CN103725710A - Self-deleting free carrier and application thereof - Google Patents
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Abstract
本发明涉及基因工程领域，具体提供了一种可自我删除游离载体，所述游离载体的核心功能区域从5’端到3’端包括：人EF1α启动子序列，loxp序列，EGFP编码序列，来源于人β干扰素基因的S/MAR序列，SV40的PolyA序列，小鼠Oct4启动子驱动Cre基因的表达盒序列，loxp序列。利用本发明所述载体进行基因打靶细胞及动物的制备，可删除筛选标记基因且不会产生基因插入突变，消除了marker基因对邻近基因表达的影响及对机体产生的潜在生物安全隐患。The present invention relates to the field of genetic engineering, and specifically provides a self-deleting episomal vector. The core functional region of the episomal vector includes from the 5' end to the 3' end: human EF1α promoter sequence, loxp sequence, EGFP coding sequence, source It is based on the S/MAR sequence of the human interferon beta gene, the PolyA sequence of SV40, the expression cassette sequence of the Cre gene driven by the mouse Oct4 promoter, and the loxp sequence. Using the carrier of the present invention to prepare gene targeting cells and animals can delete screening marker genes without generating gene insertion mutations, eliminating the influence of marker genes on the expression of adjacent genes and potential biological safety hazards to the body.
Description
Technical field
The present invention relates to genetically engineered field, specifically, relating to one can oneself 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 to the genomic modification of pig, can prepare the research model of similar human inheritance's disease, can should be used widely the research and development of pathological study and the medicine of human diseases.In addition pork is also the animal proteinum source that Chinese people democracy is wanted, and for the people's standard of living, improves the key effect of playing.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 accounts for the over half of whole world total amount according to statistics.The expanded demand in market, has promoted the fast development of China's modernization large-scale cultivation industry.But severe challenge is also proposed simultaneously.In China, main herding veriety depends critically upon import, and pig variety degree of dependence approaches 90%.This means that the source of the industry of pig-breeding---breeding stock is controlled by European & American Market completely, the serious food safety that threatens China.The pig new product of cultivating independent intellectual property right is present stage China agricultural problem in the urgent need to address.Long taking effect slowly of traditional hereditary and selection method cycle, 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 in a short time rapidly for single traits the production performance of domestic animal.Early stage genetic engineering breeding, be mainly foreign gene random integration in genome, exogenous gene expression is difficult to regulation and control, and expression amount is unstable, easily produces the insertion mutation of gene, to the very important potential safety hazard of transgenic animal generation itself.Zinc finger nuclease (ZFN), TALEN, CRISPR/Cas9 developed the molecular tool that can transform for the genome fixed point to complicated in recent years.Its preparation is simple 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 are combined the meticulous modification of genome of the pig that can fast, efficiently realize with somatic cell clone, be widely used in the future production performance improvement and the rearing new variety of pig.Because the fetal fibroblast of pig cannot be realized single cell culture, therefore when using Zinc finger nuclease (ZFN), TALEN, CRISPR/Cas9 to realize the gene knockout of pig cell, the assisting sifting that must will have marker gene, could obtain the cell clone knocking out.Current method is to contain neomycin resistance gene expression vector and Zinc finger nuclease (ZFN), TALEN or the screening of 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 is that selected marker refers to the marker gene that can make transformant (or individual) screen from numerous non-transformed cells in genetic transformation, they can produce the product certain selective agent to resistance by render transgenic cell conventionally, thereby render transgenic cell is normal growth on the substratum that adds this selection, but not transgenic cell shows the sensitivity of this selective agent because transgene efficiency is low owing to lacking resistance, therefore marker gene can screen and enrichment positive colony.But marker gene mainly contains following shortcoming: the first, and this marker gene pairs contiguous gene is expressed impact. and the second transgenic animal that contain marker gene cannot, by the Biosafety assessment of animal, hinder the commercial applications of transgenic pig.
Although marker gene also can directly 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, body is produced to potential Biosafety hidden danger, be difficult to equally the safety assessment by transgenic animal, limited 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 free carrier of can oneself deleting that can delete flag gene can not produce again gene insertion mutation.
