CN107904237A - The application of the gene order and the sequence of targeting editor's rabbit H11 locus - Google Patents
The application of the gene order and the sequence of targeting editor's rabbit H11 locus Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- 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
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/10—Mammal
- A01K2227/107—Rabbit
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- C12N2810/00—Vectors comprising a targeting moiety
- C12N2810/10—Vectors comprising a non-peptidic targeting moiety
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Abstract
The present invention provides a kind of gRNA for targeting editor's rabbit H11 locus and its application, which includes the RNA fragments and DNA fragmentation of H11 gene orders on special editor's rabbit chromosome, and the nucleotide sequences of RNA fragments is following 1) or 2)；The nucleotide sequence of DNA fragmentation is following 3) or 4).The efficient specific recognition rabbit H11 locus of gRNA energy provided by the present invention, and targeting editor is carried out to the site under the mediation of Cas9 nucleases, it is supported for follow-up H11 locus gene editor rabbits and the foreign gene H11 locus site-directed integrations transgene rabbit of preparing with important technology.
Description
Technical field
The present invention relates to a kind of animal gene technical field, and in particular to a kind of can target edits rabbit H11 locus
The application of gene order and the sequence.
Background technology
Hipp11 (H11) locus, was found and further in transgenosis by Hippenmeyer et al. in 2010 first
Verified in mouse and human stem cells.It is H11 locus in mouse, between Eif4enif1 and Drg1 genes, positioned at No. 11 dyes
Near colour solid centromere.Experiment in vivo confirms to express in the integration of H11 locus and diallele, without interference with the work of mouse
Power or fertility.Compared with the Rosa26 sites in mouse, H11 locus shows high-caliber complete identical studies have shown that
The high efficiency of body transgene expression and Chromosome recombination.2015, Jin et al. successfully will be big using CRISPR/Cas9 systems
In the H11 locus of the genetic fragment insertion pig of 9kb.Also demonstrate the gene for being inserted into the site in cell, embryo and animal
It is that altimeter reaches.Tasic etc. have selected H11 locus, be inserted into integrase, they study find the site possess relative to
The integration efficiency of Rosa26 sites higher.H11 locus does not contain any promoter, therefore can start in target gene itself
Expressed after son, such as tissue-specific promoter's specific expressed transgenosis in the tissue.It is if fixed in the genome of rabbit
Position hipp11 so safely and effectively genetic modification sites, are beneficial to stablize the technical system of transgene rabbit cultivation.
Occur many genes editing technique, including Zinc finger nuclease (ZFNs), the similar effect of activating transcription factor in recent years
The CRISPR/Cas nucleic acid enzyme systems for answering factor nucleic acid enzyme (TALENs) and RNA to instruct.First two technology is by endonuclease
Catalyst structure domain is connected with DNA binding protein component, and the side of targeting DNA double chain fracture (DSB) is induced in specific gene loci
Method.In contrast, Cas9 is by Watson-Crick base pair complementarity principles, by the tiny RNA and purpose of nuclease guiding
DNA is matched.
CRISPR-Cas is a kind of microorganism adaptive immune system, the nuclease cutting DNA guided using RNA.Due to
Its efficient gene editing efficiency, CRISPR-Cas systems be applied to it is a variety of biology on, including pig, Caenorhabditis elegans,
Chicken, rat, goat, zebra fish, bacterium and plant.The research of multiple computer MSR Information systems is all shown, compared with ZFN and TALEN, is borrowed
The gene editing of CRISPR-Cas systems is helped to test the efficiency for showing higher.By means of this technology, gene functional research and
Molecular breeding etc. will all obtain swift and violent development.
Therefore, seek can efficient selectively targeted editor's H11 locus CRISPR-Cas gRNA sequences, and
Targeting editor is carried out to the site under the mediation of Cas9 nucleases, further efficiently to prepare foreign gene in rabbit H11 genes
Technical support is provided in the transgene rabbit of seat site-directed integration.
