CN107937345B - A kind of fibroblastic method of pig for preparing while knocking out CD163 gene and CD13 gene - Google Patents

A kind of fibroblastic method of pig for preparing while knocking out CD163 gene and CD13 gene Download PDF

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CN107937345B
CN107937345B CN201711138478.0A CN201711138478A CN107937345B CN 107937345 B CN107937345 B CN 107937345B CN 201711138478 A CN201711138478 A CN 201711138478A CN 107937345 B CN107937345 B CN 107937345B
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gene
carrier
seq
pig
gene knockout
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CN107937345A (en
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李奎
牟玉莲
刘志国
李巨浪
徐奎
魏迎辉
葛长利
蔡雪辉
王龙
陈其美
邱阳
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Shandong Rongfa Biotechnology Development Co.,Ltd.
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Shandong Blue Seed Industry Ltd By Share Ltd
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    • C12N5/0602—Vertebrate cells
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    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
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    • C12N2800/80—Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites

Abstract

The present invention provides a kind of dual-gene knockout carrier system, the carrier system includes CD163 gene knockout carrier and CD13 gene knockout carrier, wherein: the CD163 gene knockout carrier includes gene editing carrier framework and the section of DNA segment being connected on the carrier framework, and the nucleotide sequence of the DNA fragmentation is as shown in SEQ ID any one of NO:1-3；The CD13 gene knockout carrier includes gene editing carrier framework and the section of DNA segment being connected on the carrier framework, and the nucleotide sequence of the DNA fragmentation is as shown in SEQ ID any one of NO:4-6；The carrier framework of the CD163 gene knockout carrier and the CD13 gene knockout carrier is selected from CRISPR/Cas9, CRISPR/Cas9n, CRISPR/Cpf1 or CRISPR/C2c2.It can simply, fast and efficiently be pinpointed by above-mentioned carrier system while knock out CD163 gene and CD13 gene.

Description

It is a kind of to prepare while knocking out CD163 gene and the pig of CD13 gene is fibroblastic Method
Technical field
The present invention relates to gene editing field, prepares in particular to a kind of while knocking out CD163 gene and CD13 base The fibroblastic method of the pig of cause.
Background technique
Pig reproduction and respiration syndrome (porcine reproductive and respiratory syndrome, It PRRS is) by pig reproduction and breath syndrome virus (porcine reproductive and respiratory syndrome Virus, PRRSV) it is caused with pig anorexia, fever, pregnant sow premature labor, late abortion, stillborn foetus, weak tire and the mummification of fetus etc. are numerous The respiratory disease and height death for growing obstacle and piglet and grower pigs are that a kind of high degree in contact of main feature infects Disease.Because the disease clinically shows as ear skin cyanosis, so being otherwise known as " blue otopathy ".The disease is in 1987 for the first time in beauty State is found, and and then breaks out in 1989 in Europe, in China's Mainland the first explosion at the bottom of nineteen ninety-five, the infection rate of swinery is up to 90%, great economic loss is brought to pig breeding industry, it has also become a kind of infectious disease for seriously endangering pig breeding industry in world wide.
Porcine alveolar macrophage (the porcine alveolar of PRRSV main infection well differentiated in vivo Macrophages, PAM).PRRSV infect target cell prerequisite be with host cell adsorb, and host cell surface by Body is that this adsorption process of completion is essential.The study found that Heparan sulfate (Heparin Sulphate, HS), saliva Plain (Sialoadhesin, Sn) and CD163 (the Cluster of Differentiation 163) molecule of liquid acid adhesion is on PAM It is existing can three important acceptor molecules in conjunction with PRRSV.Wherein, CD163 be a kind of street cleaner rich in cysteine by Body, is typical I type glycosylation albumen and a kind of antigen of macrophage differentiation, molecular size are 130kD, Gu claimed again For M130 albumen.CD163 is initially that the specificity of macrophage and monocyte is used as to identify what protein was realized, lung, Spleen, liver, aggregate nodules and thymic tissue macrophage in have expression.Some researches show that in the non-permissive cell of PRRSV Transfection expression CD163 molecule can make these cell lines infection PRRSV and generate son in the cell in system (BHK-21 and PK-15) For virion, the antibody of anti-human CD163 can block the infection of PRRSV, show that CD163 is the required receptor of the virus. CD163 protein structure domain SRCR5 is necessary to virus infected cell, and 4 SRCR and cytoplasmic tail right and wrong of aminoterminal must It needs, wherein SRCR5 structural domain is exactly as coded by the 7th exon of CD163.Therefore, research CD163 gene modification pig can be Whether CD163 receptor is that key player during PRRSV infection provides indispensable evidence.