In order to realize the object of the invention, first the present invention provides one oneself to delete free carrier, the Core Feature region of described free carrier comprises to 3 ' end from 5 ' 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 selection markers gene, and described selection markers gene is resistant gene.
The present invention also provides and has utilized aforementioned free carrier to prepare the method for the gene targeting animal of marker-free, and its concrete steps comprise:
1), by described free carrier and ZFN, TALEN or CRISPR/Cas9 cotransfection animal fetus somatocyte, the screening of medicine helper obtains gene knockout cell clone;
2), using gene knockout cell as nuclear transplantation donorcells, in vitro ovocyte, as nuclear transplantation recipient cell, obtains animal cloning embryo by nuclear transfer technology; Clone embryos starts to express Oct4 gene from two-cell stage, and the expression of mouse Oct4 promotor cre enzyme is simultaneously deleted the free carrier oneself of site specific recombination mediation of cre enzyme mediation;
3) clone embryos is moved in animal uterus and is carried out gestation by modus operandi, obtains the gene targeting animal of marker-free.
Further, described animal is pig, ox, sheep.
Medicine described in step 1) is G418.
The present invention also provides and has utilized aforementioned free carrier the present invention also to provide to utilize aforementioned free carrier in the application of preparing in marker-free zooblast.
The present invention also provides and has utilized the application of aforementioned free carrier in the gene targeting animal of preparing marker-free.
Beneficial effect of the present invention is:
The present invention forms a kind of controlled free carrier by Cre/loxP site differential recombination enzyme system and S/MAR unit construction.The people bate interferon gene that wherein S/MAR derives from, when carrier exists S/MAR sequence and can be transcribed time, carrier can self-replacation in cell, and follow cell fission to pass to daughter cell, and carrier can form genetic stability without being incorporated into cellular genome.During design vector, in the sequence upstream and downstream that includes S/MAR element, respectively insert a loxp sequence in the same way, Oct4 promotor can drive cre gene to express at clone embryos, S/MAR on carrier in embryonic cell is deleted in the site specific recombination reaction of cre enzyme mediation, losing the carrier of 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 can effectively be applied to the gene knock-out pig of transgenic pig, especially ZFN, TALEN, the CRISPR/Cas9 mediation of preparing marker-free (marker free).Because the fetus somatocyte of pig cannot be realized single cell culture (separate cell cannot be bred and be formed a cell mass), therefore when utilizing ZFN, TALEN, CRISPR/Cas9 to prepare genetic modification pig fetus somatocyte, need to express the carrier cotransfection of drug resistance gene, then with drug screening, just can obtain the cell clone of gene knockout.Although this method can obtain the pig fetus somatocyte of gene knockout, resistant gene also will be incorporated into cellular genome simultaneously.The free carrier of can oneself deleting that utilizes that we build and ZFN, TALEN, CRISPR/Cas9 cotransfection, assisting sifting (expression neomycin resistance gene) can obtain gene knockout cell clone efficiently.After body-cell neucleus transplanting, in clone embryos, Oct4 promoters driven Cre expression of enzymes is deleted the carrier for assisting sifting.Embryo transfer can obtain the gene knock-out pig of marker-free.Simultaneously the method also can be for the screening of other species, and the preparation of induced dry-cell.
Utilize carrier of the present invention 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 at present all transgenic pigs, 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 carrier collection of illustrative plates 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 are used for illustrating 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 synthetic by the raw work in Shanghai, and sequencing is completed by Beijing Hua Da.Taq enzyme, T4DNA ligase enzyme, restriction endonuclease are all from Beijing NEB company, and somatic cell clone agents useful for same is all purchased from Sigma company.The normal experiment operation stepss such as enzyme is cut, connected, recovery, conversion, pcr amplification refer to < < molecular cloning (third edition) > >.
Vector construction step is as follows, builds flow process as Fig. 1;
1.NheI and AgeI double digestion pEPI-eGFP carrier, insert first loxp sequence, obtains intermediate carrier 1.
2.PciI and NheI double digestion intermediate carrier 1, delete CMV promotor, inserts people EF1 α promotor and replace CMV promotor, obtains intermediate carrier 2.
