CN110257425A - A kind of PS Transposon System and its gene transfer method of mediation - Google Patents

Abstract

The present invention discloses the gene transfer method of a kind of PS Transposon System and its mediation, including can be inserted into the transgenic donor plasmid of two terminal repeats of target gene box and provide the transposase helper plasmid of transposition activity.Target gene is imported into acceptor gene group by PS Transposon System.Gene transfer method provided by the invention based on PS Transposon System, gene transfer can be efficiently mediated by cell and embryo's level verification, it can be applied to multiple field of biotechnology: 1) effectively target gene box can be inserted into host genome using the method provided in the present invention, improve gene transfering efficiency；2) animal gene functional study can effectively be carried out in conjunction with gene trapping；3) it can mediate and carry out human gene therapy etc..

Description

A kind of PS Transposon System and its gene transfer method of mediation

Technical field

The present invention relates to the gene transfer methods for establishing a kind of PS Transposon System mediation, while also disclosing this method and relating to And transgenic donor plasmid and transposase eukaryotic expression and the construction method that helper plasmid is transcribed in vitro, and its turn base in preparation Because of the application in animal, research gene function and human gene therapy.The invention belongs to animal genetic engineering fields.

Background technique

Gene transfer be when the important biotechnological method of previous item, can with mediate foreign gene it is stable be integrated into host In chromosome, so having important answer in the fields such as research gene function, prepare transgenosis biology and human gene therapy With value.

Transposons is the DNA sequence dna that genome the preceding paragraph can freely jump, and is by Mc.Clintock first in 20th century It is found in maize chromosome the forties, it is rear to be found in the various biologies such as bacterium, fungi and insect successively again.By to swivel base Find no matter all there are a considerable amount of transposons in prokaryotes or eucaryote after the note of son.Transposons is not With distribution between biology there are content in larger difference, especially higher eucaryote is higher, such as transposons is mammal base Because of component maximum in group, the half of human genome is almost accounted for, 80% is almost accounted in corn.DNA transposons turns The seat process mechanism that follows " shearing-stickup " is moved in the genome, thus by as efficient gene transfer tool into Row exploitation.In recent years, transposons is in human gene therapy, trangenic mice and the preparation of zebra fish isotype biology and functional genome Important progress is had been achieved in research.In addition to this, transposons has many advantages as the carrier of gene transfer: 1) structure is simple, and the TIR sequence of the only two sides of acceptor gene group, the influence to foreign gene are entered with foreign gene co-integration Very little, integration site also without find large fragment loss and chromosomal rearrangement the phenomenon that；2) it compares and viral vectors, DNA Transposon vector limits Insert Fragment smaller；3) foreign gene can be with stable integration into chromosome, and it is thin to pass through reproduction Born of the same parents passage after also can be long-term expression；4) transposase can be catalyzed the gene singly copied and accurately be inserted into portable sequence, no It will not change by the size of random integration and Insert Fragment again；5) Transposon System can be all with exposed DNA Form is given, and can also provide transposase in the form of RNA or protein by DNA and combine carry out swivel base, so its immunogene Property is lower.Most commonly used DNA transposons Sleeping Beauty (SB), PiggBac (PB) and Tol2 are wherein studied, this three Origin difference not only occurs for kind transposons, and there is also differences in terms of transposition activity, insertion Preference, therefore also have using upper It is distinguished.Such as SB preference insertion point is " TA ", but there is no Preference to gene, so SB transposons is commonly used for the mankind Gene therapy；PB preference insertion point is " TTAA ", and preference is inserted into gene internal, grinds so being commonly used for functional gene Study carefully, gene trap etc.；Tol2 then not apparent preference insertion point, insertion position is mostly upstream region of gene control region, therefore often For studying enhancer capture etc..

Microinjection has been widely used, and technology maturation is stablized, while scientists utilize transposon-mediated gene The characteristics of transfer, the further perfect method of transposon-mediated cytoplasm microinjection.Due to the swivel base in DNA transposon system Enzyme contains nuclear localization signal sequence (NLS), and target gene can smoothly be mediated to enter nucleus, and injection can not enter core, directly Tap into row cytoplasmic injection.So this technology is receiving attention, and achieves encouraging progress, this may be to break through greatly The new method of type Mammals Transgenic Technology bottleneck.

Transposons mainly includes target gene clone, gene transfer, target cell selection and clinic for human gene therapy In the stages such as experimental observation, wherein gene transfer is then the committed step of gene therapy, and DNA transposon-mediated gene transfer exists Advantage in gene therapy is exactly efficient, safety.So far, transposons is already known to most common non-viral in gene therapy Carrier.In addition, transposons can also prepare mutant, carries out gene trap, studied using with functional genomics.