Pig epidemic diarrhea (porcine epidemic diarrhea, PED) is one kind by Porcine epidemic diarrhea virus Intestines problem highly infectious caused by (porcine epidemic diarrhea virus, PEDV).The pig at any age is equal It can infect, especially serious on piglet influence, case fatality rate is very high, effectively female lacking especially for the newborn piglet in 7 ages in days Under the antibody of source, the death rate may be up to 100%；For the farrowing sow of adult, reproductive performance is affected after infection, is such as pregnant It will appear miscarriage after the sow infection of early stage, pregnancy rate reduces；Weight loss after growing and fattening pigs infection.
Recent studies have shown that PEDV with the CD13 (APN) in intestinal mucosa epithelial cell mainly by combining infection young Pig.CD13 is significant as the necessary bind receptor of PEDV intrusion cell, this is the new varieties of anti-epidemic diarrhea virus pig It cultivates and new approaches is provided, i.e., prevent infection of the PEDV to pig by the APN gene of knock-out pig.
Therefore, establish it is a kind of can quickly, the accurately and efficiently method of knock-out pig CD163 gene and CD13 gene simultaneously, The pig fibroblast or pig for obtaining knock-out pig CD163 gene and CD13 gene are in research pig blue-ear disease and pig prevalence diarrhea diarrhea And have great importance in terms of disease-resistant pig breeding.
In view of this, the present invention is specifically proposed.
Summary of the invention
The first object of the present invention is to provide a kind of dual-gene knockout carrier system, the gene knockout carrier system packet CD163 gene knockout carrier and CD13 gene knockout carrier are included, can simply, fast and efficiently be determined by above-mentioned carrier system Point knocks out CD163 gene and CD13 gene.
The second object of the present invention is to provide a kind of pig for preparing while knocking out CD163 gene and CD13 gene into fiber The method of cell, by the method can simply, fast and efficiently obtain CD163 gene and CD13 gene pure knock out and The pig fibroblast for not carrying any exogenous marker, to research pig blue-ear disease, pig epidemic diarrhea and the breeding for disease resistance of pig It is of great significance.
The third object of the present invention, which is to provide, knocks out CD163 gene and CD13 while being prepared according to the above method The pig fibroblast of gene, pig fibroblast homozygous knockout CD163 and the CD13 gene and does not carry exogenous marker, right Research pig blue-ear disease, pig epidemic diarrhea and the breeding for disease resistance of pig are of great significance.
The fourth object of the present invention is to provide a kind of gene editing prepared while knocking out CD163 gene and CD13 gene The method of pig can simply and efficiently obtain CD163 gene by this method and CD13 gene pure knocks out gene editing pig, It is of great significance to research pig blue-ear disease, pig epidemic diarrhea and the breeding for disease resistance of pig.
In order to realize above-mentioned purpose of the invention, the following technical scheme is adopted:
A kind of dual-gene knockout carrier system, the carrier system include CD163 gene knockout carrier and CD13 clpp gene Except carrier, in which:
The CD163 gene knockout carrier includes gene editing carrier framework and be connected on the carrier framework one section DNA fragmentation, the nucleotide sequence of the DNA fragmentation is as shown in SEQ ID any one of NO:1-3, it is preferable that such as SEQ ID NO:1 It is shown；
The CD13 gene knockout carrier includes gene editing carrier framework and be connected on the carrier framework one section DNA fragmentation, the nucleotide sequence of the DNA fragmentation is as shown in SEQ ID any one of NO:4-6, it is preferable that such as SEQ ID NO:4 It is shown；
The carrier framework of the CD163 gene knockout carrier and the CD13 gene knockout carrier be selected from CRISPR/Cas9, CRISPR/Cas9n, CRISPR/Cpf1 or CRISPR/C2c2.