3.SalI and MluI double digestion intermediate carrier 2, glue reclaims large fragment and links with the PCR product (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, in 2h, transport laboratory back, from intrauterine, take out fetus, with cleaning fetus containing antibiotic DPBS, transfer in Bechtop, remove fetus head, four limbs, internal organ with eye scissors, rinse with DPBS.
2) in diameter 100mm Tissue Culture Dish, with eye scissors, remainder is shredded as far as possible.
3) add a little serum, 1ml rifle head tip is cut off with scissors, leave diameter and be about the above part of 40mm, connect 1ml rifle, tissue block is transferred to the diapire of 3 T25 Tissue Culture Flasks, tissue block is evenly spread out with elbow straw.
4) by the one side that is covered with tissue block upwards, respectively add 5ml cell culture fluid, put into CO
2incubator, 39 ℃, 5%CO
2, 100% humidity is cultivated.
5), after cultivating 6-8h, by being covered with the one side upset of tissue block, make cell culture fluid submergence tissue block.
6) whether cultivate 5 days left and right tissues observed pieces has cell to climb out of around.
7), when Growth of Cells to 80% converges, go down to posterity and cultivate or carry out freezing preservation.
2.pEPI-EF1 α-EGFP-Oct4-Cre and ZFN(MSTN) cotransformation
1ug pEPI-EF1 α-EGFP-Oct4-Cre fully mixes with 1ugZFN plasmid, joins 2 × 106 cell pipettors and inhales and beat and mix, and whole operating process operation softly; Especially it should be noted that pEPI-EF1 α-EGFP-Oct4-Cre plasmid must be now with now carrying, and guarantee that plasmid superhelix ratio is more than 90%.
3.G418 drug screening cell clone
Cell after electricity is turned is on average inoculated into the Tissue Culture Dish of 6 10cm, in the Gibco that has added 10% foetal calf serum cultivates, cultivates after 48 hours, adds G418 to concentration 500ug/ml, step sizing 8 days, and every two days with changing the substratum that once adds G418.
4. knocking out the order-checking of positive colony PCR product identifies
After cell clonal formation, utilize clone's ring delimitation order cell clone and add trypsin digestion cell, and the cell that comes from monospecific polyclonal is inoculated in the same hole of 48 Tissue Culture Plates, add 500ul culture medium culturing to degree of converging to reach 90%, after cell tryptase enzymic digestion, expanding 12 orifice plates to continues to cultivate, in 48 orifice plates, remaining cell adds substratum to continue to be cultured to completely to converge, after trysinization, collecting cell extracts cellular genome with test kit again, take tag ctctaagatacaacacaata and ACCATCGGGGTAAGGTACCT as primer, carry out pcr amplification, order-checking after amplified production glue reclaims, using sequencing result and the former sequence alignment of MSTN select 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
From slaughterhouse, get ovary, put into 28 ℃-35 ℃, in the physiological saline containing penicillin and Vetstrep, in 2h, transport use for laboratory back and be furnished with the ovarian follicle of 3-6mm on the 20mL syringe pump ovary of No. 18 syringe needles.Extraction liquid is put in 50mL centrifuge tube, 37 ℃ of standing 15min of water-bath remove supernatant, add the resuspended precipitation of PVA-TL-HEPES, standing 15min again, repeat once, resuspended liquid is put into the plastic culture dish of diameter 60mm, under Stereo microscope, with mouth suction pipe, selecting ovarian cumulus wraps up more than 2 layers, fine and close, and kytoplasm uniform cumulus cell-ovocyte complex body (Cumulus-Oocyte-complexes, COCs), proceed to for 3 times in incubator at least in the cultivation drop of balance 4h (every 100 μ L drops are put 25 pieces) with maturation culture solution washing.In the plastic culture dish of each diameter 35mm, do 4 drops, with the covering of embryo's level mineral oil.
COCs is first cultivated to 20 ± 2h in maturation culture solution, then transfer to without continuing to cultivate 20 ± 2h in the ripe liquid of hCG and eCG.
2. somatocyte is prepared
Utilize serum starvation method, cell is grown to 80% while converging until it, carry out serum starvation processing, the FBS (Gibco in nutrient solution, Life Technologies) concentration from 20% be down to 0.5% continuation cultivate 2-5d, digestion according to a conventional method, centrifuge washing, finally adds 1mL micrurgy liquid re-suspended cell precipitation standby.
