CN109666646A - A kind of preparation and application of the Pig embryos fibroblast of CD163 gene editing - Google Patents
A kind of preparation and application of the Pig embryos fibroblast of CD163 gene editing Download PDFInfo
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
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- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
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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
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- C12N2810/00—Vectors comprising a targeting moiety
- C12N2810/10—Vectors comprising a non-peptidic targeting moiety
Abstract
The present invention relates to the technical fields of cytogene engineering can reduce the carrying of foreign aid's inhereditary material more particularly to the preparation and application of a kind of Pig embryos fibroblast of CD163 gene editing, and can reduce other biological afunction；The following steps are included: the expression of (1) Cas9 albumen；(2) purifying of Cas9 albumen；(3) design of sgRNA boot sequence；(4) it is transcribed in vitro；(5) Pig embryos fibroblast is Nuclear transformation；(6) cell dilutes；(7) separate screening；(8) expand culture；(9) identification is collected, cell line of the invention can be used as the donor of body-cell neucleus transplanting, the preparation for transgene pig.
Description
Technical field
The present invention relates to the technical fields of cytogene engineering, more particularly to a kind of Pig embryos of CD163 gene editing
The preparation and application of fibroblast.
Background technique
Pig breeding and respiratory disorder syndrome, also known as blue otopathy, swine fever epidemic disease etc., are that a kind of pig developed rapidly is common
Disease, the normal easy infection of piglet will lead to respiratory system collapse and final dead, and the sow infection of pregnancy can then lose cub,
The propagation of this severe disease can be prevented using the method for gene editing, existing treatment method makes to utilize CRISPR-Cas9
The plasmid vector system of genome editor knocks out CD163 whole gene, obtains the Pig embryos fibroblast of CD163 gene knockout
System, the cell line that existing method is built up may carry exogenous genetic material, and knock out CD163 whole gene and will lead to this
The other biological function missings of gene.
Summary of the invention
In order to solve the above technical problems, the present invention provides a kind of carrying that can reduce foreign aid's inhereditary material, and can be with
Reduce the preparation and application of the Pig embryos fibroblast of the CD163 gene editing of other biological afunction.
A kind of preparation of the Pig embryos fibroblast of CD163 gene editing of the invention, method include following step
It is rapid:
(1) it the expression of Cas9 albumen: by Cas9 gene cloning to pET28-b (+) carrier, is expressed in E. coliRosetta
Cas9 recombinant protein；
(2) it the purifying of Cas9 albumen: is separated using Ni Sepharose High performance with Qsepharose, in pH
For 7.5 20mM HEPES, 500mM KCl, 1mM TCEP, 10% glycerol buffer in dialyse, use Ultracel
The concentration of 100K pillar, obtains the Cas9 recombinant protein of purifying；
(3) design of sgRNA boot sequence: searching CD163 gene coded sequence in the database of Ensembl, chooses CD163
The 7th exon of the extracellular 5th structural domain SRCR5 of receptor, and sgRNA boot sequence is designed in downstream on it；
(4) it is transcribed in vitro: by the pDR274 carrier of sgRNA boot sequence insertion DraI digestion.Use MEGAcriptTM T7 High
Yield transcription Kit is transcribed in vitro, the sgRNA of acquisition, and through MEGAclearTM Kit
Purification of Transcription Reactions purifying；
(5) Pig embryos fibroblast is Nuclear transformation: mixing 90-110pmol Cas recombinant protein and 110-130pmol after purification
SgRNA is placed at room temperature for 10 minutes, is added to and has been pre-mixed 20 μ L Amaxa P3 Primary Cell 4D-
The 2 × 10 of the Nuclear transformation solution of Nucleofector Kit5In fibroblast, with program CM-138 in Amaxa 4D-
It is carried out in Nucleofector nuclear transformed；
(6) cell dilutes: the cell after conversion being diluted to 7-8 cell/mL with culture medium；
(7) separate screening: the cell after taking 200 μ L to dilute respectively is added in each hole in 96- orifice plate, makes the cell in each hole
Number is 1 to 2, and is screened under the microscope, and the hole containing individual cells is filtered out；
(8) expand culture: after there are also individual cells hole in it is unicellular grow up to cell clone after, move on in 12- orifice plate and expanded
Big culture；
(9) it collects identification: after the completion of expanding culture, collecting cell, identify gene knockout situation, obtain the bis- equipotential bases of CD163
Because of the Pig embryos fibroblast of knockout.