DNA transposon-mediated gene transfer system is the transgenic technology favored by scientists, but is had certainly The DNA transposons of main transposition activity is rarely found in vertebrate.1996, for the first time in albefaction blueness clanging or clanking sound (Oryziaslatipes) In be found to have the vertebrate transposons of natural activity, i.e. Tol2 transposons；2008, have found that second case has in goldfish There is the active Tgf2 transposons of autonomous transposon, and the transposons and green clanging or clanking sound Tol2 transposable elements are quite similar.Inventor's benefit With the means of bioinformatics, to having found passer in the Tc1/Mariner transposons superfamily Research on Mining of vertebrate (PS) family shows that it may activity with higher by insertion Analysis of age etc..On the basis of phyletic evolution comparative studies Molecular remodeling has been carried out, the sequence of two terminal repeat TIR key elements and transposase of PS transposons is obtained, and has been constructed It at a set of gene transfer vector system, is verified through cell, trangenic mice and zebra fish etc., which can effectively mediate base Because of transfer, there is very big application potential in transgenic animals preparation and gene therapy.

Summary of the invention

In order to overcome drawbacks described above, the present invention provides the gene transfer method of a kind of PS Transposon System and its mediation, adopts With bioinformatics method, PS family in Tc1/Mariner superfamily is found, carry out Molecular remodeling, obtain two ends of transposons Repetitive sequence and transposase sequence are held, and constructs PS transposons donor plasmid and expresses the helper plasmid of PS transposase, is assembled into A set of gene transfer system is named as PS Transposon System.It is mediated the purpose of the present invention is to provide a kind of PS transposon system High efficiency gene transfer method, improves the transgenosis preparation efficiency of the animals such as mouse, zebra fish, and can be efficiently applied to cytogene The research fields such as transfection integration, human gene therapy and gene trap.

PS Transposon System of the present invention is mainly made of PS transposons donor plasmid and PS transposase helper plasmid.

Transgenic donor plasmid (pLB-PS) of the present invention includes PS transposons both-side ends repetitive sequence and Duo Ke The target gene for needing to shift can be inserted in grand insertion point, polyclonal insertion point (NruI/NotI/EcoRI restriction enzyme site) Box, the sequence of the donor plasmid (pLB-PS) is as shown in SEQ ID No.1.Target gene can be more grams by any one above-mentioned In grand insertion point insertion transgenic donor plasmid.Refer in target gene insertion transgenic donor plasmid: target gene insertion Between PS5 ' TIR and PS3 ' TIR.

PS transposons both-side ends repetitive sequence of the present invention is from PS family in Tc1/Mariner superfamily Obtained by Molecular remodeling, both-side ends inverted repeats (TIR) respectively length be 28 nucleotide sequences (i.e. PS5 ' TIR and PS3 ' TIR), sequence is respectively as shown in SEQ ID No.2 and SEQ ID No.3.

Target gene box of the present invention can be reporter gene expression box or other exogenous gene expression boxes, or Person's gene trap element.

PS transposase (i.e. PS CDS, also known as PSase CDS) sequence of the present invention is to utilize biological information credit Analysis means carry out Molecular remodeling to PS swivel base sub-family and obtain transposase sequence, obtain through chemical synthesis, such as SEQ ID No.5 It is shown.

PS transposase helper plasmid pcDNA3.9-PSase of the present invention there are two types of form, first is that can in cell and The eukaryon expression plasmid directly expressed in vivo, another kind are through helper plasmid is transcribed in vitro can to carry out that formation 5 ' is transcribed in vitro The mRNA of capped PS transposase.

PcDNA3.9-PSase eukaryon expression plasmid of the present invention includes viral promoter sequence (CMV) and PS swivel base Enzyme cDNA sequence and bGH polyadenylic acid (PolyA) composition；Carrier framework comes from pcDNA3.9 carrier, the viral promotors Sequence (CMV) length is 584 nucleotide sequences, can instruct the expression of downstream gene；The PS transposase length is 1275 A nucleotide sequence；BGH polyadenylic acid (PolyA) length is 225 nucleotide sequences, which can independently express Transposase, while it includes T7 promoter, PS transposase cDNA sequence and bGH that the carrier, which can be used as in-vitro transcription helper plasmid, PolyA sequence, wherein the length of T7 promoter is the sequence of 19 nucleotide sequence (bp) carriers, and bGH PolyA sequence is whole Rotation stop record, carrier can carry out PS using mMESSAGE Mmachine T7kit (being purchased from Invitrogen company) kit and turn Seat enzyme 5 ' is capped the in-vitro transcription of mRNA, and the sequence of the carrier is as shown in SEQ ID No.4.