Traditional gene knockout system mainly based on ZFN and TALEN technology, is needed through complicated operation and flower Expense great effort could knock out target gene.And said gene knockout carrier of the present invention belongs to CRISPR gene editing system, benefit Fixed point knockout is carried out to target gene with CRISPR technology, significantly reduces the operation difficulty of gene knockout on the whole, is improved The efficiency knocked out；Meanwhile the present invention realizes dual-gene editor and no introducing exogenous marker gene, tool by once-through operation Efficient advantage high, accuracy is high, easy to operate and highly-safe.Still further aspect, the present invention to for CD163 gene and The target sequence gRNA of CD13 gene knockout is optimized, and screening, which obtains, from a plurality of target sequence knocks out the highest target sequence of efficiency Column, overcome CRISPR gene editing system high defect of off-target rate during knockout, further increase the efficiency of target practice, from And it can be quickly obtained the homozygote for knocking out target gene CD163 gene and CD13 gene in a short time.
In some embodiments, the carrier bone of the CD163 gene knockout carrier and the CD13 gene knockout carrier Frame is CRISPR/Cas9, it is preferable that the CRISPR/Cas9 be selected from pX330, pX260, pX334, pX335, pX458, PX459, pX461, pX462, pX551 and pX552, it is highly preferred that the CRISPR/Cas9 is pX330.
The invention further relates to a kind of fibroblastic method of the pig for preparing while knocking out CD163 gene and CD13 gene, The described method includes:
(1) above-mentioned dual-gene knockout carrier system is constructed；
(2) the gene knockout carrier system is transferred in pig fibroblast, identification obtain homozygous knockout CD163 and The monoclonal cell of CD13 gene.
The above method of the present invention is established based on aforementioned gene knockout carrier, by the method can it is simple, It fast and efficiently obtains CD163 gene and CD13 gene pure knocks out and do not carry the pig fibroblast of any exogenous marker, It is of great significance to research pig blue-ear disease, pig epidemic diarrhea and the breeding for disease resistance of pig.
In some embodiments, the pig fibroblast is porcine fetus fibroblasts.
In some embodiments, the step (1) specifically includes: the complementary single-stranded annealing of oligonucleotides being formed double Chain is connect with the carrier framework Jing Guo digestion, and screening obtains positive colony up to CD163 gene knockout carrier, described complementary Shown in the single-stranded sequence of oligonucleotides perhaps SEQ ID NO:9-10 as shown in SEQ ID NO:7-8 or SEQ ID NO: Shown in 11-12；The complementary single-stranded annealing of oligonucleotides is formed into double-strand, is connect with the carrier framework Jing Guo digestion, screening obtains Positive colony is up to CD13 gene knockout carrier, the single-stranded sequence of the oligonucleotides of the complementation such as SEQ ID NO:13-14 institute Show, perhaps as shown in SEQ ID NO:15-16 or as shown in SEQ ID NO:17-18.
In some embodiments, the step (2) specifically includes: by the CD13 gene knockout carrier and described CD163 gene knockout carrier is transferred to pig fibroblast by way of electrotransfection, and it is thin to screen monoclonal by limiting dilution assay Born of the same parents, and identify whether the monoclonal cell system is positive monoclonal cell that CD163 gene and CD13 gene pure knock out.