3. acceptor oocyte enucleation and donorcells nuclear transplantation
Utilize blind suction method to carry out mature oocyte stoning, i.e. ripe 40-44h porcine oocytes, selects kytoplasm even after de-ovarian cumulus, and 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 standby transfers in micrurgy drop for HEPES buffering: before nuclear transplantation, 1h carries out, (the drop 50-80 μ l of diameter 60mm Tissue Culture Dish lid central authorities, 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, be equipped with under the inverted microscope of micrurgy instrument and thermostatic platform, 40 × use fixed tube (internal diameter 25-35 μ m, external diameter 100-120 μ m) sticking ovocyte stirs ovocyte with stoning/entry needle of internal diameter 15-25 μ m, make first polar body in clock and watch 1 o ' clock position 200 × under, from 3, clock and watch, stoning/injection acupuncture is crossed to zona pellucida, sucking-off first polar body and near a little tenuigenin thereof exit stoning/entry needle, first polar body and a little kytoplasm are spued and select diameter 15-20 μ m, refractivity is strong, circular, smooth somatocyte, 200 × lower to a donorcells of stoning/entry needle absorption, from stoning inserting needle, somatocyte is expelled in all gaps of ovum under zona pellucida and presses zona pellucida with entry needle, make cytolemma every crowd of 25-30 of close contact ovocyte each other of donorcells and acceptor ooecium matter, after structure completes, the cell after end, donorcells-ooecium matter being formed is transferred in NCSU-23+4mg/ml BSA (reconstruct ovum), 39 ℃, 5%CO
2, repair 1-2h in 100% humidity incubator.
4. merge and activate
The reconstruct ovum having recovered is transferred in batches and merged balance 3min in liquid, with after fusion/activation solution washing 3 times, put into for 5 every batch and be paved with the integration slot that merges liquid, stir recombinant eggs with very thin solid glass pin that draw and most advanced and sophisticated, make that donorcells-recipient oocyte film contact surface is parallel with electrode applies 30 μ s with ECM2001 fusion instrument, activation is simultaneously merged in the electric pulse induction of 2.0kv/cm, with NCSU-23%+4mg/ml BSA washing 5 times, proceed to immediately in the embryo medium that mineral oil covers, 39 ℃, 5%CO
2, 100% humidity cultivates after 0.5h～1h and takes out, decision fusion under stereoscopic microscope.
5. embryo culture
Cultivate drop starting before micrurgy at least 4h well in advance, in super clean bench, get 35mm Micro-Organism Culture Dish, do 6-8 the large drop of 30 μ L, carefully add 2.5-3ml mineral oil to cover, put into CO after carrying out mark
2incubator inner equilibrium proceeds to after the reconstructed embryo of above-mentioned fusion is washed to 5 times with embryo medium, and the drop of each 30 μ L is cultivated 8-10 reconstructed embryo, records the spilting of an egg and Blastocyst formation result while cultivating 48h and 168h.
6. embryo transfer
With boar examination feelings or observation, press back of the body reaction, select spontaneous estrus sow, generally will be at CO on the same day of structure clone embryo
2the 1-2 cell clone embryo of cultivating 12-30h in incubator takes out, and packs in 2.5ml tubule.Package with the masking foil of sterilizing, carry out mark, be put into and in constant temperature transport case, transport to pig and transplant 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 (multiparity sow) Baoding of lying on the back (young replacement gilt) or lie on one's side to replacement gilt: below belly between the 1st pair and the 2nd pair of nipple, ventrimeson part 15 × 10cm
2the clean Mao Houxian iodine disinfection of scraping in area, rear alcohol swab takes off iodine (to multiparity sow: at the belly the part between the ribs and the hips nest 15 × 10cm of portion
2scope in clean, after scraping mao iodine disinfection, alcohol takes off iodine) before preparing moving knife incision ventrimeson, remaining above narcotic is taken the circumstances into consideration to inject 20-30ml again, along ventrimeson, cut a mouth that is about 8-10cm, open abdominal cavity, visit the hand of entering, take out after ovary and with wetting sterilizing absorbent gauze, to cover (and to multiparity sow: along flank portion, do the otch that is about 10cm vertical with ventrimeson face) and be about to take out ovary at surgical staff, when adjusting fimbriae tubae, the embryo's taking-up being contained in suction pipe is placed on hot platform, under stereoscopic microscope, embryo being packed into surgical staff and embryo transfer personnel in grafts coordinates mutually, by grafts from fimbriae tubae portion probe into 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 confirming not have, start ovary return, 10d intramuscular injection 800IU hCG after art mouth stitching embryo transfer, 13d injection 500IU PMSG.