Therefore, the gRNA for rabbit H11 genes of a kind of offer, has the characteristics that specificity and high efficiency, can carry out
The site-directed integration of foreign gene, which greatly improves efficiency and security prepared by transgene rabbit.It is meanwhile provided by the invention
Encode the DNA sequence dna of the gRNA, can by build PX330 expression vectors realize in body cell, embryonated egg or individual level it is right
Rabbit H11 genes realize target gene editor, including site-directed integration, pointed decoration, rite-directed mutagenesis.Rabbit H11 genes are carried out with this
The gene order that can target editor's rabbit H11 locus of the production of the transgene rabbit of functional analysis and fixed point integration of foreign gene
And the application of the sequence is suggested.
The content of the invention
The present invention provides the gRNA that can efficiently target editor's rabbit H11 locus.The double-stranded DNA for encoding gRNA is connected into
PX330 expression vectors, the expression vector can give expression to complete gRNA and remove H11 gene DNA sequences on identification chromosome, at the same time
The carrier can also give expression to biologically active Cas9 nucleases.2 gRNA can specifically with rabbit H11 gene portions
Sub-sequence complementary pairing, mediation Cas9 nucleases carry out rabbit H11 locus specifically, efficiently to edit.
The present invention provides a kind of gRNA for targeting editor's rabbit H11 locus and its application, the gRNA sequences to include special
Edit H11 gene order RNA fragments and DNA fragmentation on rabbit chromosome, the nucleotide sequences of RNA fragments is following 1) or 2)；DNA pieces
The nucleotide sequence of section is following 3) or 4).The efficient specific recognition rabbit H11 locus of gRNA energy provided by the present invention, and
Targeting editor is carried out to the site under the mediation of Cas9 nucleases, it prepares H11 locus gene editor rabbits and external source to be follow-up
Gene H11 locus site-directed integrations transgene rabbit is supported with important technology.
The present invention provides the RNA sequence for the gRNA that can target editor's rabbit H11 locus, it is characterised in that identification chromosome
The RNA sequences of upper rabbit H11 gene orders is following 1) or 2)：
The present invention provides the DNA sequence dna for the gRNA that can target editor's rabbit H11 locus, it is characterised in that identification chromosome
The DNA sequence dnas of upper rabbit H11 gene orders is following 3) or 4)：
The present invention provides the preparation method for the DNA sequence dna that can target editor's rabbit H11 locus gRNA.Step is as follows：
1) rabbit H11 locus DNA sequences are retrieved in gene pool, select target area design gRNA sequences；
2) according to the gRNA sequences of design, corresponding DNA sequence dna is synthesized；
3) DNA sequence dna of gRNA anneal to be formed nucleotide dimer (Coligoduplex }；
4) nucleotide dimer is connected to corresponding PX330 expression vectors, so as to obtain the expression vector of gRNA；
GRNA provided by the invention for rabbit H11 genes, has the characteristics that specificity and high efficiency, can be without any
The site-directed integration of foreign gene is carried out in the case of the promoter gene and positive-negative selection marker gene of external source, which greatly improves
Efficiency and security prepared by transgene rabbit.Meanwhile the DNA sequence dna of the coding gRNA provided by the invention, structure can be passed through
PX330 expression vectors are realized carries out target gene editor in body cell, embryonated egg or individual level to rabbit H11 genes, including
Site-directed integration, pointed decoration, rite-directed mutagenesis.Turn of rabbit H11 gene function analysis and fixed point integration of foreign gene is carried out with this
The production of gene rabbit.
Brief description of the drawings
Fig. 1 is gRNA1 sequencing detection and analysis digestion carrier result figures.
Fig. 2 is gRNA2 sequencing detection and analysis digestion carrier result figures.
Embodiment
Embodiment 1, contrived experiment 1, experiment 1 are the materials that uses of preparing experiment 2, and experiment 2 is the beneficial of the verification present invention
Effect.