In some embodiments, it identifies and specifically includes described in step (2): extracting the genomic DNA of monoclonal cell, Respectively using the primer as shown in SEQ ID NO:19-20 and SEQ ID NO:21-22 progress PCR amplification, and to amplified production into Row sequencing determines whether the monoclonal cell is the positive that CD163 gene and CD13 gene pure knock out according to sequencing result Monoclonal cell；Preferably, it is 60~62 DEG C by the annealing temperature of the PCR amplification of primer of SEQ ID NO:19-20, follows Number of rings is 32~38, it is highly preferred that 61 DEG C, 36 circulations；Using SEQ ID NO:21-22 moving back as the PCR amplification of primer Fiery temperature is 57~59 DEG C, and recurring number is 32~36, it is highly preferred that 58 DEG C, 34 circulations.
The invention further relates to the pigs of the CD163 gene being prepared according to the above method and CD13 gene knockout into fiber finer Born of the same parents.The above-mentioned pig fibroblast of the present invention knocks out CD163 and CD13 gene homozygously, and does not carry exogenous marker gene, to grinding Study carefully pig blue-ear disease, pig epidemic diarrhea and the breeding for disease resistance of pig to be of great significance.
The invention further relates to a kind of method of gene editing pig for preparing while knocking out CD163 gene and CD13 gene, institutes Method is stated using above-mentioned pig fibroblast as nuclear transfer donor cell, its nuclear transplantation is entered into non-nucleus egg mother cell, is made Standby recombinant clone embryo is simultaneously implanted into the gene editing for obtaining while knocking out CD163 gene and CD13 gene in parent through gestation Pig.
The above method of the present invention based on the pig fibroblast of aforementioned CD163 gene knockout and CD13 gene knockout to build It is vertical to form, the gene of CD163 gene knockout and CD13 homozygous knockout can quickly, be simply and efficiently obtained by the method Pig is edited, is of great significance to research pig blue-ear disease, pig epidemic diarrhea and the breeding for disease resistance of pig.
In some embodiments, the method also includes identifying the gene editing pig, the mirror Fixed step includes: to extract the genomic DNA of pig, uses nucleotide sequence such as SEQ ID NO:19-20 and SEQ ID NO:21- Upstream and downstream primer shown in 22 expands the DNA genome of extraction, carries out agarose gel electrophoresis or survey to amplified production Whether sequence determines the pig by CD163 gene and CD13 gene knockout, it is preferable that with SEQ according to electrophoresis or sequencing result ID NO:19-20 is that the annealing temperature of the PCR amplification of primer is 60~62 DEG C, and recurring number is 32~38, it is highly preferred that 61 DEG C, 36 circulations；It is 57~59 DEG C by the annealing temperature of the PCR amplification of primer of SEQ ID NO:21-22, recurring number is 32~36, it is highly preferred that 58 DEG C, 34 circulations.
Compared with prior art, the invention has the benefit that
The present invention uses CRISPR gene editing system, and the DNA sequence dna as shown in SEQ ID NO:1-6 is as target position Point constructs the carrier system of CD163 gene and CD13 gene, is to rely on to establish a kind of prepare together with the gene knockout carrier system When knock out the pig fibroblast of CD163 gene and CD13 gene, gene editing pig method.Above-mentioned carrier system and method tool Having reduces genetic manipulation difficulty, improves and knock out efficiency, in a short time quickly by the excellent of CD163 gene and CD13 gene knockout Point.And pig fibroblast or the gene of the CD163 gene and CD13 gene knockout being prepared by above-mentioned carrier and method Edit pig, CD163 gene and CD13 gene are homozygous knockout, and in the pig fibroblast or gene editing pig without Exogenous marker can provide research platform for pig blue-ear disease and pig epidemic diarrhea, while have high Breeding value.
Detailed description of the invention
It, below will be to specific in order to illustrate more clearly of the specific embodiment of the invention or technical solution in the prior art Embodiment or attached drawing needed to be used in the description of the prior art be briefly described, it should be apparent that, it is described below Attached drawing is some embodiments of the present invention, for those of ordinary skill in the art, before not making the creative labor It puts, is also possible to obtain other drawings based on these drawings.
Fig. 1 is pX330 carrier framework figure；
Fig. 2 is that part is dual-gene while knocking out the genotype of cell line.
