CN107177630A - A kind of anti-PCV2 transgene pigs preparation method without exogenous marker gene - Google Patents
A kind of anti-PCV2 transgene pigs preparation method without exogenous marker gene Download PDFInfo
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Abstract
The invention discloses a kind of anti-PCV2 transgene pigs preparation method without exogenous marker gene.The anti-PCV2 transgene pigs without exogenous marker gene are by the way that target gene (anti-PCV2 siRNA sequences) and exogenous marker gene (Neo/EGFP amalgamation and expressions gene) are built into coexpression vector, and pass through transfection, the technologies such as somatic cell clone obtain F0 for anti-PCV2 transgene pigs, and the F0 of acquisition is subjected to the breeding acquisition anti-PCV2 transgene pigs of F1 for transgene pig, PCV2 transgene pigs anti-to F1 generation are identified, filter out suitable F1 transgene pigs and extract ear skin fibroblast, progress, which is built, is, and the cell line for deleting Neo/EGFP exogenous marker genes is filtered out with HTNCre recombinases, and carry out somatic cell clone using the cell line, finally obtain the anti-PCV2 transgene pigs without exogenous marker gene.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, prepared by more particularly to a kind of anti-PCV2 transgene pigs without exogenous marker gene
Method.
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 preparation method of the anti-PCV2 without exogenous marker gene, to solve
Above mentioned problem.
According to an aspect of the invention, there is provided a kind of anti-PCV2 transgene pigs preparation side without exogenous marker gene
Method, it is implemented by the following steps：
(1), the structure of anti-PCV2 expression vectors, the carrier includes the anti-PCV2 of target gene siRNA expressing gene sequences
With marker gene sequence；
(2), screening can stable expressed vector monoclonal transgenic cell line；
(3), using the monoclonal transgenic cell line of acquisition as nuclear donor cell, carry out body-cell neucleus transplanting and obtain F0 generations
Anti- PCV2 transgene pig；
(4), obtaining the F0 of acquisition for anti-PCV2 transgene pig and sow crossbreed can stable hereditary anti-PCV2
F1 generation transgene pig；
(5), screening F1 generation transgene pig is individual and carries out exogenous origin gene integrator Locus Analysis in Shoots；
(6) the F1 generation transgene pig individual filtered out, is taken into ear skin fibroblast, and carries out ear skin fibroblast
The foundation of system；
(7) cell monoclonal for deleting exogenous marker gene, is set up, and carries out somatic cell clone and obtains F2 generations without external source
The anti-PCV2 of marker gene transgene pig.
Thus, the anti-PCV2 obtained by the above method transgene pig, can be with the anti-PCV2siRNA of specifically expressing, to pig
PCV-II disease plays epidemic prevention effect；The transgene pig is free of exogenous marker gene simultaneously, pig health will not be produced extra
Burden and influence, while transgene pig safety evaluation program can also be simplified, be conducive to the Industry Promotion of the transgene pig
And application.
In some embodiments, above-mentioned exogenous marker gene is Neo/EGFP amalgamation and expression genes, and Neo genes are a kind of
Antibiotics resistance gene, cell, which expresses the gene, can inactivate aminoglycosides antibiotics, destroy G418, and often as one kind
Riddled basins are used for the screening of transgenosis monoclonal cell；And EGFP is egfp expression gene and target gene
When common transfection builds transgenosis monoclonal cell system, it is easy to intuitively observe the selection result with microscope, to transgenic animals
Can also be by observing the expression of green fluorescent protein come the preliminary result for judging transgenosis, but EGFP green fluorescent proteins are thin
Born of the same parents' horizontal expression can produce certain toxicity to cell, and also certain shadow can be produced to the health of pig in transgene pig individual
The shadow to the non-transgenic healthy can be reduced by ringing, therefore deleting the transgene pig of Neo/EGFP amalgamation and expression exogenous marker genes
Ring, 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 anti-PCV2 transgene pigs of F1 generation be by F0 for anti-PCV2 transgene pigs and same kind,
And the consistent wild type sow crossbreed of genetic background.
In some embodiments, F0 is for anti-PCV2 transgene pigs and same kind, and the consistent wild type of genetic background is female
Pig crossbreed and the standard of F1 generation anti-PCV2 transgene pigs screening come is the anti-PCV2siRNA of efficient specifically expressing, it is and anti-
The integration site only one of which of PCV2siRNA gene orders, the anti-PCV2 transgene pigs of F1 generation thus screened both can be steady
The fixed heredity anti-PCV2siRNA of target gene, simultaneously because integration site only one of which, is easy to orientation to delete with target gene simultaneously
It is incorporated into the exogenous marker gene on transgene pig chromosome.
