CN107018955A - A kind of transgene pig of the type of resisting porcine circovirus 2 - Google Patents
A kind of transgene pig of the type of resisting porcine circovirus 2 Download PDFInfo
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- CN107018955A CN107018955A CN201710321636.XA CN201710321636A CN107018955A CN 107018955 A CN107018955 A CN 107018955A CN 201710321636 A CN201710321636 A CN 201710321636A CN 107018955 A CN107018955 A CN 107018955A
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Classifications
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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
- 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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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- 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
- C12N15/1131—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 against viruses
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- 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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- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/873—Techniques for producing new embryos, e.g. nuclear transfer, manipulation of totipotent cells or production of chimeric embryos
- C12N15/877—Techniques for producing new mammalian cloned embryos
- C12N15/8778—Swine embryos
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- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
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Abstract
The invention discloses a kind of type transgene pig of resisting porcine circovirus 2.The type siRNA gene orders of resisting porcine circovirus 2 of the type transgene pig of resisting porcine circovirus 2 are incorporated on No. 10 chromosome, can the efficient type siRNA of specifically expressing resisting porcine circovirus 2, to suppress the Expression and biological activity of porcine circovirus 2 type, so as to play a part of preventing and treating pig infection PCV-II disease.Meanwhile, the transgene pig deletes exogenous marker gene, reduces influence of the foreign gene to transgene pig individual health, while can also simplify transgene pig safety evaluation program, is conducive to the Industry Promotion and application of the transgene pig.
Description
Technical field
The present invention relates to genetic engineering field, more particularly to a kind of transgene pig of the type of resisting porcine circovirus 2.
Background technology
In the growth and disease prevention and cure of livestock animals, transgenic technology reaches its maturity, for ease of transgenic cell line
Screening and the detection of transgenic animals, while target gene is transferred to, are usually transferred to exogenous marker gene simultaneously, and EGFP is green
Color fluorescin can directly be observed under fluorescence microscope, and often as a kind of marker gene；Neo genes are a kind of antibiosis
Plain resistant gene, cell, which expresses the gene, can inactivate aminoglycosides antibiotics, destroy G418, and often as one kind screening
Marker gene is used for the screening of transgenosis monoclonal cell.Thus both genes are inevasible appears in transgenic animals
In, and these marker gene are just no longer necessary after transgenic animals successfully obtain.
Meanwhile, the presence of exogenous marker gene will will exert an adverse impact to the health of breeding transgenic livestock, and China
Transgene-safty is evaluated policy and expressly provided is equal to target gene by marker gene, and the safety for equally having to pass through complexity is commented
Valency, this popularization that undoubtedly directly affects breeding transgenic livestock and industrialization process.Therefore, in the operation for carrying out transgenosis, lead to
Often in the both sides of marker gene respectively plus an identical LoxP sequence, to be utilized after the enrichment successful cell of transgenosis
Cre/LoxP systems delete marker gene unnecessary in transgenic cell.
Pig circular ring virus, belongs to PCV-II section, is sub-thread cyclic DNA virus, is divided into 1 type and 2 types.Wherein pig circular ring virus 2
Malicious 1 type (PCV1) is not pathogenic, and porcine circovirus 2 type (PCV2) has pathogenic.In pig industry control and prevention of disease, vaccine is people
For tackling the most common means of disease.And because PCV-II inactivated vaccine and subunit vaccine cost height, frequency injection are more
The shortcomings of with physical stress is easily caused, and immune efficacy have much room for improvement, can not immunity inoculation on a large scale.In addition, epidemic disease
The use of seedling obtains some successes in control causes PMWS by PCV2 infection, but the morbidity pathogeny of this syndrome is
Various, and do not broken through in other syndromes, so anti-PCV2siRNA gene orders are introduced by transgenic approach,
More having real value is seemed to the defence of porcine circovirus 2 type using RNA perturbation techniques.
The Patent No. 201310321355.6 announced on December 25th, 2013, patent name is " anti-pig circular ring virus 2
A kind of expression of the type of resisting porcine circovirus 2 (PCV2) is disclosed in the expression vector and transgene pig and its construction method of malicious 2 types "
Carrier and transgene pig and its construction method, and the F0 generation types (PCV2) of (primary) resisting porcine circovirus 2 are obtained by this method
Transgene pig, the foreign gene that the transgene pig is integrated includes the anti-PCV2siRNA of target gene gene order and marker gene
Neo/EGFP amalgamation and expression gene orders, and the presence of marker gene, will exert an adverse impact, and influence to the transgene pig
The safety evaluation of the transgene pig, is unfavorable for popularization and the industrialization process of the transgene pig.
The content of the invention
It is an object of the invention to provide a kind of transgene pig of the type of resisting porcine circovirus 2, to solve the above problems.
According to an aspect of the invention, there is provided a kind of transgene pig of the type of resisting porcine circovirus 2, the transgene pig can
With the siRNA of the type of specifically expressing resisting porcine circovirus 2.
In some embodiments, the transgene pig of the type of resisting porcine circovirus 2 does not include exogenous marker gene, thus may be used
To reduce influence of the exogenous marker gene for transgene pig health, while transgene pig safety evaluation program can be simplified,
Be conducive to the Industry Promotion and application of the transgene pig.
In some embodiments, the type transgene pig of resisting porcine circovirus 2 does not include Neo/EGFP amalgamation and expression external sources
Marker gene, Neo genes are a kind of antibiotics resistance genes, and cell, which expresses the gene, can inactivate aminoglycosides antibiotics,
G418 is destroyed, and is used for the screening of transgenosis monoclonal cell often as a kind of riddled basins；And EGFP is glimmering for green
When photoprotein expressing gene transfects structure transgenosis monoclonal cell system jointly with target gene, it is easy to intuitively be seen with microscope
The selection result is examined, can also be by observing the expression of green fluorescent protein come the preliminary knot for judging transgenosis to transgenic animals
Really, but EGFP green fluorescent proteins are expressed in cellular level and can produce certain toxicity to cell, in transgene pig individual
Certain influence can be produced to the health of pig, therefore delete the transgene pig meeting of Neo/EGFP amalgamation and expression exogenous marker genes
The influence to the non-transgenic healthy is reduced, while transgene pig safety evaluation program can be simplified, is conducive to the transgene pig
Industry Promotion and application.