Test the structure of 1 PX330 expression plasmids
1st, rabbit H11 locus sequences, its nucleotide sequence are recalled in NCBI (US National Biotechnology Information center)
As shown in the sequence 5 in sequence table, CRISPR/Cas9 Photographing On-line instruments (http is utilized://
Tools.genomeenging.org gRNA) is designed.
GRNA target sites position 1 (being named as H11-gRNA1):CATCCTCTGCTGCGTTAGCAGGG, can target editor rabbit
The gRNA nucleotide sequences of H11 locus are as shown in sequence 1 in sequence table；In DNA sequence dna such as sequence table that the sequence can be encoded
Shown in sequence 3.
GRNA target sites position 2 (being named as H11-gRNA2):AGTGCTTAGACCATCCACATGGG, can target editor rabbit
The gRNA nucleotide sequences of H11 locus are as shown in sequence 2 in sequence table；In DNA sequence dna such as sequence table that the sequence can be encoded
Shown in sequence 4.
2nd, PX330 expression plasmids are built
It is mainly completed by skeleton of the mutational vector of the pX330 in the Zhang Feng laboratories of masschusetts, U.S.A science and engineering, its specific structure
It is as follows to build process：
(1) according to foregoing two target sequences, corresponding primer sequence is designed, by raw work bioengineering (Shanghai)
Limited company synthesizes, and particular sequence is shown in Table 1：
1 two gRNA target spot primer sequences of table
| Nucleotide title | Sequence (5 ' -3 ') |
| H11-gRNA1-F | CACCGCATCCTCTGCTGCGTTAGCA |
| H11-gRNA1-R | AAACTGCTAACGCAGCAGAGGATGC |
| H11-gRNA2-F | CACCGAGTGCTTAGACCATCCACAT |
| H11-gRNA2-R | AAACATGTGGATGGTCTAAGCACTC |
(2) formation of oligonucleotides dimer (oligoduplex)
The gRNA target spot primers for being melted into synthesis are diluted to 10uM respectively, are mixed respectively in following ratio
The mixture of mixing is subjected to water-bath, 95 DEG C, 5min of condition, natural slow cooling is to room temperature after water-bath.Annealing production
Thing is double chain DNA fragment, so as to obtain H11-gRNA1.
The mixture of mixing is subjected to water-bath, 95 DEG C, 5min of condition, natural slow cooling is to room temperature after water-bath.Annealing production
Thing is double chain DNA fragment, so as to obtain H11-gRNA2.
(3) restriction enzyme linearisation PX330 carriers are utilized
It is as follows in the reaction system of restriction enzyme：
37 DEG C of digestion condition, 30min.Product after digestion is analyzed into row agarose gel electrophoresis, cuts respective strap,
Utilize Ago-Gel QIAquick Gel Extraction Kit (TaKaRa, article No.：9762) PX330 linearized vectors are recycled.
(4) oligonucleotides dimer is inserted respectively into carrier
It is as follows in reaction system：
Reaction condition be 16 DEG C, 12 it is small when, by connection product (PX330-H11-sRNAg1) be used for subsequent transformation experiment.
Reaction condition be 16 DEG C, 12 it is small when, by connection product (PX330-H11-sRNAg2) be used for subsequent transformation experiment.
(5) convert
In defrosting DH5 α competent cells on ice, connection product that step (4) is obtained (PX330-H11-sRNAg1,
PX330-H11-sRNAg2) 5uL is separately added into the DH5 α competent cells of defrosting, and after mixing, ice bath is after 30 minutes, 42 DEG C of heat
Hit 90 seconds, stand 5 minutes on ice, add the LB fluid nutrient mediums of 1mL non-resistants, 37 DEG C, 200 revs/min, cultivate 60 minutes,
Bacterium solution 8000rpm, centrifuges 2min, removes supernatant, and remaining LB nutrient solutions have hanged bacterial precipitation, and bacterium solution is all coated on containing ammonia after mixing
In the screening flat board of benzyl, 37 DEG C of culture 15h.