In some embodiments, the step of heterogenous expression gene is deleted by HTNCre recombinases is as follows：
(1), the anti-PCV2 transgene pigs ear skin fibroblast of recovery F1 generation；
(2), when cell growth state is good, ferment treatment cell line is recombinated with HTNCre；
(3), after 24h, Secondary Culture is carried out to the cell line after processing, and select redgreen luciferase expression under the microscope
Cell monoclonal group, and carry out purifying culture, that is, obtain the cell monoclonal of no Neo/EGFP marker gene.
Thus, the LOXP sites of foreign gene both sides are utilized by HTNCre recombinases, you can to delete exogenous marker base
Cause, and the cell deleted after marker gene does not express green fluorescence, and the cell for not expressing green fluorescence is chosen in cell mass, is entered
Row purifying, which is built, is, you can to obtain the cell monoclonal of no Neo/EGFP marker gene.
In some embodiments, when HTNCre recombinates ferment treatment cell line deletion exogenous marker gene, specific requirement is such as
Under：When the anti-PCV2 transgene pigs ear skin fibroblast density of F1 generation is up to 80% or so, washed with DPBS 2 times, add OPTI-
The HTNCre recombinases of MEM dilutions, make its final concentration of 200ng/ μ L, siphon away HTNCre working solutions after being incubated 3h, 2 are washed with DPBS
Secondary, the complete medium for adding 10%FBS continues to cultivate cell, it is possible thereby to obtain optimal deletion efficiency, and obtains and can protect
Cell growth state after card deletion marker gene is preferable.
In some embodiments, select redgreen luciferase expression cell monoclonal group comprise the following steps that：Work as warp
The cell culture of HTNCre restructuring ferment treatments is aobvious being inverted when cell clone group cell number reaches 200 or so to 10 days or so
Cell clone group position is irised out in Tissue Culture Dish bottom under the observation of micro mirror, and is irised out again without miscellaneous thin in cloning cluster periphery
The safety zone of born of the same parents, while having unstressed configuration with fluorescence microscope cell clone group, carries out mark, to ensure that picking is purer
Unstressed configuration cell monoclonal.Thus, it is possible to the accurate cell mass for picking out redgreen fluorescent protein expression, build after being culture,
Ensure in cell line without the heteroproteose cell for not deleting marker gene.
In some embodiments, comprising the following steps that for the cell monoclonal without Neo/EGFP marker gene is obtained：Mark
Remember after unstressed configuration cell monoclonal, siphoned away cell culture complete medium, washed with DPBS 2 times, it is carefully accurate to clone ring with tweezers
It is placed on the cell clone made marks, adding two in each clone's ring drips 0.25% pancreatin, observes under the microscope to cell
Become bowlder, the FBS for adding 50 μ L or so terminates digestion, and cell is carefully blown afloat with pipettor and is transferred in 24 orifice plates, by 24 holes-
6 hole -60mm order carries out Secondary Culture, to obtain the cell monoclonal of marker-free luciferase expression.Thus, it is possible to ensure
Cell line ensures that the cell line growth state filtered out is preferable without the heteroproteose cell for not deleting marker gene.
In some embodiments, the final concentration of HTNCre recombinases can also be 100ng/ when deleting exogenous marker gene
μ L, 150ng/ μ L or 300ng/ μ L isoconcentrations, simply preferred selection 200ng/ μ L.
The above-mentioned anti-PCV2 transgene pigs preparation method without exogenous marker gene, has the advantages that：Without external source
The anti-PCV2 transgene pigs of marker gene are to contain target gene (anti-PCV2siRNA genes) and exogenous marker gene by building
The coexpression vector of (Neo/EGFP amalgamation and expressions gene), carries out transfection prepare transgenosis monoclonal cell system, then with this list
Cloned cell line as somatic cell clone technique confession somatic cell, to obtain the F0 containing target gene and exogenous marker gene
For anti-PCV2 transgene pigs, then obtained by the F0 generation wild type Duluke's sow crossbreeds consistent with kind, genetic background
Can stable heredity anti-PCV2 F1 generation transgene pig, it is high to filter out anti-PCV2siRNA expression quantity, and only one of which integration position
The F1 generation transgene pig individual of point carries out follow-up study, takes the ear skin of the F1 generation transgene pig individual that this filters out into fiber finer
Born of the same parents carry out culture, and HTNCre restructuring ferment treatments are carried out to the ear skin fibroblast of acquisition, and filter out and delete mark
Remember the cell monoclonal of gene, using this cell monoclonal as the donor cell of somatic cell clone technique, carry out somatic cell clone
Anti- PCV2 transgene pig of the F2 generations without exogenous marker gene is obtained, and the F2 is detected for transgene pig, it was demonstrated that mark
Gene has been deleted, but anti-PCV2siRNA gene orders are retained.The resisting without exogenous marker gene obtained by the above method
PCV2 transgene pigs can be with the anti-PCV2siRNA of specifically expressing, to suppress PCV2 Expression and biological activity, so as to play preventing and treating pig
Infect the effect of PCV-II disease.Meanwhile, the transgene pig deletes exogenous marker gene, reduces foreign gene to turning base
Because of the influence of pig individual health, while transgene pig safety evaluation program can also be simplified, be conducive to the industry of the transgene pig
Change and promote and apply.