In some embodiments, the resisting porcine circovirus 2 type siRNA gene order is located on No. 10 chromosome,
Thus, the resisting porcine circovirus 2 type siRNA expression quantity is high, can preferably suppress the expression of porcine circovirus 2 type gene, rises
The effect of circovurus type 2 disease is infected to preventing and treating pig.
In some embodiments, the resisting porcine circovirus 2 type siRNA gene order is located on No. 10 chromosome,
And positioned at two functional gene CUGBP Elav-like family member of No. 10 chromosome and trans-acting T-
Between cell-specific transcription factor GATA-3, the type of resisting porcine circovirus 2 in the site is incorporated into
SiRNA gene order, with higher expression quantity and bioactivity, can preferably suppress the table of porcine circovirus 2 type gene
Reach, play a part of preventing and treating pig infection circovurus type 2 disease.
In some embodiments, the type transgene pig of resisting porcine circovirus 2 is implemented by the following steps：
(1), the structure of the type expression vector of resisting porcine circovirus 2, the carrier includes the type of target gene resisting porcine circovirus 2
SiRNA expressing gene sequences, marker gene Neo/EGFP amalgamation and expression gene orders；
(2), by the expression vector of the type of resisting porcine circovirus 2 of acquisition and transposase plasmids mixing cotransfection pig fetus into fibre
Tie up cell line, screening can stable expressed vector monoclonal transgenic cell line；
(3), the monoclonal transgenic cell line of acquisition is expelled in egg mother cell as nuclear donor cell, weight is built
Structure embryo, is cultivated by somatic cell clone technique, and using traditional breeding method, that is, obtains F0 for resisting porcine circovirus 2
The transgene pig of type；
(4), by the F0 of acquisition for the transgene pig of the type of resisting porcine circovirus 2 wild type consistent with kind, genetic background
Sow crossbreed obtains the F1 generation transgene pig for the type of resisting porcine circovirus 2 that can stablize heredity；
(5), to the type transgene pig genetic test of F1 generation resisting porcine circovirus 2 of acquisition, F1 generation moderate resistance pig circular ring virus 2 is screened
Malicious 2 type siRNA expression quantity are high, and the F1 generation transgene pig individual of only one of which foreign gene insertion point；
(6) the F1 generation transgene pig filtered out individual, is subjected to exogenous origin gene integrator Locus Analysis in Shoots；
(7) the F1 generation transgene pig individual filtered out, is taken into ear skin fibroblast, and carries out ear skin fibroblast
The foundation of system；
(8), recombinate the ear skin fibroblast of ferment treatment F1 generation transgene pig to delete Neo/EGFP using HTNCre
Marker gene, and cell monoclonal culture is carried out, screening deletes the cell monoclonal of Neo/EGFP marker gene；
(9), it regard the cell monoclonal for deleting marker gene filtered out as the nuclear donor cell of somatic cell clone, note
It is mapped in egg mother cell, builds reconstruct embryo, is cultivated by somatic cell clone technique, and using traditional breeding method, i.e.,
Obtain the transgene pig of resisting porcine circovirus 2 type of the F2 generations without Neo/EGFP marker gene.
In some embodiments, using HTNCre recombinate ferment treatment F1 generation transgene pig ear skin fibroblast come
The step of deleting Neo/EGFP marker gene is as follows：
(1), the type transgene pig ear skin fibroblast of recovery F1 generation resisting porcine circovirus 2 is in Tissue Culture Dish, after covering with
6 orifice plates are passaged to, condition of culture is 37 DEG C, 5%CO2；
(2), when cell density is up to 80% or so, washed with DPBS 2 times, add the HTNCre of OPTI-MEM dilutions, make it
Final concentration of 200ng/ μ L, siphon away HTNCre working solutions after being incubated 3h, are washed with DPBS 2 times, add 10%FBS complete medium
Continue to cultivate；
(3), after 24h, with 0.25% trypsin digestion cell, cell is counted, reached by 200 cell/wares
In 100mm Tissue Culture Dish, changed a not good liquor every 3 days and observe the formational situation of cell monoclonal；
(4), when cell culture was to 10 days or so, when cell clone group cell number reaches 200 or so, in inverted microscope
Observation under in Tissue Culture Dish bottom iris out cell clone group position, and cloning cluster periphery iris out no heteroproteose cell again
Safety zone, while having unstressed configuration with fluorescence microscope cell clone group, carries out mark, with ensure picking it is purer without glimmering
Photo-cell monoclonal；
(5) complete medium, is siphoned away, is washed with DPBS 2 times, made marks thin is carefully accurately placed on by ring is cloned with tweezers
Two are added on born of the same parents clone, in each clone's ring and drips 0.25% pancreatin, when observing under the microscope to cell rounding, add 50 μ L left
Right FBS terminates digestion, and cell is carefully blown afloat with pipettor and is transferred in 24 orifice plates, is entered by the hole -60mm of 24 hole -6 order
Row Secondary Culture, to obtain the cell monoclonal of marker-free luciferase expression.
In some embodiments, it can also be 100ng/ μ L, 150ng/ μ L or 300ng/ μ L that HTNCre, which recombinates enzyme concentration,
Isoconcentration.