Specific embodiment
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the invention.It is not specified in embodiment specific Condition person carries out according to conventional conditions or manufacturer's recommended conditions.Reagents or instruments used without specified manufacturer is The conventional products that can be obtained by commercially available purchase.
Porcine fetus fibroblasts (porcine embryonic fibroblast, PEF) in following embodiments according to Following method preparation: by 35 day Embryos of Large White, head, tail, four limbs, internal organ and the bone of fetus are removed, and blood is cleared up Completely.It persistently shears fetus 30min guarantee with elbow eye scissors sufficiently to shred, by the blue electron gun head of the fetal tissue's haircut shredded It is drawn in 15mL centrifuge tube, 5mL complete medium is added, removes solution above after several minutes of natural subsidence, and in lower layer's group It knits and a few drop fetal calf serums is added in block, be sucked out with 15cm glass Pasteur's pipe curved at the 1cm of tip, be laid in two T75 cultures In bottle, bottom of bottle is placed upward, and 15mL full nutrient solution is added in opposite side, and culture bottle is carefully turned over after 6-8h, tissue block is soaked Enter in culture solution, changes a not good liquor within every two days, frozen after cell covers with T75 culture bottle spare.Wherein, Large White is Chinese agriculture The pig of academy of sciences Beijing animal and veterinary research institute base pig farm raising.
Embodiment 1 targets the building of the CRISPR/Cas9 targeting vector of CD163 gene and CD13 gene
1, the exon of locking coding pig CD163 gene and the exon of CD13 gene first is target practice region, and utilization is soft Part separately designs multiple gRNA for CD163 and CD13, i.e. target practice site.Wherein, it is respectively for the target practice site of CD163 CD163-gRNA1:ggaaacccaggctggttgga (SEQ ID NO:1)；CD163-gRNA2: Ggaggggacattccctgctc (SEQ ID NO:2) and CD163-gRNA3:ggtcgtgttgaagtacaaca (SEQ ID NO:3).It is respectively as follows: CD13-gRNA1:gcatcctcctcggcgtgg (SEQ ID NO:4) for the target practice site of CD13； CD13-gRNA2:caagggattctacatttcca (SEQ ID NO:5) and CD13-gRNA3:ttctacatttccaaggccct (SEQ ID NO:6).
2, according to the oligonucleotide of above-mentioned gRNA sequent synthesis complementary pairing, as shown in the table, lowercase is digestion Site.
The oligonucleotides of table 1 and gRNA sequence complementary pairing
3, the CRISPR/Cas9 targeting vector of building targeting CD163 gene and CD13 gene,
PX330 carrier framework is as shown in Figure 1.Specific construction method is as follows:
(1) 6 pairs of oligonucleotides for synthesizing table 1 handle 10min under the conditions of 98 DEG C respectively, after cooled to room temperature It anneals；
(2) with restriction enzyme Bbs I to digestion under the conditions of 37 DEG C of the pX330 skeleton carrier containing Cas9 sequence 2h, gel extraction linearized fragment；
(3) after, by linearized fragment with annealing after oligonucleotide connect 1h at 16 DEG C, then conversion Top10 or DH5 α competent cell is coated on the LB plated growth of the benzyl containing ammonia；
(4) picking single colonie, which expands, cultivates and is sequenced, sequencing primer U6-FWD.Sequence is correct, expands culture；
(5) method for going the big extraction reagent kit of endotoxin (EndoFree Plasmid Maxi Kit) to provide with plasmid is extracted Plasmid, the plasmid mentioned are used for the transfection of cell.
Embodiment 2 knocks out the foundation of the Large White fetal fibroblast cell line of CD163 gene and CD13 gene simultaneously
1, cell transfecting
The day before transfection recovers primary Large White fetal fibroblast into 6cm plate, when cell reaches 70-80% Cell transfecting can be carried out when convergence degree.Transfection procedure is in strict accordance with Basic Primary Fibroblasts Nucleofector Kit (Lonza) kit specification is operated.