Brief description of the drawings
Fig. 1 is the moderate resistance PCV2 transgene pig F1 generation EGFP exogenous marker gene green fluorescence expressions of embodiment 1；
Fig. 2 is that (the Southern Blot testing results of transgene pig F1 generation, wherein MW is DNA to the moderate resistance PCV2 of embodiment 2
Molecular weight standard, Transgenic is the anti-PCV2 transgene pigs sample of F1 generation, and Wild-type is wild type control pig sample；
Fig. 3 is siRNA relative expression's situations between the anti-PCV2 transgene pigs of different F1 generations in embodiment 3, wherein " 3 " are identified
TG3 individuals；
Fig. 4 is the individual target gene integration site sequencing comparison diagrams of the anti-PCV2 transgene pigs TG3 of F1 generation in embodiment 4；
Fig. 5 is that F1 generation anti-PCV2 transgene pigs TG3 ears skin fibroblast sets up result figure in embodiment 5；
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 glimmering for FCM analysis before and after the HTNCre processing anti-PCV2 transgene pigs TG3 cell lines of F1 generation in embodiment 7
Light result figure；
Fig. 8 is the front and rear luciferase expression detection knot of the anti-PCV2 transgene pigs TG3 cell clones deletion mark of F1 generation in embodiment 7
Fruit is schemed；
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 expression result figures of the anti-PCV2 transgene pigs without exogenous marker gene in embodiment 10；
Figure 12 is the PCR testing result figures of the anti-PCV2 transgene pigs without exogenous marker gene in embodiment 11.
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 anti-PCV2 transgene pigs of signified F1 generation are by Patent No. 201310321355.6 in the present invention, and patent name is
The anti-pig circular ring virus 2 that the method that " expression vector and transgene pig and its construction method of the type of resisting porcine circovirus 2 " is announced is obtained
Obtained by the wild type Duluke's sow crossbreed consistent with kind, genetic background of malicious 2 type transgene clone pigs (F0 generations).And specially
Profit number is 201310321355.6, and patent name is " expression vector of the type of resisting porcine circovirus 2 and transgene pig and its structure side
SiRNA of the type transgene clone pig (F0 generations) of resisting porcine circovirus 2 that method " is announced comprising the type of resisting porcine circovirus 2, screening base
Because of Neo, the foreign gene such as Green Fluorescent Protein gene EGFP, wherein Neo and EGFP gene amalgamation and expression, the F0 is for anti-pig
Circovurus type 2 transgene pig derives from this school.
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
(Hind III), then designs the control of 1 copy number, with F0 for anti-PCV2 transgene pigs genomic DNA with pEGFP-sh2 plasmids
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) by reagent each component in thawing 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 on ice
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 20 × Taqman of qPCR siRNAAssay 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-PCV2siRNA 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 Hind III 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, the wherein TRE of PB 3 ' primary and CMV primary is a pair, is turned for expanding
Stand 3 ' is held and the genome sequence near integration site；PB 5 ' TRE primary and Neo primary are a pair, for expanding
Increase the genome sequence near the end of transposons 5 ' and 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 the α of DH 5 impressions
State 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, 1MNaCl, 1mM EDTA, pH8.0 buffer solution for gradient elution, collect elution fraction.
(4) Superdex 200column 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 anti-PCV2 transgene pig.Embodiment 10：Surveyed using bioluminescence lamp inspection without Neo/EGFP marker gene
The EGFP expression of anti-PCV2 transgene clones pig.
