CN103725710B - One oneself can delete free carrier and application thereof - Google Patents
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Abstract
The present invention relates to genetically engineered field, specifically provide one and oneself can delete free carrier, the Core Feature region of described free carrier comprises from 5 ' end to 3 ' end: people EF1 α promoter sequence, loxp sequence, EGFP encoding sequence, derives from the S/MAR sequence of HuIFN-β, the PolyA sequence of SV40, the expression cassette sequence of mouse Oct4 promoters driven Cre gene, loxp sequence.Utilize carrier of the present invention to carry out the preparation of gene targeting cell and animal, can riddled basins be deleted and gene insertion mutation can not be produced, eliminate the impact that marker gene pairs contiguous gene is expressed and the potential source biomolecule potential safety hazard that body is produced.
Description
Technical field
The present invention relates to genetically engineered field, specifically, relating to one oneself can delete free carrier and application thereof.
Background technology
The features such as pig physiological characteristic and life cycle similar to the mankind is long, are considered to desirable animal model.Therefore the research model of similar human inheritance's disease can be prepared to the genomic modification of pig, the pathological study of human diseases and the research and development of medicine can be should used widely.In addition pork is also the main animal protein sources of our people, and the standard of living for the people improves and plays key effect.Along with China's expanding economy, the raising of people's lives life, Chinese's pork demand is also increasing, and the pork amount of the annual consumption of China according to statistics accounts for the over half of whole world total amount.The expanded demand in market, facilitates the fast development of China's modernization large-scale cultivation industry.But also severe challenge is proposed simultaneously.In China, main herding veriety depends critically upon import, and pig variety degree of dependence is close to 90%.This means the source of the industry of pig-breeding---breeding stock is controlled by European & American Market completely, the serious food safety threatening China.Cultivating the pig new product of independent intellectual property right is present stage China agricultural problem in the urgent need to address.Traditional hereditary and selection method cycle length takes effect slow, enters to be difficult to cultivate in a short time to have high productivity energy pig new variety.After Cloning Mammals from Somatic Cells in 1997 occurs, the genetic engineering breeding of domestic animal is developed rapidly, and it can improve the production performance of domestic animal in a short time rapidly for single traits.Early stage genetic engineering breeding, mainly foreign gene random integration is in genome, and exogenous gene expression is difficult to regulation and control, and expression amount is unstable, easily produces the insertion mutation of gene, produces very important potential safety hazard to transgenic animal itself.Zinc finger nuclease (ZFN), TALEN, CRISPR/Cas9 are the molecular tools developing the genome fixed point transformation that may be used for complexity in recent years.It is prepared simply compared with traditional gene targeting, and efficiency is high, is widely used in the meticulous modification of genome of people and mouse cell.At present, these editing techniques are also applied to the genome pointed decoration of large animal.Zinc finger nuclease (ZFN), TALEN, CRISPR/Cas9 gene editing technology be combined with somatic cell clone can fast, the meticulous modification of genome of the pig of effective implemention, be widely used in production performance improvement and the rearing new variety of pig in the future.Because the fetal fibroblast of pig cannot realize single cell culture, therefore when use Zinc finger nuclease (ZFN), TALEN, CRISPR/Cas9 realize the gene knockout of pig cell, necessarily there is the assisting sifting of marker gene, the cell clone knocked out could be obtained.Current method is screened containing neomycin resistance gene expression vector and Zinc finger nuclease (ZFN), TALEN or CRISPR/Cas9 cotransfection with one section, although can obtain the cell clone of gene knockout, marker gene is also incorporated in the genome of cell simultaneously.Marker gene and selected marker refer to the marker gene that transformant (or individual) can be made to screen from numerous non-transformed cells in genetic transformation, they can produce the product certain selective agent to resistance by render transgenic cell usually, thus render transgenic cell normal growth on the substratum adding this selection, Nontransgenic cells then shows to the sensitivity of this selective agent because transgene efficiency is low owing to lacking resistance, and therefore marker gene can screen and enrichment positive colony.But marker gene mainly contains following shortcoming: the first, this marker gene pairs contiguous gene is expressed impact. the second transgenic animal containing marker gene cannot be assessed by the Biosafety of animal, hinder the commercial applications of transgenic pig.