The preparation of the Pig embryos fibroblast of a kind of CD163 gene editing of the invention, in the step (1)
Cas9 recombinant protein contains His, FLAG label and nuclear localisation signal, His the and FLAG label is located at Cas9 albumen
Aminoterminal, the nuclear localisation signal are located at the both ends of Cas9 albumen.
A kind of application of the Pig embryos fibroblast of CD163 gene editing of the invention, can be used as somatic cell nuclear
The donor of transplanting, the preparation for transgene pig.
Compared with prior art the invention has the benefit that
1, the Cas9 recombinant protein purified can be assembled into sgRNA-Cas9 nucleic acid-protein from different sgRNA, can be used for not jljl
The CRISPR-Cas9 genome editor of kind；
2, reached by introducing the nucleic acid-protein complex that sgRNA and Cas9 recombinant protein forms to Pig embryos fibroblast
The purpose of CD163 gene knockout, can be improved gene knockout efficiency and specificity.CRISPR-Cas9 gene editing technology can be not
Gene modification is carried out in the case where introducing any foreign gene, the Knockout cells system of acquisition can be used as body-cell neucleus transplanting
Donor, the preparation etc. for transgene pig；
3, porcine reproductive and respiratory syndrome is virosis, clinically without specific medicament, is mainly connect to the prevention and control of the disease at present
Kind inactivated vaccine/Attenuate vaccine.The present invention knocks out the virus and disseminates relevant host receptor gene, cuts off the approach of infection, acquisition
Knockout cells system can infect from the disease.This opens new thinking for PRRSV isolation, uses such genome editor
Technology can reduce heavy economic losses caused by PRRS in agricultural production.
Specific embodiment
With reference to embodiment, the embodiment of the present invention is furthur described in detail.Following embodiment is used for
Illustrate the present invention, but is not intended to limit the scope of the invention.
Embodiment 1
The expression and purifying of Cas9 albumen:
By the plasmid Transformed E .coli Rosetta containing Cas9 gene, picking monoclonal is incubated overnight.Bacterium is by 1:100's overnight
Ratio is transferred in big bottle LB culture medium, 37 DEG C of 200rpm cultures to OD600When=0.6, IPTG is added to final concentration of
0.5mM, 22 DEG C of 160 rpm receive bacterium after continuing culture 16 hours and extract albumen.Following steps carry out at low temperature: receiving bacterium
6000rpm, 15min centrifugation, remove supernatant, by precipitating molecular sieve buffer (20mM Tris, 50mM NaCl, pH 7.5,1
MM TCEP) it suspends, it is collected in bottle, ultrasound cracking on ice (ultrasonic 6s, interval 12s, 120 times, 300W or so).
15000rpm, 20min centrifugation, removal precipitating, by 0.22 μm of filter filtration sterilization of supernatant.Supernatant Ni Sepharose
High performance(GE) separation, obtained eluent is separated again with Qsepharose(Sigma), then in 20mM
HEPES(pH7.5), dialyse in the buffer of 500mM KCl, 1mM TCEP, 10%glycerol, with Ultracel 100K pillar
(Millipore) it is concentrated, obtains the Cas9 albumen of purifying.
As a result: obtaining the Cas9 albumen of high-purity high-concentration.
The design and in-vitro transcription of sgRNA boot sequence:
In Ensembl(www.ensembl.org) database in search CD163 gene coded sequence, choose CD163 receptor born of the same parents
The exon 7 upstream and downstream of outer 5th structural domain SRCR5 designs sgRNA boot sequence.SgRNA boot sequence is inserted into DraI enzyme
The pDR274 carrier cut.Use MEGAcriptTMT7 High Yield transcription Kit (invitrogen) is carried out
It is transcribed in vitro, the sgRNA of acquisition is through MEGAclearTM Kit Purification of Transcription Reactions
(invitrogen) it purifies.
As a result: obtaining the sgRNA of in-vitro transcription.