Gene transfer method provided by the invention based on PS Transposon System, can by cell and embryo's level verification Efficiently mediate gene transfer, can be applied to multiple field of biotechnology: 1) using the method provided in the present invention can effectively by Target gene box is inserted into host genome, improves gene transfering efficiency；2) it can effectively be opened in conjunction with gene trapping Open up animal gene functional study；3) it can mediate and carry out human gene therapy etc..

Detailed description of the invention

Fig. 1: pLB-PSase plasmid electrophoretogram；

Fig. 2: pcDNA3.9-PSase plasmid electrophoretogram；

Fig. 3: pcDNA3.9-PSase plasmid map；

Fig. 4: pLB-PS plasmid schematic diagram；

Fig. 5: PS-TIR annealed product electrophoretogram；

Fig. 6: PS-TIR purified product electrophoretogram；

Fig. 7: pLB-PS plasmid electrophoretogram；

Fig. 8: pLB-PS plasmid enzyme restriction product electrophoretogram；

Fig. 9: pPS-PGK-NEO plasmid electrophoretogram；

Figure 10: pPS-PGK-NEO plasmid enzyme restriction product electrophoretogram；

Figure 11: pPS-PGK-NEO plasmid map；

Figure 12: PS-FAG-GFP PCR switchback product；

Figure 13: pPS-FAG-GFP plasmid electrophoretogram；

Figure 14: pPS-FAG-GFP restriction enzyme digestion and electrophoresis figure, 1:pPS-FAG-GFP digestion；2:pPS-FAG-GFP plasmid；

Figure 15: pPS-FAG-GFP plasmid map；

Figure 16: pZB-RT-bGH restriction enzyme digestion and electrophoresis figure；

Figure 17: pPS-PGK-NEO restriction enzyme digestion and electrophoresis figure；

Figure 18: PS frame, Tyr-TYR-bGHpA segment switchback electrophoretogram, 1:PS frame switchback electrophoretogram；2:Tyr-TYR- BGHpA segment switchback electrophoretogram；

Figure 19: pPS-Tyr plasmid electrophoretogram；

Figure 20: pPS-Tyr restriction enzyme digestion and electrophoresis figure, 1-6:pPS-Tyr digestion；7:pPS-Tyr plasmid；

Figure 21: pPS-Tyr plasmid map；

Figure 22: Hela cell G418 resistance screening positive colony figure；

Figure 23: PS compared with SB transposition activity figure；

Figure 24: PS transposon system prepare transgenosis mouse figure；

Figure 25: different times microinjection zebrafish embryo fluorogram；

Figure 26: PS transposons microinjection zebra fish positive rate compares figure.

Specific embodiment

In order to better illustrate the present invention, it is easy to understand technical solution of the present invention, of the invention is typical but unrestricted Embodiment is as follows:

The experimental method mentioned in following embodiments is conventional method unless otherwise instructed.

The building of embodiment I, transposase expression vector pcDNA3.9-PSase

1, transposase PSase carrier synthesizes.

PSase transposase sequence is cloned on LB carrier after chemical synthesis, constructs p LB-PSase carrier, Plasmid DNA (such as Fig. 1) is detected with 1% agarose gel electrophoresis.

2, the building of transposase eukaryon expression plasmid and in-vitro transcription helper plasmid pcDNA3.9-PSase

Transposase CDS is cut out from pLB-PSase carrier with restriction enzyme BamHI and XhoI, Ago-Gel Recycle the CDS sequence of 1296bp.BamHI and XhoI double digestion pcDNA3.9 carrier, gel extraction 3.9kb segment conduct are used simultaneously Carrier frame.After transposase CDS is connected with pcDNA3.9 carrier frame, transformed competence colibacillus cell Top10 chooses single bacterium and drops down onto liquid Body culture medium LA culture, upgrading grain carry out electroresis appraisal (such as Fig. 2), and positive colony person is named as pcDNA3.9-PSase, plasmid Map is as shown in Figure 3.

PcDNA3.9-PSase carrier sequence is as shown in SEQ ID No.4, including CMV Enhancer promoter 235-818、T7promoter 863-881、PSase CDS 919-2193、Sp6promoter 2224-2242、bGH poly (A)signal 2268-2492、F1Origin 2538-2896、SV40poly(A)signal 2897-2998、Ampicillin Resistance gene 4208-5068。

The building of embodiment II, transposons expression vector

1, transgenic donor plasmid pLB-PS vector construction

The synthesis of two end inverted repeat elements (PS 3 ' and 5 ') of 1.1PS

PS transposons in Tc1/Marinier family is carried out to several species and carries out the analysis such as insertion age, and in phyletic evolution Molecular remodeling is carried out on the basis of comparative studies, it is determined that the reverse complemental sequence of highly conserved 5 ' and 3 ' TIR, respectively 28bp Column.And this sequence is added into restriction endonuclease sites, send Huada gene company to synthesize, sequence is shown in Table 1.