(6) verify
Respectively choose 3 bacterial clumps and shake bacterium, extraction Plasmid DNA is sequenced.Sequencing primer is：ATGGACTATCATATGCTT
ACCGTA, obtains the sequencing result of PX330-H11-sRNAg1 and PX330-H11-gRNA2, and above-mentioned sequencing result is shown in sequence table
Sequence 6 and sequence 7. should the result shows that, by aforesaid operations can smoothly will encode gRNA DNA sequence dna (i.e. target site 1 and target
The sequence in site 2) it is inserted into PX330 carrier frameworks.
Test the efficiency verification of 2 gRNA expression plasmids
1st, rabbit fetal fibroblast is separated
The New Zealand mother rabbit of selection pregnancy 15-20d or so is put to death, and is taken out uterus, is placed in culture dish.Taken under aseptic condition
Go out fetus, be placed in clean culture dish, aseptically, cut off head, tail, four limbs with sterile scissors and remove internal organs,
Fetal skin is removed with tweezers, with containing dual anti-sterile phosphate buffer (Gbico, article No.：10010-023) by above-mentioned pre- place
The portion of tissue of reason rinses 4 times, and partial skin tissue is cut into the small of 1mm3 or so with eye scissors in sterile culture dish
Block, is uniformly planted by 0.5cm spacing, treats the adherent completion of all skin histologies, by culture dish is placed in 37 DEG C, 5.0%CO2 satisfies
After adhere-wall culture 2-4h in humidified incubator, complete culture solution is added, 37 DEG C is placed in, is cultivated in 5%CO2 incubators.First three
It adds nutrient solution height is advisable in tissue block 1/3, avoids tissue block from suspending as far as possible.After three days, add nutrient solution and highly flood
Tissue block, continues to cultivate, while the cell growth status around tissues observed block.Treat that cell confluency is in blocks, remove tissue block.Obtain
Obtain primary fibroblast.
2nd, consideration convey contaminates
The recombinant plasmid PX330-H11-sg1 obtained by embodiment 1 and each 4ug of PX330-H11-sg2 are passed through into consideration convey
The mode of dye transfects rabbit fibroblast, obtains recombinant cell.Utilize zooblast consideration convey instrument (Amaxa, model：AAD-
1001S) and transfection reagent box (Amaxa, article No.：VPI-1002) transfected.The detailed step of transfection is as follows：When in 6 orifice plates
When the cell growth in every hole is to logarithmic phase, phosphate buffer (Gbico, article No. are used：10010-023) clean 2 times, use
0.1% trypsase (Gbico, article No.：Attached cell 610-5300AG) is digested, ensures that culture dish bottom is all covered, disappears
Change cell 1 minute or so.Micro- Microscopic observation cell, when the cell being digested nearly all becomes round, uses hyclone
(Gbico, article No.：Digestion 16000-044) is terminated, adds after complete culture solution is blown and beaten repeatedly and moves in the centrifuge tube of 1.5mL,
1000rpm centrifuges 8min；Supernatant liquid is discarded, nutrient solution is added and cell is resuspended.Cell is counted using red blood cell count(RBC) plate
Number.Collect 2 × 105~1 × 106A cell, removes supernatant, be separately added into 5 μ g except endotoxic PX330-H11-sRNAg1 plasmids with
PX330-H11-gRNA2 plasmids, add transfection reagent, use program T-016 transfectional cells.
3rd, the extraction of DNA
The cell that step 2 is obtained is cultivated, 37 DEG C of cultures of condition of culture, when co-cultivation 48 is small, then with 0.1% pancreas
Protease collects cell.Detailed step is as follows：With 0.1% trypsase (Gbico, article No.：610-5300AG) digest adherent thin
Born of the same parents 1-2 minutes, trypsase dosage, which is subject to, covers whole cells.Add hyclone (Gbico, article No.：16000-044) eventually
Only cell dissociation, adds after complete culture solution is blown and beaten repeatedly and moves in the centrifuge tube of 1.5mL, 1000rpm centrifugations 8min；Discard
Layer liquid, adds 200 microlitres of cell pyrolysis liquid GA (component in TIANGEN companies DNA extraction kit DP304).With reference to examination
Agent box specification step extracts 3 kinds of cell STb genes respectively.