By the transgene pig of the type of resisting porcine circovirus 2 obtained with upper type, have the advantages that：This anti-pig circle
The type transgene pig of circovirus virus 2 is to contain target gene (the type siRNA genes of resisting porcine circovirus 2) and exogenous marker base by building
Because of the coexpression vector of (Neo/EGFP amalgamation and expressions gene), transfection prepare transgenosis monoclonal cell system is carried out, then with this
Monoclonal cell system as somatic cell clone technique confession somatic cell, to obtain containing target gene and exogenous marker gene
Then F0 hybridizes numerous for the type transgene pig of resisting porcine circovirus 2 by the F0 generations wild type sow consistent with kind, genetic background
The F1 generation transgene pig for obtaining the type of resisting porcine circovirus 2 that can stablize heredity is educated, the type siRNA tables of resisting porcine circovirus 2 are filtered out
It is high up to amount, and the F1 generation transgene pig individual of only one of which integration site carries out follow-up study, takes the F1 generation filtered out to turn
The ear skin fibroblast of gene pig individual carries out culture, and HTNCre restructuring is carried out to the ear skin fibroblast of acquisition
Ferment treatment, and the cell monoclonal for deleting marker gene is filtered out, somatic cell clone technique is used as using this cell monoclonal
Donor cell, carries out the type transgene pig of resisting porcine circovirus 2 that somatic cell clone obtains F2 generations and is free of exogenous marker gene, and right
The F2 is detected for transgene pig, it was demonstrated that marker gene has been deleted, but the type siRNA gene order quilts of resisting porcine circovirus 2
Retain.The type transgene pig of resisting porcine circovirus 2 obtained by the above method can be with the type of specifically expressing resisting porcine circovirus 2
SiRNA, to suppress the Expression and biological activity of porcine circovirus 2 type, so as to play the work that preventing and treating pig infects PCV-II disease
With.Meanwhile, the transgene pig deletes exogenous marker gene, reduces influence of the foreign gene to transgene pig individual health,
Transgene pig safety evaluation program can also be simplified simultaneously, be conducive to the Industry Promotion and application of the transgene pig.
Brief description of the drawings
Fig. 1 is that embodiment 1 moderate resistance porcine circovirus 2 type (PCV2) transgene pig F1 generation EGFP exogenous marker gene greens are glimmering
Light expression；
Fig. 2 ties for the Southern Blot detections of embodiment 2 moderate resistance porcine circovirus 2 type (PCV2) transgene pig F1 generation
Really, wherein MW is DNA molecular amount standard, and Transgenic is the type transgene pig sample of F1 generation resisting porcine circovirus 2, Wild-
Type is wild type control pig sample；
Fig. 3 is siRNA relative expressions between different type (PCV2) transgene pigs of F1 generation resisting porcine circovirus 2 in embodiment 3
Situation, wherein " 3 " mark TG3 individuals；
Fig. 4 is the individual target gene integration site sequencings of the type transgene pig TG3 of F1 generation resisting porcine circovirus 2 in embodiment 4
Comparison diagram；
Fig. 5 is the foundation knot of the type transgene pig TG3 ear skin fibroblasts of F1 generation resisting porcine circovirus 2 in embodiment 5
Fruit is schemed；
Fig. 6 is the expression and purification SDS-PAGE analysis result figures of HTNCre recombinases in embodiment 6, and wherein MW is albumen point
Sub- amount standard；1~3, the 5 lysate rough segmentation to induce；The 4 lysate rough segmentation not induce；6 lysate to induce
Supernatant；7 precipitate for the lysate of induction；8 be product after purification；
Fig. 7 is streaming before and after the type transgene pig TG3 cell lines of HTNCre processing F1 generations resisting porcine circovirus 2 in embodiment 7
Cell detection fluorescence results figure；
Fig. 8 is the front and rear fluorescence of the type transgene pig TG3 cell clones of F1 generation resisting porcine circovirus 2 deletion mark in embodiment 7
Detection of expression result figure；
Fig. 9 is deletes the PCR testing result figures of the transgene pig TG3 cell monoclonals of mark in embodiment 8, wherein MW is
DNAmarker；Nagtive is negative control；W is water；WT is wild type pig；Positive is positive control；P is pEGFP-
Sh2 plasmids；- Cre is without HTNCre processing；Mix is the cell mixing after HTNCre is handled；C1~C7 is 7 cell Dan Ke
It is grand；
Figure 10 is that the deletion reaction principle of HTNCre recombinase-mediateds in embodiment 8 and PCR primer (530bp) sequencing are tested
Demonstrate,prove result figure；
Figure 11 is the EGFP expressions of the type transgene pig of resisting porcine circovirus 2 without exogenous marker gene in embodiment 10
Result figure；
Figure 12 is the PCR testing results of the type transgene pig of resisting porcine circovirus 2 without exogenous marker gene in embodiment 11
Figure.
Embodiment
Invention is described in further detail below in conjunction with the accompanying drawings.
Unless otherwise specified, the reagent used in following examples derive from it is commercially available, operating method be existing routine
Operating method.
The signified type of F1 generation resisting porcine circovirus 2 (PCV2) transgene pig passes through Patent No. in the present invention
201310321355.6, patent name is " expression vector and transgene pig and its construction method of the type of resisting porcine circovirus 2 " institute
The type transgene clone pig (F0 generations) of resisting porcine circovirus 2 that the method for announcement the is obtained wild type consistent with kind, genetic background
Obtained by sow crossbreed.And Patent No. 201310321355.6, patent name is " expression of the type of resisting porcine circovirus 2 is carried
The type transgene clone pig (F0 generations) of resisting porcine circovirus 2 that body and transgene pig and its construction method " is announced is justified comprising anti-pig
The foreign genes, wherein Neo and EGFP such as siRNA, the screening-gene Neo of the type of circovirus virus 2, Green Fluorescent Protein gene EGFP
Gene fusion expression, the F0 derives from this school for the type transgene pig of resisting porcine circovirus 2.
Embodiment 1：The EGFP expression of the anti-PCV2 transgene pigs of F1 generation is observed using bioluminescence lamp.
Under blue photo-excitation conditions, visually through corresponding optical filter it is observed that EGFP albumen presented it is green glimmering
Light.Based on this principle, the anti-PCV2 of F1 generation that can express EGFP albumen with Preliminary Identification body tissue using bioluminescence lamp turns base
Because of pig.Concrete operations are as follows：Transgene pig and wild type pig are placed in same place, respectively in identical blue light background and in vain
Observe and take pictures under light background.Wherein, camera acquisition parameter is under blue light background：Aperture priority, aperture F2.8, ISO4000, hand
Dynamic focusing；Camera acquisition parameter is under white light background：Aperture priority, aperture F5.6, ISO100, manual focus.Similarly, to turning base
Because pig and the taken partial organ of wild type pig dissection organize to be observed and are taken pictures.