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 anti-PCV2siRNA bases of target gene
Because sequence is retained, also further demonstrate that screening be used to preparing the cell monoclonal of unmarked anti-PCV2 transgene clones pig compared with
It is pure and successfully delete marker gene, it is consistent with green fluorescence testing result.Show to obtain no external source by the above method
The anti-PCV2 transgene clones pig of 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 (9)
1. a kind of anti-PCV2 transgene pigs preparation method without exogenous marker gene, it is characterised in that comprise the following steps：
(1), build anti-PCV2 expression vectors, described siRNA expressing gene sequence of the carrier comprising the anti-PCV2 of target gene and
Marker gene sequence；
(2), screening can stable expressed vector monoclonal transgenic cell line；
(3), using the monoclonal transgenic cell line of acquisition as nuclear donor cell, carry out body-cell neucleus transplanting and obtain F0 for anti-
PCV2 transgene pig；
(4), described F0 is obtained to the F1 for the anti-PCV2 that can stablize heredity for anti-PCV2 transgene pig and sow crossbreed
For transgene pig；
(5), screen described F1 generation transgene pig individual and carry out exogenous origin gene integrator Locus Analysis in Shoots；
(6) ear skin fibroblast, is taken to the F1 generation transgene pig individual filtered out, and carries out ear skin fibroblast
Set up；
(7) cell exogenous marker gene, is deleted, cell monoclonal is resettled, and carry out somatic cell clone acquisition F2 generations without external source
The anti-PCV2 of marker gene transgene pig.
2. the anti-PCV2 transgene pigs preparation method according to claim 1 without exogenous marker gene, it is characterised in that institute
The exogenous marker gene stated is Neo/EGFP amalgamation and expression genes.
3. the anti-PCV2 transgene pigs preparation method according to claim 1 without exogenous marker gene, it is characterised in that step
Suddenly the sow described in (4) be with described F0 for anti-PCV2 transgene pig is with kind and the consistent wild type of genetic background is female
Pig.
4. the anti-PCV2 transgene pigs preparation method according to claim 1 without exogenous marker gene, it is characterised in that step
Suddenly the F1 generation transgene pig individual of screening mainly has following feature in (5)：Can the anti-PCV2 siRNA of efficient specifically expressing；Purpose
The anti-PCV2 siRNA sequences integration site of gene is single and can stable heredity.
5. the anti-PCV2 transgene pigs preparation method according to claim 1 without exogenous marker gene, it is characterised in that step
Suddenly cell exogenous marker gene is deleted in (7) to realize using HTNCre recombinases, concretely comprise the following steps：
(1), the anti-PCV2 transgene pigs ear skin fibroblast of recovery F1 generation；
(2), when cell growth state is good, ferment treatment cell line is recombinated with HTNCre；
(3), after 24h, Secondary Culture is carried out to the cell line after processing, and select the thin of redgreen luciferase expression under the microscope
Born of the same parents' monoclonal group, and carry out purifying culture, that is, obtain the cell monoclonal of no Neo/EGFP marker gene.
6. the anti-PCV2 transgene pigs preparation method according to claim 5 without exogenous marker gene, it is characterised in that institute
State HTNCre restructuring ferment treatment cell line method be：Treat that the anti-PCV2 transgene pigs ear skin fibroblast density of F1 generation reaches
When 80% or so, washed with DPBS 2 times, add the HTNCre recombinases of OPTI-MEM dilutions, described HTNCre recombinases are dense eventually
Spend for 100ng/ μ L, 150ng/ μ L, 200ng/ μ L or 300ng/ μ L, siphon away HTNCre working solutions after being incubated 3h, 2 are washed with DPBS
Secondary, the complete medium for adding 10%FBS continues to cultivate cell.
7. the anti-PCV2 transgene pigs preparation method according to claim 5 without exogenous marker gene, it is characterised in that institute
State select redgreen luciferase expression cell monoclonal group method be：When through HTNCre recombinate ferment treatment cell culture extremely
10 days or so, when cell clone group cell number reaches 200 or so, in Tissue Culture Dish bottom under the observation of inverted microscope
Cell clone group position is irised out, and the safety zone of no heteroproteose cell is irised out again in cloning cluster periphery, while using fluorescence microscopy
There is unstressed configuration in sem observation cell clone group, carries out mark, to ensure unstressed configuration cell monoclonal that picking is purer.
8. the anti-PCV2 transgene pigs preparation method according to claim 5 without exogenous marker gene, it is characterised in that institute
The method of cell monoclonal of the acquisition stated without Neo/EGFP marker gene is：Mark after unstressed configuration cell monoclonal, siphoned away
Cell culture complete medium, is washed 2 times with DPBS, is carefully accurately placed on the cell clone made marks with tweezers by ring is cloned, often
Two are added in individual clone's ring and drips 0.25% pancreatin, when observing under the microscope to cell rounding, the FBS for adding 50 μ L or so is terminated
Digestion, cell is carefully blown afloat with pipettor and is transferred in 24 orifice plates, and Secondary Culture is carried out by the hole -60mm of 24 hole -6 order,
To obtain the cell monoclonal of marker-free luciferase expression.
9. the anti-PCV2 transgene pigs preparation method according to claim 6 without exogenous marker gene, it is characterised in that institute
The final concentration of 200ng/ μ L for the HTNCre recombinases stated.
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