7. blastomere counting
Take out reconstructed embryo blastaea, fixing again 10min after 3 times the blastaea after fixing transferred to containing 10 μ g/ml Hoechst33342(bisbenzimide containing washing in the DPBS of 3.7% paraformaldehyde) DPBS in after incubated at room 10min dyeing finishes in the magazine of lucifuge, blastaea is transferred on slide glass, lack carry liquid as far as possible, use as early as possible 9:1(3:1) Vaseline/paraffin oil at four posts of its four angle point, covered, careful pressing cover slide under stereomicroscope, make expand a little but be unlikely to be broken for suitable after ovum pressurized, use nail varnish mounting, Nikon E800 microscope, under ultraviolet excitation, observe, take a picture, counting.
The extraction of embodiment 4 clone pig tissue gene group DNA and PCR order-checking are identified
Tissue block is shredded or cell precipitation is put into 1.5ml centrifuge tube, add 700 μ l DNA extraction buffers, 20 μ l Proteinase Ks (20mg/ml), more than mixing 56 ℃ of water-bath digestion 18h, until tissue block or cell precipitation completely dissolve, intermittently shake, to obtain better effect, adds the saturated phenol of 700 μ l Tris-, gentle shake 12min, the centrifugal 12min of 12000rpm.Upper phase is transferred in another new centrifuge tube, repeats previous step, upper phase is transferred in another new centrifuge tube, add 700 μ l phenol: imitative (1:1), gentle shake 12min, the centrifugal 12min of 12000rpm.Upper phase is transferred in another new centrifuge tube, adds 700 μ l chloroforms, gentle shake 12min, the centrifugal 12min of 12000rpm.Upper phase is transferred in another new centrifuge tube, adds 2 times of volume dehydrated alcohols, after putting upside down and mixing, the centrifugal 10min of 12000rpm.Abandon supernatant, with 70% ethanol, wash and precipitate and tube wall, tipping ethanol, drop is raffinate to the greatest extent, room temperature lower open mouth is placed 20～30min, ethanol is volatilized completely, add appropriate (l) TE damping fluid of approximately 100 μ, dissolving DNA in 56 ℃ of water-baths, 0.7% agarose gel electrophoresis detects genomic dna concentration, measure the concentration and the purity that under 260/280nm wavelength, detect genomic dna, be adjusted to proper concn,-20 ℃ of preservations, carry out PCR sequenced genes simultaneously and pound out situation, result shows the MSTN gene of 5 clone pigs, all producer is deleted sudden change, and carrier sequence and neomycin resistance gene (see figure 3) do not detected.Result shows that oneself deletes the Zinc finger nuclease of carrier in conjunction with 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.
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| CN103993027A (en) * | 2014-04-17 | 2014-08-20 | 中国农业大学 | Transgenic pig screening marker gene knockout method |
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| US11071289B2 (en) | 2014-08-14 | 2021-07-27 | Biocytogen Boston Corp | DNA knock-in system |
| US10660316B2 (en) | 2016-11-04 | 2020-05-26 | Akeagen, Inc. | Genetically modified non-human animals and methods for producing heavy chain-only antibodies |
| CN109385405A (en) * | 2018-11-07 | 2019-02-26 | 北京赛贝生物技术有限公司 | Maternal, its construction method and its application using the SuperH cell of the low immunocyte system of gene editing screening system |
| CN110004181A (en) * | 2019-03-20 | 2019-07-12 | 东北农业大学 | An episomal CRISPR/Cas9 expression vector and its construction method and application |
| CN113462720A (en) * | 2020-11-04 | 2021-10-01 | 北京可瑞生物科技有限公司 | Efficient cell line gene knockout system |
| 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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