Testing result as shown in Figure 1, the nose and mouth of the anti-PCV2 transgene pigs of F1 generation and organ-tissue kidney, lung,
The heart is it is observed that obvious green fluorescence is expressed, and negative control fails to observe green fluorescence under similarity condition.This table
Bright, the anti-PCV2 transgene pigs of F1 generation can stablize heredity and high efficient expression is incorporated into foreign genes of the F0 for transgene pig genome.
Embodiment 2：Integration of the foreign gene in the anti-PCV2 transgene pigs of F1 generation is detected using Southern Blot.
The copy number that foreign gene is integrated in transgene pig genome can be identified by Southern Blot, it is former
Reason is substantially：Restriction enzyme site analysis first is carried out to pEGFP-sh2 carrier sequences, a proper single endonuclease digestion site is selected
(HindIII) control of 1 copy number, is then designed with pEGFP-sh2 plasmids, with F0 for anti-PCV2 transgene pigs genomic DNA
It is about 3 × 10 as positive control, and with mammalian genome DNA sizes9Individual base-pair (bp) is used as foundation, by following public affairs
Formula calculates the plasmid amount required for copy control：
Plasmid amount (μ g)=insertion plasmid fragments size (bp)/3*109(bp) × genomic DNA amount (μ g)
So as to estimate that the anti-PCV2 transgene pigs of F1 generation insert the copy number of foreign gene according to result.Concrete operation step is such as
Under：
(1) PCR methods prepare probe needed for Southern Blot
Probe needed for Southern Blot is prepared using PCR labelling methods, and its principle is：In expression purpose siRNA plasmid
Fragment two ends separately design primer, are marked using PCR DIG Probe Synthesis Kit, expand obtained 650bp
PCR primer is the probe marked.Primer sequence and amplified production length are shown in Table as follows：
Reaction system is formulated as follows：
Concrete operation step is：
(1) reagent each component is thawed on ice, short centrifugation is mixed；
(2) sequentially added by the amount of each component in upper table by large volume to small size in reaction tube, all operations are in ice
It is upper to carry out, mix, short centrifugation；
(3) reaction tube is placed in PCR instrument and carries out amplified reaction, program is as follows：95℃-2min；95 DEG C of -30s, 60 DEG C -
30s, 72 DEG C of -40s；30cycles；72℃-7min；
(4) detection probe labeling effciency：The Ago-Gel progress electrophoresis that isometric PCR primer is splined on 2% is drawn,
If it can be seen that purpose band size is correct, and DIG label probes are slightly slower than unmarked control probe migration velocity, then show institute
Label probe is successfully prepared, and -20 DEG C save backup
(2) Southern Blot hybridize flow
Southern Blot used kits are DIG High Prime DNA Labeling and Detection
Starter Kit II, concrete operation step is carried out according to kit specification.
Testing result as shown in Figure 2, shows that the anti-PCV2 transgene pigs of 6 F1 generations of detection have 1-4 integration site,
The equal only one of which integration sites of wherein TG3 and TG4, and wild type control pig then there is no Southern Blot results, show
The prepared probe of this experiment is special, and the anti-PCV2 transgene pigs of F1 generation specifically incorporate target gene and exogenous marker base
Cause.
Embodiment 3：Expression of the siRNA in the anti-PCV2 transgene pigs of F1 generation is detected using Real-time qPCR.
(1) Total RNAs extraction
RNA organizes sample to be soaked in RNAlater and is stored in -80 DEG C.Using MirVana miRNA isolation
Kit extracts total serum IgE, and concrete operation step is carried out according to specification.
(2) specific siRNA reverse transcription
According to target gene siRNA and reference gene miR16 primers, with same RNA templates respectively at PCR pipe
Middle carry out reverse transcription reaction, cDNA is obtained after being expanded through primer.Primer sequence is as follows：
Reverse transcription (7.5 μ L reaction systems) Master Mix systems are formulated as follows：
The process for preparation of reverse transcription Master Mix systems is as follows：
I. gently mix, be sure not to be vortexed, centrifuge to ttom of pipe, be then placed on ice；
II. for each 7.5 μ L reaction system, 1 μ L 5ng~10ng total serum IgE is added；
III. gently mix, be sure not to be vortexed, short centrifugation is operated on ice to ttom of pipe；
IV. 3 μ L 50nM primer is added, primer is mixed and short centrifugation using preceding；
V. mix, be sure not to be vortexed, short centrifugation is incubated 5min on ice to ttom of pipe；
VI. reverse transcription program is：16 DEG C, 30min；42 DEG C, 30min；85 DEG C, 5min；4 DEG C of preservations.
(3) specific siRNA Real-time qPCR detections
I. target gene siRNA and reference gene miR16 qPCR detection primers and probe are as follows：
II.siRNA qPCR 20 × Taqman siRNA Assay 100 μ L systems are formulated as follows：
III.siRNA qPCR reaction systems are formulated as follows：
Mentioned reagent dissolves on ice, mixes, short centrifugation to ttom of pipe.QRCR response procedures are：95 DEG C, 10min；95
DEG C, 15s, 60 DEG C, 1min；40cycles.
Testing result shows that the expression of siRNA between different F1 generation transgene pigs has differences as shown in Figure 3, as a result,
Its expression quantity is how many not strict corresponding with the external source target gene copy number learnt in Southern Blot results, thus it is speculated that this
May be relevant with integration position of the external source target gene on genome；And foreign gene is the individual TG3 of single copy table
It it is 4 times or so of TG4 up to amount.
Shown according to the result of embodiment 2 and embodiment 3, TG3 transgene pigs individual is integrated in foreign gene only one of which
Site, and with higher anti-PCV2 siRNA expression quantity, thus selection filters out TG3 individual progress follow-up studies.
Embodiment 4：Determine that foreign gene is whole in the anti-PCV2 transgene pigs genomes of TG3 using IPCR (inverse PCR)
Close site.
Tentatively judge that foreign gene after integration, is further adopted in TG3 genes of individuals groups by Southern Blot
The integration site of foreign gene is found with IPCR method, Southern Blot result on the one hand can be verified, on the other hand
Influence of the integration position to transgene pig of foreign gene on chromosome can be analyzed, on the other hand can also be marked to delete
Gene is prepared.By the result for analyzing Southern Blot, it can be determined that HindIII restriction enzymes are equally applicable to
IPCR.IPCR concrete operation steps are as follows：
(1) extraction of genomic DNA
With reference to the step in embodiment 2.