SgRNA-Cas9 nucleic acid-protein In vitro digestion:
Pair of primers CD163 Primer F is designed in the exon 7 upstream and downstream of the extracellular 5th structural domain SRCR5 of CD163 receptor
With CD163 Primer R, Pig embryos fibroblast genomic DNA is extracted, with primer CD163 Primer F and CD163
Primer R expands target fragment (1001bp), and PCR product is used as target DNA after purification.It is carried out according to following system anti-
It answers: 60ng target DNA, 500 ng Cas9,100 ng sgRNA, 0.3 μ L 10mg/mL BSA, 3 μ L 10xNEB
Buffer3 adds DEPC-H2O to 30 μ L of total volume.37℃ 1h；80℃ 2min.SgRNA-Cas9 nucleic acid is detected through 1.5% glue
Albumen In vitro digestion situation.
As a result: the purpose band cut is consistent with expected size, demonstrates the work of the high-purity C as9 albumen of acquisition
Property.
Pig embryos fibroblast is Nuclear transformation:
90pmol Cas9 albumen and 110pmol sgRNA are mixed, 10min is placed at room temperature for, is added to and has been pre-mixed 20 μ L
The 2 × 10 of the Nuclear transformation solution of Amaxa P3 Primary Cell 4D-Nucleofector Kit5In fibroblast, use
Program CM-138 carries out nuclear transformed in Amaxa 4D-Nucleofector.
Cell dilution clone:
It is diluted to 7-8 cell/mL by cell is transfected with culture medium, 200 μ L is taken to be added in each hole in 96- orifice plate, so that
The cell number in each hole is 1 to 2；Under the microscope, confirm the hole containing individual cells.After it is unicellular grow up to cell mass after, move
Make to expand culture into 12- orifice plate, collect cell, identifies gene knockout situation.
As a result: obtaining multiple monoclonal cell systems.
The case where CD163 gene knockout, is detected:
The monoclonal cell for extracting screening rolls into a ball genomic DNA, is expanded with primer CD163 Primer F and CD163 Primer R
It detects and whether there is about 500bp segment in genome, carrier T is connected after target fragment is recycled, extract plasmid and company is sent to be sequenced.
As a result: 7 sequence of monoclonal cell system genomic deletion CD163 gene extron of acquisition.
Embodiment 2
The expression and purifying of Cas9 albumen:
By the plasmid Transformed E .coli Rosetta containing Cas9 gene, picking monoclonal is incubated overnight.Bacterium is by 1:100's overnight
Ratio is transferred in big bottle LB culture medium, 37 DEG C of 200rpm cultures to OD600When=0.6, IPTG is added to final concentration of
0.5mM, 22 DEG C of 160 rpm receive bacterium after continuing culture 16 hours and extract albumen.Following steps carry out at low temperature: receiving bacterium
6000rpm, 15min centrifugation, remove supernatant, by precipitating molecular sieve buffer (20mM Tris, 50mM NaCl, pH 7.5,1
MM TCEP) it suspends, it is collected in bottle, ultrasound cracking on ice (ultrasonic 6s, interval 12s, 120 times, 300W or so).
15000rpm, 20min centrifugation, removal precipitating, by 0.22 μm of filter filtration sterilization of supernatant.Supernatant Ni Sepharose
High performance(GE) separation, obtained eluent is separated again with Qsepharose(Sigma), then in 20mM
HEPES(pH7.5), dialyse in the buffer of 500mM KCl, 1mM TCEP, 10%glycerol, with Ultracel 100K pillar
(Millipore) it is concentrated, obtains the Cas9 albumen of purifying.
As a result: obtaining the Cas9 albumen of high-purity high-concentration.
The design and in-vitro transcription of sgRNA boot sequence:
In Ensembl(www.ensembl.org) database in search CD163 gene coded sequence, choose CD163 receptor born of the same parents
The exon 7 upstream and downstream of outer 5th structural domain SRCR5 designs sgRNA boot sequence.SgRNA boot sequence is inserted into DraI enzyme
The pDR274 carrier cut.Use MEGAcriptTMT7 High Yield transcription Kit (invitrogen) is carried out
It is transcribed in vitro, the sgRNA of acquisition is through MEGAclearTM Kit Purification of Transcription Reactions
(invitrogen) it purifies.
As a result: obtaining the sgRNA of in-vitro transcription.