Although marker gene also directly can be deleted by cre/loxp system, but delete marker gene and in genome, leave loxp sequence simultaneously, the loxp sequence of radom insertion probably produces the insertion mutation of gene, potential Biosafety hidden danger is produced to body, be difficult to the safety assessment by transgenic animal equally, limit the production application of transgenic animal.
Summary of the invention
In order to solve problems of the prior art, the object of this invention is to provide a kind of can delete flag gene can not produce again gene insertion mutation can oneself delete free carrier.
In order to realize the object of the invention, first the present invention provides one oneself can delete free carrier, the Core Feature region of described free carrier comprises from 5 ' end to 3 ' end: people EF1 α promoter sequence, loxp sequence, EGFP encoding sequence, derives from the S/MAR sequence of HuIFN-β, the PolyA sequence of SV40, the expression cassette sequence of mouse Oct4 promoters driven Cre gene, loxp sequence.
Further, described free carrier contains riddled basins, and described riddled basins is resistant gene.
Present invention also offers and utilize aforementioned free carrier to prepare the method for the gene-targeted animals of marker-free, its concrete steps comprise:
1) by described free carrier and ZFN, TALEN or CRISPR/Cas9 cotransfection foetal animal somatocyte, the screening of medicine helper obtains Knockout cells clone;
2) using Knockout cells as nuclear transfer donor cell, in vitro ovocyte, as nuclear transplantation recipient cell, obtains animal cloning embryo by nuclear transfer technology; Clone embryos expresses Oct4 gene from two-cell stage, simultaneously the expression of mouse Oct4 promotor cre enzyme, and the site specific recombination that cre enzyme is mediated mediation free carrier oneself delete;
3) clone embryos is moved in animal uterus by modus operandi and carries out gestation, obtains the gene-targeted animals of marker-free.
Further, described animal is pig, ox, sheep.
Medicine described in step 1) is G418.
Present invention also offers and utilize aforementioned free carrier present invention also offers to utilize aforementioned free carrier preparing the application in marker-free zooblast.
Present invention also offers and utilize the application of aforementioned free carrier in the gene-targeted animals preparing marker-free.
Beneficial effect of the present invention is:
Cre/loxP site differential recombination enzyme system and S/MAR unit construction are formed a kind of controlled free carrier by the present invention.The wherein people bate interferon gene that derives from of S/MAR, when carrier exist S/MAR sequence and can transcribed time, carrier can self-replacation in cell, and pass to daughter cell with cell fission, and namely carrier can form genetic stability without the need to being incorporated into cellular genome.A loxp sequence in the same way is respectively inserted in the sequence upstream and downstream including S/MAR element during design vector, Oct4 promotor can drive cre gene to express at clone embryos, S/MAR on carrier in embryonic cell deletes by the site specific recombination reaction of cre enzyme mediation, the carrier losing S/MAR element cannot self-replacation and form free episome state again, finally causes carrier deleted from cell.The present invention finds that this carrier effectively can be applied to the transgenic pig preparing marker-free (marker free), the gene knock-out pig of especially ZFN, TALEN, CRISPR/Cas9 mediation.Fetal somatic cell due to pig cannot realize single cell culture (separate cell cannot breed formation cell mass), therefore when utilizing ZFN, TALEN, CRISPR/Cas9 to prepare genetic modification pig fetal somatic cell, need and the carrier cotransfection of expressing drug resistance gene, more just can obtain the cell clone of gene knockout with drug screening.Although this method can obtain the pig fetal somatic cell of gene knockout, resistant gene also will be incorporated into cellular genome simultaneously.The free carrier that can self-ly delete utilizing us to build and ZFN, TALEN, CRISPR/Cas9 cotransfection, assisting sifting (expression neomycin resistance gene) can obtain Knockout cells clone efficiently.After body-cell neucleus transplanting, in clone embryos, Oct4 promoters driven Cre expression of enzymes deletes the carrier being used for assisting sifting.Embryo transfer can obtain the gene knock-out pig of marker-free.The method also may be used for the screening of other species simultaneously, and the preparation of induced dry-cell.