(2) digestion and purifying of genomic DNA
With reference to the step in embodiment 2, DNA consumptions are 2 μ g.
(3) connection and purifying of digestion products
Take the μ g of μ g of DNA sample 0.2 that above-mentioned digestion reclaims~2 to be placed in 0.5mL centrifuge tubes, sequentially add coupled reaction
Reagent, is mixed, and reaction tube is placed in 16 DEG C of constant-temperature metal baths and reacts 12h-16h by short centrifugation, and final purification is reclaimed, specific pure
Change step as above.
(4) IPCR is detected
IPCR is reacted using the connection product for purifying recovery as template, and the primer sequence is as follows：
In 4 primer sequences of the above, wherein PB3 ' TRE primary and CMV primary is a pair, for expanding swivel base
Genome sequence near the end of son 3 ' and integration site；PB5 ' TRE primary and Neo primary are a pair, for expanding
Transposons 5 ' is held and the genome sequence near integration site.
(5) IPCR products gel extraction
IPCR products are all entered into row agarose gel electrophoresis, each band is separated as much as possible so as to gel extraction.Cut
Glue reclaim uses E.Z.N.A.Gel Extraction Kit, and concrete operation step is carried out according to explanation.
(6) T-A is cloned
The end of IPCR products 3 ' through gel extraction carry A bases, can directly with InsTAclone PCR Cloning
T-Vector in Kit carries out T-A cloning reactions, and reaction system is as follows：
Above-mentioned mixed liquor is mixed, short centrifugation, as 22 DEG C of reaction 1h, the product after connection is used for converting DH5 α competence
Cell.
(7) convert
I. the competent cell for being stored in -80 DEG C is taken out to be placed in and thawed on ice；
II. 50 μ L competent cells are moved in the centrifuge tube of sterilization treatment, add above-mentioned connection product, gently mix,
Ice bath 30min；
III.42 DEG C of thermal shock 45s~60s, is then transferred quickly in ice place 2min, this process should not shake centrifuge tube；
IV. the LB culture mediums of 500 μ L, 37 DEG C of preheatings are added, Tempeerature-constant air shaking table, 37 DEG C, 220rpm, rejuvenation 1h is placed in；
V. take the bacterium solution after appropriate rejuvenation to be coated on the LA solid mediums through IPTG and X-Gal processing, and be placed on
37 DEG C are absorbed to liquid, are inverted, 37 DEG C of culture 12h~16h.
(8) blue hickie screening and bacterium solution PCR identifications
Picking white monoclonal bacterium colony, is placed in 1mL LA fluid nutrient mediums, 37 DEG C on above-mentioned LA culture plates,
220rpm, cultivates 4h；Bacterium solution PCR identifications are carried out using IPCR detection primers.
(9) bacterium solution sequencing and comparison analysis
The above-mentioned PCR monoclonal bacterium solutions for being accredited as the positive are sent into Hua Da gene to be sequenced, and the sequence that success is sequenced
It is listed on NCBI and is compared with pEGFP-sh2 carrier sequences and pig whole genome sequence, integration site is found in analysis.
As shown in Figure 4, sequencing and comparison result show that in TG3 individuals exogenous origin gene integrator is the 10th to testing result
On number chromosome, positioned at No. 10 chromosome two functional gene CUGBP Elav-like family member and trans-
Between acting T-cell-specific transcription factor GATA-3, the TG3 transgene pigs are not interfered with
The expression of normal gene, while exogenous origin gene integrator has higher expression effect in the site.
Embodiment 5：The foundation of the ear skin fibroblast of the anti-PCV2 transgene pigs of TG3.
After the anti-PCV2 transgene pigs births of TG3, identified by PCR and Southern Blot, be defined as follow-up need deeply
The individual of research, adopts its ear skin tissue, and it is that method is as follows to carry out ear skin fibroblast to build in laboratory：
(1) with the tincture of iodine position to be sampled and ear are lacked pincers, tweezers, etc. carry out disinfection, removing ear epidermis as much as possible
The dirt in face, the tissue block of size as one piece of little finger of toe nail of clip is placed in bubble 1min~2min in the tincture of iodine, is then transferred to 75%
Ethanol disinfection liquid carries out de- iodine and simultaneously sterilizes about 30s, then is clamped with tweezers and to be organized in containing rinsing 3 times in 2% dual anti-DPBS, finally
It is quick to be put into equipped with being covered tightly in the 50mL centrifuge tubes containing 2% dual anti-serum-free DMEM, it is placed in ice chest and takes back laboratory as early as possible
The processing of next step is carried out, to ensure the activity of ear chrotoplast；
(2) tissue is taken out from pipe and be placed in Tissue Culture Dish, washed 3~5 times, wiped out with containing 2% dual anti-DPBS
Hair stubble, epidermal cell is gently scraped off with knife blade, retains skin corium, then tissue is steeped in tincture of iodine 30s, 75% ethanol disinfection liquid
De- iodine 30s, rinses 3~5 times containing 2% dual anti-DPBS, ear skin tissue is shredded into fritter with eye scissors；
(3) with being resuspended after complete medium cleansing tissue powder 2 times with 1mL serum, it is then transferred in new culture dish
And it is uniformly distributed, it is put into 37 DEG C, in the cell culture incubator of 5%CO2 and saturated humidity, 5h~7h to be cultivated is to organizing fritter
Complete medium is added after being attached at culture dish bottom；
(4) liquid was changed every 2 days once and tissues observed block peripheral cell climbs out of situation, treats cell length to 80%~90% remittance
Secondary Culture is carried out when right and is frozen.
Testing result is shown in accompanying drawing 5, as a result shows that TG3 ear skin fibroblasts successfully build the cell attachment for being normally, raw
Long speed is good, in spindle shape or irregular shape, rich in third dimension, there is stronger green fluorescence expression.
Embodiment 6：The expression and purification of HTNCre recombinases.