SgRNA-Cas9 nucleic acid-protein In vitro digestion:
Pair of primers CD163 Primer F is designed in the exon 7 upstream and downstream of the extracellular 5th structural domain SRCR5 of CD163 receptor
With CD163 Primer R, Pig embryos fibroblast genomic DNA is extracted, with primer CD163 Primer F and CD163
Primer R expands target fragment (1001bp), and PCR product is used as target DNA after purification.It is carried out according to following system anti-
It answers: 60ng target DNA, 500 ng Cas9,100 ng sgRNA, 0.3 μ L 10mg/mL BSA, 3 μ L 10xNEB
Buffer3 adds DEPC-H2O to 30 μ L of total volume.37℃ 1h；80℃ 2min.SgRNA-Cas9 nucleic acid is detected through 1.5% glue
Albumen In vitro digestion situation.
As a result: the purpose band cut is consistent with expected size, demonstrates the work of the high-purity C as9 albumen of acquisition
Property.
Pig embryos fibroblast is Nuclear transformation:
100pmol Cas9 albumen and 120pmol sgRNA are mixed, 10min is placed at room temperature for, is added to and has been pre-mixed 20 μ L
The 2 × 10 of the Nuclear transformation solution of Amaxa P3 Primary Cell 4D-Nucleofector Kit5In fibroblast, use
Program CM-138 carries out nuclear transformed in Amaxa 4D-Nucleofector.
Cell dilution clone:
It is diluted to 7-8 cell/mL by cell is transfected with culture medium, 200 μ L is taken to be added in each hole in 96- orifice plate, so that
The cell number in each hole is 1 to 2；Under the microscope, confirm the hole containing individual cells.After it is unicellular grow up to cell mass after, move
Make to expand culture into 12- orifice plate, collect cell, identifies gene knockout situation.
As a result: obtaining multiple monoclonal cell systems.
The case where CD163 gene knockout, is detected:
The monoclonal cell for extracting screening rolls into a ball genomic DNA, is expanded with primer CD163 Primer F and CD163 Primer R
It detects and whether there is about 500bp segment in genome, carrier T is connected after target fragment is recycled, extract plasmid and company is sent to be sequenced.
As a result: 7 sequence of monoclonal cell system genomic deletion CD163 gene extron of acquisition.
Embodiment 3
The expression and purifying of Cas9 albumen:
By the plasmid Transformed E .coli Rosetta containing Cas9 gene, picking monoclonal is incubated overnight.Bacterium is by 1:100's overnight
Ratio is transferred in big bottle LB culture medium, 37 DEG C of 200rpm cultures to OD600When=0.6, IPTG is added to final concentration of
0.5mM, 22 DEG C of 160 rpm receive bacterium after continuing culture 16 hours and extract albumen.Following steps carry out at low temperature: receiving bacterium
6000rpm, 15min centrifugation, remove supernatant, by precipitating molecular sieve buffer (20mM Tris, 50mM NaCl, pH 7.5,1
MM TCEP) it suspends, it is collected in bottle, ultrasound cracking on ice (ultrasonic 6s, interval 12s, 120 times, 300W or so).
15000rpm, 20min centrifugation, removal precipitating, by 0.22 μm of filter filtration sterilization of supernatant.Supernatant Ni Sepharose
High performance(GE) separation, obtained eluent is separated again with Qsepharose(Sigma), then in 20mM
HEPES(pH7.5), dialyse in the buffer of 500mM KCl, 1mM TCEP, 10%glycerol, with Ultracel 100K pillar
(Millipore) it is concentrated, obtains the Cas9 albumen of purifying.
As a result: obtaining the Cas9 albumen of high-purity high-concentration.
The design and in-vitro transcription of sgRNA boot sequence:
In Ensembl(www.ensembl.org) database in search CD163 gene coded sequence, choose CD163 receptor born of the same parents
The exon 7 upstream and downstream of outer 5th structural domain SRCR5 designs sgRNA boot sequence.SgRNA boot sequence is inserted into DraI enzyme
The pDR274 carrier cut.Use MEGAcriptTMT7 High Yield transcription Kit (invitrogen) is carried out
It is transcribed in vitro, the sgRNA of acquisition is through MEGAclearTM Kit Purification of Transcription Reactions
(invitrogen) it purifies.
As a result: obtaining the sgRNA of in-vitro transcription.