Carrier of the present invention is utilized to prepare gene knock-out pig in conjunction with Zinc finger nuclease (ZFN), TALEN, CRISPR/Cas9 gene editing technology, except accurately being modified of goal gene, its genome is without other any changes, this point is better than the marker free method of transgenic pigs all at present, the present invention simultaneously also can be used to produce ox, other domestic animal transgenic breedings such as sheep.
Accompanying drawing explanation
Fig. 1 is the free vector construction schema that oneself of the present invention deletes.
Fig. 2 is the free Vector map that oneself of the present invention deletes.
Fig. 3 is the gene sequencing qualification result of gene knock-out pig in the embodiment of the present invention 4.
Embodiment
Following examples for illustration of the present invention, but are not used for limiting the scope of the invention.
In embodiment, carrier design is completed by this seminar, and the carrier of use all comes from Agricultural biotechnologies National Key Laboratory of China Agricultural University, and oligoDNA is synthesized by the raw work in Shanghai, and sequencing is completed by Beijing Hua Da.Taq enzyme, T4DNA ligase enzyme, restriction endonuclease all from Beijing NEB company, the equal available from Sigma of somatic cell clone agents useful for same.The normal experiment operation stepss such as enzyme is cut, connect, reclaim, transform, pcr amplification refer to " molecular cloning (third edition) ".
Embodiment 1 oneself deletes the structure of carrier
Vector construction step is as follows, builds flow process as Fig. 1;
1.NheI and AgeI double digestion pEPI-eGFP carrier, inserts first loxp sequence, obtains intermediate carrier 1.
2.PciI and NheI double digestion intermediate carrier 1, deletes CMV promoter, inserts people EF1 α promotor and replaces CMV promoter, obtain intermediate carrier 2.
3.SalI and MluI double digestion intermediate carrier 2, glue reclaims large fragment and links with the PCR primer (containing AscI and NotI restriction enzyme site) of SV40PolyA, obtains intermediate carrier 3.
4.AscI and NotI double digestion pSP72-mOct4-GFPNEO, glue reclaims object fragment and inserts intermediate carrier 3, obtains final carrier pEPI-EF1 α-EGFP-Oct4-Cre(as Fig. 2), carrier sequence is as shown in SEQ ID No.1.
Embodiment 2MSTN knocks out the acquisition of cell
1. long white fetal fibroblast is set up
1) get the landrace of pregnant 30d, after execution, get uterine tube uterus, wrapping outlet, transport laboratory back in 2h, take out fetus from intrauterine, clean fetus with containing antibiotic DPBS, transfer in Bechtop, remove fetus head, four limbs, internal organ with eye scissors, rinse with DPBS.
2) with eye scissors, remainder is shredded as far as possible in diameter 100mm Tissue Culture Dish.
3) add a little serum, 1ml rifle head tip scissors is cut off, leaves diameter and be about 40mm with upper part, connect 1ml rifle, tissue block is transferred to the diapire of 3 T25 Tissue Culture Flasks, with elbow straw, tissue block is evenly spread out.
4) will the one side of tissue block be covered with upwards, respectively add 5ml cell culture fluid, put into CO
2incubator, 39 DEG C, 5%CO
2, 100% humidity is cultivated.
5) after cultivation 6-8h, will the one side upset of tissue block be covered with, make cell culture fluid submergence tissue block.
6) whether cultivation has cell to climb out of for about 5 days around tissues observed block.
7) when Growth of Cells to 80% converges, carry out Secondary Culture or carry out freezen protective.
2.pEPI-EF1 α-EGFP-Oct4-Cre and ZFN(MSTN) cotransformation
1ug pEPI-EF1 α-EGFP-Oct4-Cre fully mixes with 1ugZFN plasmid, and join 2 × 106 cell pipettors and inhale and beat and mix, whole operating process operation softly; Especially it should be noted that pEPI-EF1 α-EGFP-Oct4-Cre plasmid now with now carrying, and must guarantee that plasmid superhelix ratio is more than 90%.