(1) HTNCre recombinase prokaryotic expression plasmids are converted in BL21 (DE3) bacterial strain, 37 DEG C of overnight incubations.In flat board
On choose (contain 50 μ g/mL Amp), 37 DEG C, 250rmp shaken cultivations to OD600=in monoclonal access 3mL LA fluid nutrient mediums
After 0.5~0.6, (a) adds 37 DEG C of induction 3h of 0.5mM IPTG；(b) 0.1mM 16 DEG C of overnight inductions of IPTG are added；SDS-
PAGE detection expression.
(2) BL21 (DE3) of recombinant plasmid transformed expresses inoculation in 100mL LA fluid nutrient mediums, 37 DEG C of cultures
Overnight, it is transferred in 1LLA fluid nutrient mediums within second day, 37 DEG C, after 250rmp shaken cultivations to OD600=1.0, adds 0.1mM
16 DEG C of overnight inductions of IPTG, receive bacterium.
(3) SP-FF column 20mM PB, 200mM NaCl, 1mM EDTA, pH8.0 buffer solution are balanced, with 20mM
PB, 1M NaCl, 1mM EDTA, pH8.0 buffer solution for gradient elution, collect elution fraction.
(4) column of Superdex 200 are with PBS, 10%glycerol, pH7.4 buffer solutions balance, SP-FF column
Elution is collected after component loading, and with PBS, 10%glycerol, pH7.4 buffer solution stepwise elutions are collected elution fraction, are concentrated to give
To protein product, protein concentration is detected.
Testing result as shown in Figure 6, from the PAGE gel electrophoresis detection in accompanying drawing 6, inducible strain BL21
(DE3) overexpression product is about to occur the of a relatively high obvious band of expression quantity at 40kDa in molecular size range, with theory meter
The size 42.76kDa of calculation is consistent, and tentatively judges the band for purpose recombinant protein band.This experiment obtains solubility expression, but
Expression is not obvious, collects lysate supernatant by a large amount of, soluble HTNCre recombinases are finally concentrated and purified to obtain from supernatant
1.2mg, concentration is 0.6mg/mL, is stored in PBS, 10%glycerol, pH7.4 buffer solution, and tubule packing is long after -80 DEG C
Phase is saved backup, and multigelation is avoided as far as possible.
Embodiment 7：The individual ear skin fibroblast Neo/EGFP exogenous marker genes of TG3 are deleted using HTNCre.
(1) the individual ear skin fibroblasts of recovery TG3 are passaged to 6 orifice plates, cultivate bar in 60mm Tissue Culture Dish after covering with
Part is 37 DEG C, 5%CO2.
(2) when cell density is up to 80% or so, washed with DPBS 2 times, add the HTNCre of OPTI-MEM dilutions, make its end
Concentration is 200ng/ μ L, siphons away HTNCre working solutions after being incubated 3h, is washed with DPBS 2 times, adds 10%FBS complete medium
(dual anti-containing 1%) continues to cultivate.
(3) after 24h, with 0.25% trypsin digestion cell, cell is counted with cell counter, it is thin by 200
Born of the same parents/ware is reached in 100mm Tissue Culture Dish, was changed a not good liquor every 3 days and is observed the formational situation of cell monoclonal.
(4) when cell culture was to 10 days or so, and cell clone group cell number reaches 200 or so, with marker
Cell clone group position is irised out in Tissue Culture Dish bottom under the observation of inverted microscope, and is irised out again in cloning cluster periphery
Safety zone without heteroproteose cell, while having unstressed configuration with fluorescence microscope cell clone group, carries out mark, to ensure picking
Purer unstressed configuration cell monoclonal.
(5) complete medium is siphoned away, is washed with DPBS 2 times, made marks thin is carefully accurately placed on by ring is cloned with tweezers
Two are added on born of the same parents clone, in each clone's ring and drips 0.25% pancreatin, when observing under the microscope to cell rounding, add 50 μ L left
Right FBS terminates digestion, and cell is carefully blown afloat with pipettor and is transferred in 24 orifice plates, is entered by the hole -60mm of 24 hole -6 order
Row Secondary Culture, can be divided into two in succeeding generations, and portion is used for PCR identifications, and portion freezes standby.Often pass a generation all need into
Row Fluirescence observation, is further ensured that in the cell monoclonal screened without the heteroproteose cell for not deleting fluorescence labeling.
Testing result recombinates the anti-PCV2 transgene pigs TG3 ears of ferment treatment F1 generation with the HTNCre of expression and purification in embodiment 6
Skin fibroblast, because green fluorescent protein has just existed in HTNCre recombinases before processing, deletes in HTNCre and marks
Its metabolism needs the regular hour after note, so the deleted effect of cell line mark can not be observed substantially in 24h,
Cell has visible green fluorescence all the time.But with the continuation merisis of cell, green fluorescence is more and more weaker.Such as the institute of accompanying drawing 7
Show, TG3 cell lines carry out flow cytometry in latter 7th day in HTNCre processing, find the thin of TG3 expression green fluorescences
Born of the same parents are from marking 99.8% before not deleting to be changed into deleting 4.8% after mark, and mark deletion efficiency is up to 95%.
Testing result is as shown in Figure 8：HTNCre handles the anti-PCV2 transgene pigs TG3 ears skin fibroblast of F1 generation
Afterwards, dilution is cultivated and carries out cell monoclonal screening with clone's ring, finds to delete the cell monoclonal of mark without HTNCre
The stronger green fluorescence (40 ×) of expression, and by HTNCre deletion marks then not it is observed that there is green fluorescence expression.
Simultaneously as marker gene Neo and green fluorescence protein gene EGFP are amalgamation and expression, and positioned at two LoxP it
Between, thus EGFP be deleted while, Neo genes can also be deleted simultaneously.
In other embodiments, HTNCre, which recombinates enzyme concentration, to be 100ng/ μ L, 150ng/ μ L or 300ng/ μ L etc.
Concentration, the effect can with the deletion exogenous marker gene of greater efficiency, simply during preferred use 200ng/ μ L, is deleted
Effect is best.
Embodiment 8：The cell monoclonal for deleting marker gene is detected using PCR.
