2020-07-09 CN CN202010656897.9A patent/CN111793653B/en active Active
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN107988268A (en) * | 2017-12-18 | 2018-05-04 | 湖南师范大学 | A kind of method of gene knockout selection and breeding tcf25 Gene Deletion zebra fish |
WO2019210268A2 (en) * | 2018-04-27 | 2019-10-31 | The Broad Institute, Inc. | Sequencing-based proteomics |
CN109825569A (en) * | 2019-01-14 | 2019-05-31 | 阅尔基因技术(苏州)有限公司 | PCR primer group, kit, amplification system and the detection method of gene DPY19L2 exon |
CN110066805A (en) * | 2019-04-26 | 2019-07-30 | 湖南师范大学 | The method of gene knockout breeding adgrf3b Gene Deletion zebra fish |
Non-Patent Citations (3)
Title |
---|
Aleksandra Shcherbakova等.Distinct C-mannosylation of netrin receptor thrombospondin type 1 repeats by mammalian DPY19L1 and DPY19L3.《PNAS》.2017,第114卷(第10期),第2574–2579页. * |
Pasquier J等.probable C-mannosyltransferase DPY19L1 [Danio rerio].GenBank数据库.2018,NP_001002868.1. * |
贾丽苑 ; 冯娟涛 ; 崔继红 ; .糖基化对斑马鱼发育及再生作用的研究进展.生物化学与生物物理进展.2017,(第10期),第95-105页. * |
Also Published As
Publication number | Publication date |
---|---|
CN111793653A (en) | 2020-10-20 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN107475300B (en) | Construction method and application of Ifit3-eKO1 gene knockout mouse animal model | |
CN110551759A (en) | Composition and method for improving recombination efficiency of transgenic cells | |
CN106282231B (en) | Construction method and application of mucopolysaccharide storage disease type II animal model | |
CN110684777B (en) | Application of isolated nucleotide sequence in construction of zebra fish with reduced intramuscular stings | |
CN112980880B (en) | Method for constructing Fzd6-Q152E fixed-point mutation mouse model based on CRISPR/Cas9 and application | |
CN111154758A (en) | Method for knocking out zebra fish slc26a4 gene | |
CN111575320B (en) | Construction method of prep gene deletion type zebra fish | |
CN110643636A (en) | Megalobrama amblycephala MSTNa & b gene knockout method and application | |
CN109280666A (en) | A kind of method of gene knockout breeding bai2 Gene Deletion zebra fish | |
CN116083492A (en) | Preparation method of csde1 gene deletion zebra fish mutant and construction method of zebra fish hematopoietic stem cell development defect model | |
CN110894510A (en) | Method for breeding Lgr6 gene-deleted zebra fish through gene knockout | |
CN108866102B (en) | Construction method of Adgrv1 gene Y6236fsX1 mutant animal model | |
CN108893495B (en) | Construction method of Pdzd7 gene mutation animal model | |
CN110066805A (en) | The method of gene knockout breeding adgrf3b Gene Deletion zebra fish | |
CN113234756A (en) | Construction method of LAMA3 gene knockout animal model based on CRISPR/Cas9 technology | |
CN112342214A (en) | sgRNA sequence for targeted knockout of channel catfish zbtb38 gene and screening method thereof | |
CN111893119A (en) | Method for obtaining SCD1 gene editing goat embryo by CRISPR/Cas9 system and microinjection | |
CN111793653B (en) | Construction method of dpy19l1l gene deletion type zebra fish | |
CN111549070A (en) | Method for editing X chromosome multi-copy gene to realize animal sex control | |
CN114480497B (en) | Construction and application method of ep400 gene knockout zebra fish heart failure model | |
CN114934073B (en) | Construction method and application of hoxa a gene knockout zebra fish mutant | |
CN110894511A (en) | Method for breeding ppm1g gene mutant zebra fish by gene editing | |
CN110669795A (en) | Technology for realizing precise fixed-point RNA shearing in fish embryo | |
CN115029352A (en) | Method for breeding adgrg1 gene-deleted zebra fish through gene knockout | |
CN109694885B (en) | Method for preparing PI3K gamma whole-body knockout mode mouse based on CRISPR/Cas9 technology, application thereof and kit |
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 |