3.G418 drug screening cell clone
Cell after being turned by electricity is on average inoculated into the Tissue Culture Dish of 6 10cm, and cultivate 48 hours in the Gibco of the foetal calf serum that with the addition of 10% cultivates after, add G418 to concentration 500ug/ml, step sizing 8 days, every two days with changing the substratum once adding G418.
4. knock out positive colony PCR primer order-checking qualification
After cell clonal formation, utilize clone's ring delimitation order cell clone and add trypsin digestion cell, and the cell coming from monospecific polyclonal is inoculated in the same hole of 48 Tissue Culture Plates, add 500ul culture medium culturing and reach 90% to degree of converging, expand 12 orifice plates after cell tryptase enzymic digestion to continue to cultivate, in 48 orifice plates, remaining cell adds substratum and continues to be cultured to and converge completely, after trysinization, collecting cell extracts cellular genome with test kit again, with tagctctaagatacaacacaata and ACCATCGGGGTAAGGTACCT for primer carries out pcr amplification, order-checking after amplified production glue reclaims, sequencing result and the former sequence alignment of MSTN are selected delete or the cell clone of insertion mutation as next step somatic cell clone donor.
The embryonic nucleus transplanting of embodiment 3MSTN gene knock-out pig and the preparation of clone pig
1. in-vitro maturity of porcine oocytes
Get ovary from slaughterhouse, put into 28 DEG C-35 DEG C, containing in the physiological saline of penicillin and Vetstrep, in 2h, transport the ovarian follicle of 3-6mm on 20mL syringe pump ovary that use for laboratory is furnished with No. 18 syringe needles back.Extraction liquid is put in 50mL centrifuge tube, 37 DEG C of water-baths leave standstill 15min and remove supernatant, add the resuspended precipitation of PVA-TL-HEPES, leave standstill 15min again, repeat once, re-suspension liquid is put into the plastic culture dish of diameter 60mm, under Stereo microscope, select ovarian cumulus with mouth suction pipe and wrap up more than 2 layers, fine and close, and kytoplasm uniform cumulus cell-oocyte complex (Cumulus-Oocyte-complexes, COCs), wash to proceed to for 3 times with maturation culture solution in incubator, at least balanced (every 100 μ L drops put 25 pieces) in the cultivation drop of 4h.Do 4 drops in the plastic culture dish of each diameter 35mm, cover with embryo's level mineral oil.
COCs is first cultivated 20 ± 2h in maturation culture solution, then transfers in the ripe liquid without hCG and eCG and continue cultivation 20 ± 2h.
2. somatocyte prepares
Utilize serum starvation method, by cell until its grow to 80% converge time, carry out serum starvation process, namely the FBS (Gibco in nutrient solution, Life Technologies) concentration from 20% be down to 0.5% continuation cultivate 2-5d, digest according to a conventional method, centrifuge washing, finally add 1mL micrurgy liquid re-suspended cell precipitation for subsequent use.
3. receptor oocytes stoning and donorcells nuclear transplantation
Utilize blind suction method to carry out mature oocyte stoning, i.e. ripe 40-44h porcine oocytes, select kytoplasm even after de-ovarian cumulus, all gaps of ovum are clear, and the ovocyte that after birth is complete is put into without Ca
2+, Mg
2+nCSU-23 is for subsequent use transfers in micrurgy drop for HEPES buffering: before nuclear transplantation, 1h carries out, diameter 60mm Tissue Culture Dish lid central authorities (drop 50-80 μ l, 2-3mm is wide, 8-10mm is long, paraffin oil covers), effect 15-30min, proceeds to donorcells and mature oocyte wherein in 39%, 5%CO simultaneously
2, 100% moisture equilibrium 15min, micro-fixed tube and stoning/entry needle are installed, regulate operating system position, under the inverted microscope being equipped with micrurgy instrument and thermostatic platform, 40 × use fixed tube (internal diameter 25-35 μm, external diameter 100-120 μm) stoning/entry needle of sticking ovocyte internal diameter 15-25 μm stirs ovocyte, make first polar body be in clock and watch 1 o ' clock position 200 × under, from 3, clock and watch, zona pellucida is crossed in stoning/injection acupuncture, sucking-off first polar body and neighbouring a little tenuigenin thereof exit stoning/entry needle, first polar body and a little kytoplasm are spued and selects diameter 15-20 μm, refractivity is strong, circular, smooth somatocyte, 200 × lower stoning/entry needle draws a donorcells, the ovum week be expelled to by somatocyte under zona pellucida from stoning inserting needle presses zona pellucida with entry needle in gap, make that the cytolemma of donorcells and acceptor ooecium matter is intimate contact with one another often criticizes 25-30 ovocyte, after structure completes, after end, the cell that donorcells-ooecium matter is formed is transferred in NCSU-23+4mg/ml BSA to (reconstructed eggs), 39 DEG C, 5%CO
2, repair 1-2h in 100% humidified incubator.