(1) cell DNA is extracted with reference to the step in embodiment 2；
(2) sequence between Neo/EGFP amalgamation and expressions marker gene and two LoxP is detected using PCR, the primer is such as
Under：
The Neo/EGFP of the anti-PCV2 transgene pigs TG3 cell monoclonals of F1 generation with PCR to screening is marked and two
Flag sequence between LoxP is detected that as shown in Figure 9, the C1~C7 redgreen fluorecytes monoclonal screened is not
Detect the flag sequence between Neo/EGFP marks and LoxP, and plasmid in positive control, after untreated and processing
Cell mixing in then can detect, illustrate in the cell monoclonal screened mark by HTNCre recombinase-mediateds
Delete reaction to delete, its PCR primer sequence verification deleted reaction principle and deleted after mark is shown in accompanying drawing 10, i.e., mark is deleted
Except the sequence between the Neo/EGFP marks of preceding energy amplification to 980bp and 3211bp two LoxP, and mark and expand after deletion
Increase the marks of the Neo/EGFP less than 980bp and can only expand deleted latter remaining to sequence between two LoxP of 530bp
Section.
Embodiment 9：The anti-PCV2 transgene clones pig without Neo/EGFP marker gene is prepared using SCNT.
The individual ear skin fibroblasts of the TG3 without Neo/EGFP marker gene obtained in embodiment 7 are supplied as core
Body cell, is expelled in egg mother cell, builds reconstruct embryo and carries out somatic cell clone (SCNT), and is entered using traditional breeding method
Row is cultivated, you can obtain the transgene pig of the type of resisting porcine circovirus 2.
Embodiment 10：The EGFP without the anti-PCV2 transgene clones pig of Neo/EGFP marker gene is surveyed using bioluminescence lamp inspection
Expression.
Detection method be the same as Example 1.
EGFP expression is carried out without Neo/EGFP marker gene anti-PCV2 transgene clones pig to acquisition with bioluminescence lamp
Detection, the similar expression EGFP of selection size pig is as control, the visible obvious EGFP expression of control pig, especially in mouth
Hoof, and do not observe EGFP expression then without the anti-PCV2 transgene clones pig of Neo/EGFP marks, such as accompanying drawing 11 judges to pass through
What embodiment 9 was obtained marks anti-PCV2 transgene clones pig its EGFP mark to be deleted without Neo/EGFP, due to Neo genes with
EGFP gene amalgamation and expression, when EGFP gene is not expressed, Neo genes will not also be expressed.
Embodiment 11：The anti-PCV2 transgene clones pig without Neo/EGFP marker gene is detected using PCR.
Detection method be the same as Example 8.Further detect that as a result testing result such as accompanying drawing 12 shows, in embodiment 9 through PCR
The anti-PCV2 transgenosis piggy obtained successfully deletes Neo/EGFP exogenous marker genes, and target gene resisting porcine circovirus 2
Type siRNA gene orders are retained, and also further demonstrate that screening is used to prepare the thin of unmarked anti-PCV2 transgene clones pig
Born of the same parents' monoclonal is purer and successfully deletes marker gene, is consistent with green fluorescence testing result.Show to obtain by the above method
Obtained the anti-PCV2 transgene clones pig of no external source Neo/EGFP marker gene.
Above-described is only some embodiments of the present invention.For the person of ordinary skill of the art, not
On the premise of departing from the invention design, various modifications and improvements can be made, these belong to the protection domain of invention.
Claims (8)
1. a kind of transgene pig of the type of resisting porcine circovirus 2, it is characterised in that the transgene pig of the described type of resisting porcine circovirus 2
Can be with the siRNA of the type of specifically expressing resisting porcine circovirus 2.
2. the transgene pig of the type of resisting porcine circovirus 2 according to claim 1, it is characterised in that described anti-pig circular ring virus 2
The transgene pig of malicious 2 types does not include exogenous marker gene.
3. the transgene pig of the type of resisting porcine circovirus 2 according to claim 2, it is characterised in that described exogenous marker base
Because Neo/EGFP amalgamation and expression genes.
4. the transgene pig of the type of resisting porcine circovirus 2 according to claim 3, it is characterised in that described anti-pig circular ring virus 2
The type siRNA of resisting porcine circovirus 2 of malicious 2 type transgene pigs gene order is incorporated into No. 10 chromosome of the transgene pig
On.
5. the transgene pig of the type of resisting porcine circovirus 2 according to claim 4, it is characterised in that described anti-pig circular ring virus 2
2 type siRNA of poison gene order is located at two functional gene CUGBP Elav-like family member of No. 10 chromosome
Between trans-acting T-cell-specific transcription factor GATA-3.
6. the transgene pig of the type of resisting porcine circovirus 2 according to claim any one of 1-5, it is characterised in that described is anti-
Porcine circovirus 2 type transgene pig is implemented by the following steps：
(1) the type expression vector of resisting porcine circovirus 2, is built, the carrier includes the siRNA of the type of target gene resisting porcine circovirus 2
Expressing gene sequence, marker gene Neo/EGFP amalgamation and expression gene orders；
(2), by the expression vector of the type of resisting porcine circovirus 2 of acquisition and transposase plasmids mixing cotransfection pig fetus into fiber finer
Born of the same parents be, and screen can stable expressed vector monoclonal transgenic cell line；
(3), the monoclonal transgenic cell line of acquisition is expelled in egg mother cell as nuclear donor cell, so as to build weight
Structure embryo, and embryo is cultivated by somatic cell clone technique and traditional breeding method, F0 is obtained for resisting porcine circovirus 2
The transgene pig of type；
(4), by the F0 of acquisition for the transgene pig of the type of the resisting porcine circovirus 2 wild type sow consistent with kind, genetic background
Crossbreed obtains the transgene pig for the type of F1 generation resisting porcine circovirus 2 that can stablize heredity；
(5), the type transgene pig of F1 generation resisting porcine circovirus 2 of acquisition is detected, and screens the wherein type of resisting porcine circovirus 2
SiRNA expression quantity is high, and the F1 generation transgene pig individual of only one of which foreign gene insertion point；
(6) the F1 generation transgene pig filtered out individual, is subjected to exogenous origin gene integrator Locus Analysis in Shoots；
(7) the F1 generation transgene pig individual filtered out, is taken into ear skin fibroblast, and sets up ear skin fibroblast；
(8), recombinate the ear skin fibroblast of ferment treatment F1 generation transgene pig using HTNCre to delete Neo/EGFP marks
Gene, and cell monoclonal culture is carried out, so as to screen and delete the cell monoclonal of Neo/EGFP marker gene；
(9) it is, that the nuclear donor of the cell monoclonal for deleting Neo/EGFP marker gene as the somatic cell clone that filter out is thin
Born of the same parents, are expelled in egg mother cell, so that reconstruct embryo is built, then by somatic cell clone technique and traditional breeding method to embryo
Tire is cultivated, and obtains the transgene pig of resisting porcine circovirus 2 type of the F2 generations without Neo/EGFP marker gene.