SgRNA-Cas9 nucleic acid-protein In vitro digestion:
Pair of primers CD163 Primer F is designed in the exon 7 upstream and downstream of the extracellular 5th structural domain SRCR5 of CD163 receptor
With CD163 Primer R, Pig embryos fibroblast genomic DNA is extracted, with primer CD163 Primer F and CD163
Primer R expands target fragment (1001bp), and PCR product is used as target DNA after purification.It is carried out according to following system anti-
It answers: 60ng target DNA, 500 ng Cas9,100 ng sgRNA, 0.3 μ L 10mg/mL BSA, 3 μ L 10xNEB
Buffer3 adds DEPC-H2O to 30 μ L of total volume.37℃ 1h；80℃ 2min.SgRNA-Cas9 nucleic acid is detected through 1.5% glue
Albumen In vitro digestion situation.
As a result: the purpose band cut is consistent with expected size, demonstrates the work of the high-purity C as9 albumen of acquisition
Property.
Pig embryos fibroblast is Nuclear transformation:
110pmol Cas9 albumen and 130pmol sgRNA are mixed, 10min is placed at room temperature for, is added to and has been pre-mixed 20 μ L
The 2 × 10 of the Nuclear transformation solution of Amaxa P3 Primary Cell 4D-Nucleofector Kit5In fibroblast, use
Program CM-138 carries out nuclear transformed in Amaxa 4D-Nucleofector.
Cell dilution clone:
It is diluted to 7-8 cell/mL by cell is transfected with culture medium, 200 μ L is taken to be added in each hole in 96- orifice plate, so that
The cell number in each hole is 1 to 2；Under the microscope, confirm the hole containing individual cells.After it is unicellular grow up to cell mass after, move
Make to expand culture into 12- orifice plate, collect cell, identifies gene knockout situation.
As a result: obtaining multiple monoclonal cell systems.
The case where CD163 gene knockout, is detected:
The monoclonal cell for extracting screening rolls into a ball genomic DNA, is expanded with primer CD163 Primer F and CD163 Primer R
It detects and whether there is about 500bp segment in genome, carrier T is connected after target fragment is recycled, extract plasmid and company is sent to be sequenced.
As a result: 7 sequence of monoclonal cell system genomic deletion CD163 gene extron of acquisition.
The present invention is converted to Pig embryos fibroblast by electric shocking method and introduces the nucleic acid egg that sgRNA and Cas9 albumen forms
White complex achievees the purpose that CD163 gene knockout, and gene knockout efficiency and specificity, the clpp gene that the present invention obtains can be improved
Except cell line is free of any foreign gene, the donor of body-cell neucleus transplanting can be used as, the preparation etc. for transgene pig.
The present invention is sgRNA-Cas9 nucleic acid-protein (RNP) the conversion skill using CRISPR-Cas9 genome editor's component
Art knocks out CD163 receptor extracellular domain SRCR5, that is, the 7th exon of CD163 gene is knocked out rather than whole gene, by Dan Ke
Grand screening obtains the Pig embryos fibroblast of CD163 diallele editor.The cell line can be used as body-cell neucleus transplanting
Donor, the preparation for transgene pig；Since its offspring is free of any exogenous DNA, group expanding is expected as new kind later
Or strain, it is directly used in the cultivation and pig production of breed system；Pig can be increased after CD163 gene editing to resist blue otopathy,
Huge economic benefit is generated to pig production.
Wherein:
E.coliRosetta: competent cell；
Ni Sepharose High performance: high-performance Ni agarose；
Qsepharose: quinalphos；
HEPES: 4- hydroxyethyl piperazineethanesulfonic acid；
TCEP: trichloroethyl phosphate；
Glycerol: glycerol, glycerine；
Ultracel: supercapacitor；
Ensembl: genomic sequence data library；
MEGAcriptTMT7 High Yield transcription Kit:T7 high yield transcript reagent box；
MEGAclearTMKit Purification of Transcription Reactions: for the pure of responsive transcription
Change kit
Amaxa P3 Primary Cell 4D-Nucleofector Kit:P3 primary cell 4D is nucleated kit.