4. merge and activate
The reconstructed eggs recovered is transferred to merge in liquid in batches and balance 3min, after washing 3 times with fusion/activation solution, often criticize 5 and put into the integration slot being paved with and merging liquid, with draw and recombinant eggs stirred by the solid glass pin that tip is very thin, donorcells-recipient oocyte film contact surface and electrode runs parallel ECM2001 fusion instrument is made to apply 30 μ s, the electric pulse induced fusion of 2.0kv/cm activates simultaneously, wash 5 times with NCSU-23%+4mg/ml BSA, proceed to immediately mineral oil cover embryo medium in, 39 DEG C, 5%CO
2, 100% humidity cultivates after 0.5h ~ 1h and takes out, decision fusion under stereoscopic microscope.
5. embryo culture
Before beginning micrurgy, at least 4h well in advance cultivates drop, gets 35mm Micro-Organism Culture Dish in super clean bench, does the drop that 6-8 30 μ L are large, carefully adds 2.5-3ml mineral oil and covers, put into CO after carrying out mark
2incubator inner equilibrium proceeds to after the reconstructed embryo embryo medium of above-mentioned fusion is washed 5 times, and the drop of each 30 μ L cultivates 8-10 reconstructed embryo, records the spilting of an egg and Blastocyst formation result when cultivating 48h and 168h.
6. embryo transfer
Try feelings with boar or observe the reaction of the pressure back of the body, selecting spontaneous estrus sow, generally will at CO on the same day of structure clone embryo
2the 1-2 cell clone embryo cultivating 12-30h in incubator takes out, and loads in 2.5ml tubule.Package with the masking foil of sterilizing, carry out mark, be put in constant temperature transport case and transport to pig transplanting place, use 40-60ml(1mg/100kg body weight) physiological saline solution 1-1.5mg Thiopental Sodium and diazepam, ear vein injection 20ml, until the preliminary fiber crops of acceptor pig after, lifted on operation bracket (Suprapubic arch sling) Baoding of lying on the back (young replacement gilt) or lie on one's side to replacement gilt: below belly the 1st to and the 2nd pair of nipple between, ventrimeson local 15 × 10cm
2clean in area and scrape Mao Houxian iodine disinfection, rear alcohol swab takes off iodine (to Suprapubic arch sling: at belly the part between the ribs and the hips nest portion 15 × 10cm
2scope in clean, after scraping mao iodine disinfection, alcohol takes off iodine) before preparation moving knife cuts ventrimeson, narcotic remaining is above taken the circumstances into consideration to inject 20-30ml again, the mouth that one is about 8-10cm is cut along ventrimeson, open abdominal cavity, visit a hand of entering, take out after ovary and cover (and to Suprapubic arch sling: do the otch that is about 10cm vertical with ventrimeson face along flank portion) at surgical staff by taking out ovary with wetting sterilized non-fat gauze, when adjusting fimbriae tubae, the embryo be contained in suction pipe being taken out is placed in thermal station, under stereoscopic microscope, embryo is loaded surgical staff and embryo transfer personnel in grafts mutually to coordinate, grafts is probeed into from fimbriae tubae portion uterine tube at least 5cm be substantially to ampulla-isthmus junction, pushing syringe on one side, remove grafts transplanting so that outer after, whether stereoscopic Microscopic observation embryo is still trapped in grafts, after confirmation does not have, start ovary return, 10d intramuscular injection 800IU hCG after art mouth stitching embryo transfer, 13d injects 500IU PMSG.