7. the transgene pig of the type of resisting porcine circovirus 2 according to claim 6, it is characterised in that described in step (8)
Recombinate the ear skin fibroblast of ferment treatment F1 generation transgene pig to delete the step of Neo/EGFP marker gene using HTNCre
It is rapid as follows：
(1), the type transgene pig ear skin fibroblast of recovery F1 generation resisting porcine circovirus 2 is passed in Tissue Culture Dish after covering with
To 6 orifice plates, condition of culture is 37 DEG C, 5%CO2；
(2), when cell density is up to 80% or so, washed with DPBS 2 times, add the HTNCre recombinases of OPTI-MEM dilutions, make
Final concentration of 100ng/ μ L, 150ng/ μ L, 200ng/ μ L or 300ng/ the μ L of HTNCre recombinases, are incubated after 3h and siphon away HTNCre weights
Group enzyme working solution, is washed 2 times with DPBS, and the complete medium for adding 10%FBS continues to cultivate；
(3), after 24h, with 0.25% trypsin digestion cell, cell is counted, it is thin by 200 cells/ware reaches 100mm
In born of the same parents' culture dish, changed a not good liquor every 3 days and observe the formational situation of cell monoclonal；
(4), when cell culture was to 10 days or so, when cell clone group cell number reaches 200 or so, in the sight of inverted microscope
Examine down and iris out cell clone group position in Tissue Culture Dish bottom, and the safety of no heteroproteose cell is irised out again in cloning cluster periphery
Region, while having unstressed configuration with fluorescence microscope cell clone group, carries out mark, to ensure that the purer unstressed configuration of picking is thin
Born of the same parents' monoclonal；
(5) complete medium, is siphoned away, is washed with DPBS 2 times, clone's ring is carefully accurately placed on to the cell gram made marks with tweezers
Two are added on grand, in each clone's ring and drips 0.25% pancreatin, when observing under the microscope to cell rounding, add 50 μ L's or so
FBS terminates digestion, and cell is carefully blown afloat with pipettor and is transferred in 24 orifice plates, is passed by the hole -60mm of 24 hole -6 order
It is commissioned to train foster, to obtain the cell monoclonal of marker-free luciferase expression.
8. the transgene pig of the type of resisting porcine circovirus 2 according to claim 7, it is characterised in that described HTNCre weights
Group enzyme concentration is 200ng/ μ L.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108795935A (en) * | 2018-05-23 | 2018-11-13 | 中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心) | The siRNA of 1 genes of Schistosoma japonicum SjELAV-like and its application |
| CN108795934A (en) * | 2018-05-23 | 2018-11-13 | 中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心) | The siRNA of 2 genes of Schistosoma japonicum SjELAV-like and its application |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101550427A (en) * | 2009-02-28 | 2009-10-07 | 吉林大学 | Transgene report pig detecting Cre site special recombinase active and culturing method |
| CN101760473A (en) * | 2008-12-25 | 2010-06-30 | 北京济普霖生物技术有限公司 | Method for deleting exogenous gene in transgenic cell at fixed point |
| CN102226201A (en) * | 2011-05-09 | 2011-10-26 | 新疆农垦科学院 | Expression vector, and its construction and application |
| CN102851311A (en) * | 2012-02-24 | 2013-01-02 | 浙江大学 | Construction method of transgenic pig resisting porcine circovirus type 2 |
| CN103468733A (en) * | 2013-07-26 | 2013-12-25 | 吴珍芳 | Expression vector resisting porcine circovirus type 2 (PCV2) and transgenic pig, and construction methods thereof |
| CN104419719A (en) * | 2013-09-02 | 2015-03-18 | 中国农业大学 | Method for knocking out selective marker gene of transgenic pig |
-
2017
- 2017-05-09 CN CN201710321636.XA patent/CN107018955A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101760473A (en) * | 2008-12-25 | 2010-06-30 | 北京济普霖生物技术有限公司 | Method for deleting exogenous gene in transgenic cell at fixed point |
| CN101550427A (en) * | 2009-02-28 | 2009-10-07 | 吉林大学 | Transgene report pig detecting Cre site special recombinase active and culturing method |
| CN102226201A (en) * | 2011-05-09 | 2011-10-26 | 新疆农垦科学院 | Expression vector, and its construction and application |
| CN102851311A (en) * | 2012-02-24 | 2013-01-02 | 浙江大学 | Construction method of transgenic pig resisting porcine circovirus type 2 |
| CN103468733A (en) * | 2013-07-26 | 2013-12-25 | 吴珍芳 | Expression vector resisting porcine circovirus type 2 (PCV2) and transgenic pig, and construction methods thereof |
| CN104419719A (en) * | 2013-09-02 | 2015-03-18 | 中国农业大学 | Method for knocking out selective marker gene of transgenic pig |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108795935A (en) * | 2018-05-23 | 2018-11-13 | 中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心) | The siRNA of 1 genes of Schistosoma japonicum SjELAV-like and its application |
| CN108795934A (en) * | 2018-05-23 | 2018-11-13 | 中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心) | The siRNA of 2 genes of Schistosoma japonicum SjELAV-like and its application |
| CN108795935B (en) * | 2018-05-23 | 2022-06-21 | 中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心) | SiRNA of SjELAV-like 1 gene of schistosoma japonicum and application thereof |
| CN108795934B (en) * | 2018-05-23 | 2022-06-21 | 中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心) | SiRNA of schistosoma japonicum SjELAV-like 2 gene and application thereof |
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Application publication date: 20170808 |