4th, PCR amplification
The genomic DNA of step cell in extraction, as pcr template, PCR amplification is carried out with primer H11-F and H11-R,
The pcr amplification product for recycling about 616bp (is obtained using the DNA for transfecting recombinant plasmid PX330-H11-gRNA1 cell extractions as template
PCR product be PCR-1, produced using transfecting the DNA of recombinant plasmid PX330-H11-gRNA2 cell extractions as the PCR that template obtains
Thing is PCR-2, using the DNA of the cell extraction of untransfected recombinant plasmid PX330-H11-gRNA1 or PX330-H11-gRNA2 as mould
The PCR product that plate obtains is PCR-3).
H11-F:5’-GCCCTGATTGCTTCTAAACT-3’
H11-R:5’-GTTACCTGGCTCCTCGTGTT-3’
5th, T7 endonucleases I digestion verifications knock out efficiency
It is as follows in reaction system：
Said mixture is mixed, reaction condition is：After 95 DEG C of 3min, 95 DEG C of slow coolings are to 25 DEG C, then at 16 DEG C
Lower processing 5min, finally obtains annealed product -1.
Said mixture is mixed, reaction condition is：After 95 DEG C of 3min, 95 DEG C of slow coolings are to 25 DEG C, then at 16 DEG C
Lower processing 5min, finally obtains annealed product -2.
Said mixture is mixed, reaction condition is：After 95 DEG C of 3min, 95 DEG C of slow coolings are to 25 DEG C, then at 16 DEG C
Lower processing 5min, finally obtains annealed product -3.
Above-mentioned annealed product is separately added into 0.5uL T7E1 enzymes, 37 DEG C of 30min of reaction condition.Reaction product is carried out
2% agarose gel electrophoresis, uses full automatic gel imaging system (BIO-RAD, model：GelDoc EZ) detection and analysis enzyme
Cut result.Experimental result shows that the gene editing efficiency of PX330-H11-sg1 is the gene editing of 23%, PX330-H11-sg2
Efficiency is 41%.
The studies above is the result shows that the two gRNA can efficiently identify rabbit H11 locus, and in Jie of Cas9 nucleases
Lead and lower targeting cutting and editor is carried out to H11 genes.These results are subsequently efficiently to prepare foreign gene in rabbit H11 locus
Technical support is provided in the transgene rabbit of site-directed integration.
<110>Shihezi Univ
<120>The application of the gene order and the sequence of targeting editor's rabbit H11 locus
<130>
<160> 10
<170> PatentIn version 3.3
<210> 1
<211> 20
<212> RNA
<213>Artificial sequence
<400> 1
ugcuaacgcagcagaggaug 20
<210> 2
<211> 20
<212> RNA
<213>Artificial sequence
<400> 2
auguggauggucuaagcacu 20
<210> 3
<211> 20
<212> DNA
<213>Artificial sequence
<400> 3
catcctctgctgcgttagca 20
<210> 4
<211> 20
<212> DNA
<213>Artificial sequence
<400> 4
agtgcttagaccatccacat 20
<210> 5
<211> 68
<212> DNA
<213>Artificial sequence
<400> 5
ccacttgtga accagctccc tgctaacgca gcagaggatg gtccaagtgc ttagaccatc 60
cacatggg 68
<210> 6
<211> 480
<212> DNA
<213>Artificial sequence
<400> 6
tttcttggct ttatatatct tgtggaagga cgaaacaccg catcctctgc tgcgttagca 60
gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt 120
ggcaccgagt cggtgctttt ttgttttaga gctagaaata gcaagttaaa ataaggctag 180
tccgttttta gcgcgtgcgc caattctgca gacaaatggc tctagaggta cccgttacat 240
aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa 300
tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg 360
cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg 420
acggtaaatg gcccgcctgg cattgtgccc agtacatgac cttatgggac tttcctactt 480
<210> 7
<211> 480
<212> DNA
<213>Artificial sequence
<400> 7
tttcttggct ttatatatct tgtggaagga cgaaacaccg agtgcttaga ccatccacat 60
gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt 120
ggcaccgagt cggtgctttt ttgttttaga gctagaaata gcaagttaaa ataaggctag 180
tccgttttta gcgcgtgcgc caattctgca gacaaatggc tctagaggta cccgttacat 240
aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa 300
tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg 360
cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg 420
acggtaaatg gcccgcctgg cattgtgccc agtacatgac cttatgggac tttcctactt 480
<210> 8
<211> 24
<212> DNA
<213>Artificial sequence
<400> 8
atggactatcatatgcttaccgta 24
<210> 9
<211> 20
<212> DNA
<213>Artificial sequence
<400> 9
gccctgattgcttctaaact 20
<210> 10
<211> 20
<212> DNA
<213>Artificial sequence
<400> 10
gttacctggctcctcgtgtt 20
Claims (5)
- A kind of 1. RNA sequence for the gRNA that can target editor's rabbit H11 locus, it is characterised in that rabbit H11 bases on identification chromosome Because the RNA sequence of sequence is following 1) or 2).
- 2. the DNA sequence dna of the gRNA of editor's rabbit H11 locus can be targeted, it is characterised in that rabbit H11 gene sequences on identification chromosome The DNA sequence dnas of row is following 3) or 4).
- 3. the preparation method of the DNA sequence dna according to claim 1 or 2 that editor's rabbit H11 locus gRNA can be targeted.
- 4. applications of the special gRNA in specific recognition and targeting editor's rabbit H11 genes described in claim 1.
- 5. the special gRNA described in claim 1 is in structure foreign gene in the transgene rabbit of rabbit H11 locus site-directed integrations Application.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104531685A (en) * | 2014-11-27 | 2015-04-22 | 中国农业科学院北京畜牧兽医研究所 | sgRNA specifically recognizing pig H11 site, and coding DNA and application of sgRNA |
| CN104560995A (en) * | 2014-11-27 | 2015-04-29 | 中国农业科学院北京畜牧兽医研究所 | SgRNA (small guide ribonucleic acid) pair for specifically identifying porcine H11 locus, and coding DNA (deoxyribonucleic acid) and application thereof |
-
2017
- 2017-11-09 CN CN201711095596.8A patent/CN107904237A/en not_active Withdrawn
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104531685A (en) * | 2014-11-27 | 2015-04-22 | 中国农业科学院北京畜牧兽医研究所 | sgRNA specifically recognizing pig H11 site, and coding DNA and application of sgRNA |
| CN104560995A (en) * | 2014-11-27 | 2015-04-29 | 中国农业科学院北京畜牧兽医研究所 | SgRNA (small guide ribonucleic acid) pair for specifically identifying porcine H11 locus, and coding DNA (deoxyribonucleic acid) and application thereof |
Non-Patent Citations (4)
| Title |
|---|
| JINXUE RUAN ET AL: "Highly efficient CRISPR/Cas9-mediated transgene knockin at the H11 locus in pigs", 《SCIENTIFIC REPORTS》 * |
| 吴曦 等: "高效切割Hippl1敲入位点的sgRNA设计及活性验证", 《实验动物科学》 * |
| 尤双: "利用CRISPR/Cas9系统建立兔基因编辑技术的初步研究", 《中国优秀硕士学位论文全文数据库（电子期刊）基础科学辑》 * |
| 阮进学: "利用基因组编辑技术针对猪H11位点的高效定点整合系统的研究", 《中国博士学位论文全文数据库（电子期刊）基础科学辑》 * |
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