2, the detection of target practice efficiency
After cell culture 48h after electrotransfection, a part is used for bed board, in addition collection part cell, extracts cytogene Group carries out PCR amplification, to detect target practice efficiency.The result shows that: the target practice efficiency of 3 gRNA of CD163 gene is respectively 9%, 5% and 4%；The target practice efficiency of 3 gRNA of CD13 gene is respectively 17%, 12% and 7%.Choose highest two gRNA of efficiency Subsequent experimental is carried out, and the two carriers are respectively designated as pX330-CD163 and pX330-CD13.
Using the cellular genome of extraction as template, PCR, the pcr amplification primer are carried out with Pre mix Taq archaeal dna polymerase Object is as follows:
Wherein the primer of CD163 gene is CD163-F:5 '-aagcccactgtaggcagaa-3 ' (SEQ ID BO:19) And CD163-R:5 '-gtggtttccctcctgggg-3 ' (SEQ ID BO:20).Amplification condition is 95 DEG C, 5min；95 DEG C, 30s；61 DEG C, 30s；72 DEG C, 30s；72 DEG C, 10min；36 circulations, 2% agarose gel electrophoresis observe band.
Wherein the amplimer of CD13 gene is (the SEQ ID of CD13-F:5 ' tacccagttcagtgaccttcgtc 3 ' ) and CD13-R:5 ' agaagaacaagaatgccgagca 3 ' (SEQ ID NO:22) BO:21.Amplification condition is 95 DEG C, 5min； 95 DEG C, 30s；58 DEG C, 30s；72 DEG C, 30s；72 DEG C, 10min；34 circulations, 2% agarose gel electrophoresis observe band.
3, the screening of positive monoclonal cell line
Electricity turns when cell confluency degree after 48h is about 90% or so with suitable density bed board, the primary culture of replacement in every 3 days Liquid.Cell after bed board is about cultivated 10 days or so, it can be observed that the clone of suitable size puts to be formed.By monoclonal cell into Row expands culture, while taking part cell for extracting genome identification genotype.
4, the identification of positive monoclonal cell line
The cell monoclonal of institute's picking is identified: using the cellular genome of extraction as template, with Pre mix Taq Archaeal dna polymerase carries out PCR.
Wherein the amplified fragments of CD163 gene are 300bp, primer CD163-F:5 ' aagcccactgtaggcagaa 3 ' (SEQ ID NO:19) and CD163-R:5 ' gtggtttccctcctgggg 3 ' (SEQ ID NO:20).Amplification condition is 95 DEG C, 5min；95 DEG C, 30s；61 DEG C, 30s；72 DEG C, 30s；72 DEG C, 10min；36 circulations.
The target fragment that CD13 gene magnification goes out is 286bp, and primer is Forward primer:5 ' Tacccagttcagtgaccttcgtc 3 ' (SEQ ID NO:21) and Reverse primer:5 ' Agaagaacaagaatgccgagca 3 ' (SEQ ID NO:22).Amplification condition is 95 DEG C, 5min；95 DEG C, 30s；58 DEG C, 30s；72 DEG C, 30s；72 DEG C, 10min；34 circulations.
2% agarose gel electrophoresis observes band, and PCR product send Beijing Tian Yihuiyuan company to be sequenced, according to sequencing result, Screen donorcells of the cell line dual-gene while that frameshift mutation occurs as nuclear transfer when.
5, experimental result
Sequencing result shows, we successfully obtain pig fetus that more plants of CD163 genes and CD13 gene all knock out at Fibrocyte system, it is as shown in Figure 2 that part of diallele knocks out cell line genotype.