Present invention cell line obtained can be used as the donor for cultivating the body-cell neucleus transplanting of anti-pig blue-ear disease pig, this feature
CD163 cell line protected as a whole, the later period of the invention in research and production application also in protection scope
Within, the present inventor possesses ownership to it.Above content is merely illustrative of the technical solution of the present invention and specific implementation method,
Rather than limiting the scope of the invention.On the basis of the present invention, technological improvement and retouching that those skilled in the art are done,
It is accordingly to be regarded as protection scope of the present invention.
Claims (3)
1. a kind of preparation of the Pig embryos fibroblast of CD163 gene editing, which comprises the following steps:
(1) it the expression of Cas9 albumen: by Cas9 gene cloning to pET28-b (+) carrier, is expressed in E.coliRosetta
Cas9 recombinant protein；
(2) it the purifying of Cas9 albumen: is separated using Ni Sepharose High performance with Qsepharose, in pH
For 7.5 20mM HEPES, 500mM KCl, 1mM TCEP, 10%glycerol buffer in dialyse, use Ultracel
The concentration of 100K pillar, obtains the Cas9 recombinant protein of purifying；
(3) design of sgRNA boot sequence: searching CD163 gene coded sequence in the database of Ensembl, chooses CD163
The 7th exon of the extracellular 5th structural domain SRCR5 of receptor, and sgRNA boot sequence is designed in downstream on it；
(4) it is transcribed in vitro: by the pDR274 carrier of sgRNA boot sequence insertion DraI digestion, using MEGAcriptTM T7 High
Yield transcription Kit is transcribed in vitro, the sgRNA of acquisition, and through MEGAclearTM Kit
Purification of Transcription Reactions purifying；
(5) Pig embryos fibroblast is Nuclear transformation: mixing 90-110pmol Cas recombinant protein and 110-130pmol after purification
SgRNA is placed at room temperature for 10 minutes, is added to and has been pre-mixed 20 μ L Amaxa P3 Primary Cell 4D-
The 2 × 10 of the Nuclear transformation solution of Nucleofector Kit5In fibroblast, with program CM-138 in Amaxa 4D-
It is carried out in Nucleofector nuclear transformed；
(6) cell dilutes: the cell after conversion being diluted to 7-8 cell/mL with culture medium；
(7) separate screening: the cell after taking 200 μ L to dilute respectively is added in each hole in 96- orifice plate, makes the cell in each hole
Number is 1 to 2, and is screened under the microscope, and the hole containing individual cells is filtered out；
(8) expand culture: after there are also individual cells hole in it is unicellular grow up to cell clone after, move on in 12- orifice plate and expanded
Big culture；
(9) it collects identification: after the completion of expanding culture, collecting cell, identify gene knockout situation, obtain the bis- equipotential bases of CD163
Because of the Pig embryos fibroblast of knockout.
2. a kind of preparation of the Pig embryos fibroblast of CD163 gene editing as described in claim 1, feature exist
In the Cas9 recombinant protein in the step (1) contains His, FLAG label and nuclear localisation signal, the His and FLAG
Label is located at the aminoterminal of Cas9 albumen, and the nuclear localisation signal is located at the both ends of Cas9 albumen.
3. a kind of application of the Pig embryos fibroblast of CD163 gene editing, which is characterized in that can be used as somatic cell nuclear
The donor of transplanting, the preparation for transgene pig.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108753832A (en) * | 2018-04-20 | 2018-11-06 | 中山大学 | A method of editing Large White CD163 genes using CRISPR/Cas9 |
| CN108823248A (en) * | 2018-04-20 | 2018-11-16 | 中山大学 | A method of Luchuan pigs CD163 gene is edited using CRISPR/Cas9 |
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| CN102715132A (en) * | 2012-05-04 | 2012-10-10 | 吉林大学 | Porcine reproductive and respiratory syndrome virus receptor CD163 knock-out swine and cultivation method thereof |
| CN107177595A (en) * | 2017-06-07 | 2017-09-19 | 浙江大学 | Targeting sgRNA, modification carrier for pig CD163 gene editings and its preparation method and application |
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| CN108753832A (en) * | 2018-04-20 | 2018-11-06 | 中山大学 | A method of editing Large White CD163 genes using CRISPR/Cas9 |
| CN108823248A (en) * | 2018-04-20 | 2018-11-16 | 中山大学 | A method of Luchuan pigs CD163 gene is edited using CRISPR/Cas9 |
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