7. blastomere counting
Take out reconstructed embryo blastaea, fix 10min in containing the DPBS of 3.7% paraformaldehyde after washing 3 times again the blastaea after fixing is transferred to containing 10 μ g/ml Hoechst33342(bisbenzimide) DPBS in lucifuge magazine in after incubated at room 10min dyeing terminates, blastaea is transferred on slide glass, lack carry liquid as far as possible, use 9:1(3:1 as early as possible) Vaseline/paraffin oil at its four angle point four posts, covered, carefully cover glass is pressed under stereomicroscope, expand a little after making ovum pressurized but be unlikely to be broken for suitable, use nail varnish mounting, Nikon E800 microscope, observe under ultraviolet excitation, take a picture, counting.
The extraction of embodiment 4 clone pig tissue gene group DNA and PCR order-checking qualification
Tissue block to be shredded or cell precipitation puts into 1.5ml centrifuge tube, add 700 μ l DNA extraction buffers, 20 μ l Proteinase Ks (20mg/ml), mix 56 DEG C of water-bath digestion more than 18h, until tissue block or cell precipitation completely dissolve, interval shake, to obtain better effect, adds the saturated phenol of 700 μ lTris-, the centrifugal 12min of gentle shake 12min, 12000rpm.Upper phase is transferred in another new centrifuge tube, repeats previous step, upper phase is transferred in another new centrifuge tube, adds 700 μ l phenol: imitative (1:1), the centrifugal 12min of gentle shake 12min, 12000rpm.Upper phase is transferred in another new centrifuge tube, adds 700 μ l chloroforms, the centrifugal 12min of gentle shake 12min, 12000rpm.Upper phase is transferred in another new centrifuge tube, adds 2 times of volume dehydrated alcohols, after putting upside down mixing, the centrifugal 10min of 12000rpm.Abandon supernatant, precipitation and tube wall is washed with 70% ethanol, tipping ethanol, drop is raffinate to the greatest extent, room temperature lower open mouth places 20 ~ 30min, ethanol is volatilized completely, add appropriate (about 100 μ l) TE damping fluid, dissolving DNA in 56 DEG C of water-baths, 0.7% agarose gel electrophoresis detects genomic dna concentration, concentration and the purity of genomic dna is detected under measuring 260/280nm wavelength, be adjusted to proper concn,-20 DEG C of preservations, carry out PCR sequenced genes simultaneously and pound out situation, result shows the MSTN gene of 5 clone pigs, all producer deletes sudden change, and carrier sequence and neomycin resistance gene (see figure 3) do not detected.Result shows that oneself deletes carrier in conjunction with the Zinc finger nuclease of MSTN, prepares the MSTN knock-out pig of marker-free efficiently.
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.
Claims (3)
1. oneself can delete free carrier for one kind, it is characterized in that, the Core Feature region of described free carrier comprises from 5 ' end to 3 ' end: people EF1 α promoter sequence, loxp sequence, EGFP encoding sequence, derives from the S/MAR sequence of HuIFN-β, the PolyA sequence of SV40, the expression cassette sequence of mouse Oct4 promoters driven Cre gene, loxp sequence.
2. dissociate carrier according to claim 1, and it is characterized in that, described free carrier contains riddled basins.
3. free carrier according to claim 1 and 2 is preparing the application in marker-free zooblast.
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| CN105002213A (en) * | 2014-04-24 | 2015-10-28 | 中国科学院上海巴斯德研究所 | Unconformable deletable novel non-viral vector, and construction method and use thereof |
| CN104131036B (en) * | 2014-07-28 | 2017-02-22 | 同济大学 | Construction method and application of conditional ivermectin receptor IVMR transgenic mouse model |
| WO2016025759A1 (en) | 2014-08-14 | 2016-02-18 | Shen Yuelei | Dna knock-in system |
| CN109385405B (en) * | 2018-11-07 | 2021-09-07 | 北京赛贝生物技术有限公司 | SuperH cell mother line for screening low-immune cell line by using gene editing system, and construction method and application thereof |
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| CN113462720B (en) * | 2020-11-04 | 2022-03-25 | 北京可瑞生物科技有限公司 | Efficient cell line gene knockout system |
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