The method that 3 somatic cell nuclear transfer technique of embodiment prepared while knocking out the gene editing pig of CD163 and CD13 gene
It is female thin with the young pig ovum of maturation in vitro 40h using the positive cell that embodiment 2 obtains as nuclear transfer donor cell Born of the same parents are nuclear transfer recipient cell, and nuclear transfer donor cell is moved into non-nucleus egg mother cell, through electro' asion and activation, are built into weight Group clone embryos select the good clone recombination embryo of developmental condition with modus operandi immigration spontaneous estrus and are produced large white sow Gestation is in utero carried out, modus operandi embryo transfer step is ventilator anesthesia, and maintains to anaesthetize with 2% chloraldurate, in hand It lies on the back on art frame Baoding, ventrimeson does the operative incision for being about 10cm, exposes ovary, fallopian tubal and uterus to the open air, moved with embryo It plants glass tube and enters about 5cm along fimbriae tubae portion, developmental condition is well cloned to recombination embryo and is transplanted to ampulla of uterine tube- Isthmus junction.After embryo transfer, technical staff notices that observing it returns feelings situation, periodically uses B-type ultrasonography receptor sow Pregnancy.
Experimental result: successfully obtaining while knocking out the gene editing pig of CD163 gene and CD13 gene.
Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention., rather than its limitations；To the greatest extent Present invention has been described in detail with reference to the aforementioned embodiments for pipe, but those skilled in the art should understand that: its It is still possible to modify the technical solutions described in the foregoing embodiments, or to some or all of the technical features It is equivalently replaced；And these are modified or replaceed, various embodiments of the present invention skill that it does not separate the essence of the corresponding technical solution The range of art scheme.
Sequence table
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ggaaacccag gctggttgga 20
<210> 2
<211> 20
<212> DNA
<213>artificial sequence ()
<400> 2
ggaggggaca ttccctgctc 20
<210> 3
<211> 20
<212> DNA
<213>artificial sequence ()
<400> 3
ggtcgtgttg aagtacaaca 20
<210> 4
<211> 18
<212> DNA
<213>artificial sequence ()
<400> 4
gcatcctcct cggcgtgg 18
<210> 5
<211> 20
<212> DNA
<213>artificial sequence ()
<400> 5
caagggattc tacatttcca 20
<210> 6
<211> 20
<212> DNA
<213>artificial sequence ()
<400> 6
ttctacattt ccaaggccct 20
<210> 7
<211> 24
<212> DNA
<213>artificial sequence ()
<400> 7
caccggaaac ccaggctggt tgga 24
<210> 8
<211> 24
<212> DNA
<213>artificial sequence ()
<400> 8
aaactccaac cagcctgggt ttcc 24
<210> 9
<211> 24
<212> DNA
<213>artificial sequence ()
<400> 9
caccggaggg gacattccct gctc 24
<210> 10
<211> 24
<212> DNA
<213>artificial sequence ()
<400> 10
aaacgagcag ggaatgtccc ctcc 24
<210> 11
<211> 24
<212> DNA
<213>artificial sequence ()
<400> 11
caccggtcgt gttgaagtac aaca 24
<210> 12
<211> 24
<212> DNA
<213>artificial sequence ()
<400> 12
aaactgttgt acttcaacac gacc 24
<210> 13
<211> 22
<212> DNA
<213>artificial sequence ()
<400> 13
caccgcatcc tcctcggcgt gg 22
<210> 14
<211> 22
<212> DNA
<213>artificial sequence ()
<400> 14
aaacccacgc cgaggaggat gc 22
<210> 15
<211> 24
<212> DNA
<213>artificial sequence ()
<400> 15
cacccaaggg attctacatt tcca 24
<210> 16
<211> 24
<212> DNA
<213>artificial sequence ()
<400> 16
aaactggaaa tgtagaatcc cttg 24
<210> 17
<211> 24
<212> DNA
<213>artificial sequence ()
<400> 17
caccttctac atttccaagg ccct 24
<210> 18
<211> 24
<212> DNA
<213>artificial sequence ()
<400> 18
aaacagggcc ttggaaatgt agaa 24
<210> 19
<211> 19
<212> DNA
<213>artificial sequence ()
<400> 19
aagcccactg taggcagaa 19
<210> 20
<211> 18
<212> DNA
<213>artificial sequence ()
<400> 20
gtggtttccc tcctgggg 18
<210> 21
<211> 23
<212> DNA
<213>artificial sequence ()
<400> 21
tacccagttc agtgaccttc gtc 23
<210> 22
<211> 22
<212> DNA
<213>artificial sequence ()
<400> 22
agaagaacaa gaatgccgag ca 22

Claims (10)

1. a kind of dual-gene knockout carrier system, which is characterized in that the carrier system include CD163 gene knockout carrier and CD13 gene knockout carrier, in which:
The CD163 gene knockout carrier includes gene editing carrier framework and the section of DNA that is connected on the carrier framework Segment, the nucleotide sequence of the DNA fragmentation is as shown in SEQ ID NO:1；
The CD13 gene knockout carrier includes gene editing carrier framework and the section of DNA piece being connected on the carrier framework Section, the nucleotide sequence of the DNA fragmentation is as shown in SEQ ID NO:4；
The carrier framework of the CD163 gene knockout carrier and the CD13 gene knockout carrier is CRISPR/Cas9.
2. carrier system according to claim 1, which is characterized in that the CRISPR/Cas9 be selected from pX330, pX260, PX334, pX335, pX458, pX459, pX461, pX462, pX551 and pX552.
3. carrier system according to claim 2, which is characterized in that the CRISPR/Cas9 is pX330.
4. a kind of fibroblastic method of pig for preparing while knocking out CD163 gene and CD13 gene, which is characterized in that described Method includes:
(1) any one of building claim 1 ~ 3 dual-gene knockout carrier system；
(2) the gene knockout carrier system is transferred in pig fibroblast, by screening and identifying acquisition homozygous knockout The monoclonal cell of CD163 gene and CD13 gene.
5. according to the method described in claim 4, it is characterized in that, the pig fibroblast is porcine fetus fibroblasts.
6. according to the method described in claim 4, it is characterized in that, the step (1) specifically includes: by complementary oligonucleotides Single-stranded annealing forms double-strand, connect with the carrier framework Jing Guo digestion, and screening obtains positive colony up to CD163 gene knockout load Body, the single-stranded sequence of the oligonucleotides of the complementation is as shown in SEQ ID NO:7-8；By the complementary single-stranded annealing shape of oligonucleotides It at double-strand, is connect with the carrier framework Jing Guo digestion, screening obtains positive colony up to CD13 gene knockout carrier, the complementation The single-stranded sequence of oligonucleotides as shown in SEQ ID NO:13-14.
7. according to the method described in claim 4, it is characterized in that, the step (2) specifically includes: by the CD13 clpp gene Except carrier and the CD163 gene knockout carrier are transferred to pig fibroblast by way of electrotransfection, pass through limiting dilution assay Monoclonal cell is screened, and identifies whether the monoclonal cell system is the positive that CD163 gene and CD13 gene pure knock out Monoclonal cell.
8. according to the method described in claim 4, being specifically included it is characterized in that, being identified described in step (2): extracting monoclonal The genomic DNA of cell carries out PCR expansion using the primer as shown in SEQ ID NO:19-20 and SEQ ID NO:21-22 respectively Increase, and amplified production is sequenced, determines whether the monoclonal cell is CD163 gene and CD13 base according to sequencing result Because of the positive monoclonal cell of homozygous knockout.
9. according to the method described in claim 8, it is characterized in that, in step (2), using SEQ ID NO:19-20 as primer The annealing temperature of the PCR amplification is 60 ~ 62 DEG C, and recurring number is 32 ~ 38；Using SEQ ID NO:21-22 as the PCR of primer The annealing temperature of amplification is 57 ~ 59 DEG C, and recurring number is 32 ~ 36.
10. according to the method described in claim 9, it is characterized in that, in step (2), using SEQ ID NO:19-20 as primer The annealing temperature of the PCR amplification is 61 DEG C, recurring number 36；Using SEQ ID NO:21-22 as the PCR amplification of primer Annealing temperature is 58 DEG C, recurring number 34.
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