The polymeric enzymatic amplification of 1.2PS transposable element

By above-mentioned chemically synthesized single-stranded nucleotide chain PS5 ' t (inverted repeat element and digestion position including the end PS5 ' Point, restriction enzyme site is for being inserted into target gene) and PS3 ' t (inverted repeat element and restriction enzyme site including the end PS3 ', digestion Site is for being inserted into target gene) first anneal, then extended with Klenow polymerase, form double-strand.Reaction system is 50 μ L, including 1ul PS5 ' t (100uM), 1ul PS3 ' t (100uM), 23ul ultrapure water, 95 DEG C of denaturation 5min, then with 0.1 DEG C of temperature per second 25 DEG C are down to, 5min is kept.Add 5 μ L Klenow Buffer, 2 μ L Klenow polymerase, 5 μ L 4dNTP, 13 μ L ultrapure water, 37 DEG C of 1.5h, 80 DEG C of 20min are down to room temperature, obtain target sequence (PS TIR).It is then pure with Qiagen PCR Change kits (such as Fig. 5,6), after nucleic acid concentration analyzer measures concentration, -20 DEG C are saved backup.Plasmid schematic diagram is as schemed Shown in 4, gene insertion site, that is, multiple cloning sites (restriction enzyme site) in Fig. 4.

1.3 p LB-PS vector constructions

P LB-PS carrier structure schematic diagram is as shown in figure 4, by PS transposon fragment (the i.e. above-mentioned 1.2 obtained mesh of purifying Mark sequence PS TIR) it is connect with zero background carrier pLB of Tiangeng with T4 ligase, transformed competence colibacillus cell Top10 chooses single bacterium and drops down onto Fluid nutrient medium LB culture containing Amp, upgrading grain carry out electrophoresis and digestion identification (such as Fig. 7,8), and positive colony person is named as pLB-PS.PLB-PS carrier sequence is as shown in SEQ ID No.1, including T7promoter 305-323；PS 5'TIR 380- 407(SEQ ID No.2)；PS 3'TIR 428-455(SEQ ID No.3)；Ori 1257-1845；Ampicillin Resistance gene 2016-2876.In SEQ ID No.1, the base of 408-427 is multiple cloning sites (digestion position Point), for being inserted into target gene.

2, the building of transposon vector pPS-PGK-NEO

With restriction enzyme Nru1 digestion PGK-NEO-bGHpA-TA cloning vector, (PGK-NEO-bGHpA-TA clone is carried Body is that this laboratory saves carrier), the PGK-NEO-bGHpA expression cassette of gel extraction 1659bp (its as a purpose box gene). With 1 digestion LB-PS carrier of Nru, the carrier frame of 3066bp is recycled, expression cassette is connect with carrier, transformed competence colibacillus cell Top10 chooses single bacterium and drops down onto the LB of fluid nutrient medium containing Amp culture, and upgrading grain carries out electrophoresis and digestion identification, screens size and insertion Person (such as Fig. 9,10) in the right direction, send Hua Da gene sequencing, plasmid map is as shown in figure 11.

3, the building of transposon vector pPS-FAG-GFP

Using pZB-FAG-GFP Plasmid DNA as template, by the primer amplification FAG- of the amplification PS-FAG-GFP enumerated in table 2 GFP genetic fragment (the FAG-GFP genetic fragment of amplification as a purpose box gene).Reaction system is 50 μ l, 10 5 × SF of μ L Buffer, 1 μ l dNTP, 2 μ L, 10 μM of primer PS-FAG-GFP-F, 2 μ l, 10 μM of primer PS-FAG-GFP-R are (see primer sequence Table 2), 1 μ l Phanta^TMSuper-Fidelity, 1 μ l pZB-FAG-GFP Plasmid DNA template add ultrapure water (high to 50 μ l Fidelity enzyme is purchased from Nuo Weizan company).PCR amplification program: 94 DEG C of 40s, 55 DEG C of 40s, 72 DEG C of 1m30s are recycled 30 times.PCR expands Volume increase object is detected with 1% agarose gel electrophoresis.PCR product Ago-Gel is recycled into (such as Figure 12), is connect with p LB carrier, TA clone is carried out, positive colony, upgrading grain and digestion identification (such as Figure 13,14) is filtered out, is saved after sequencing comparison is correct under supplying The experiment of one step is used, and pPS-FAG-GFP, plasmid map such as Figure 15 are named as.

4, the building of transposon vector pPS-Tyr

With restriction enzyme NheI digestion carrier pZB-RT-bGH (such as Figure 16), Tyr-TYR-bGHpA segment is recycled (2555bp) (such as Figure 18), filling-in save for use after purification；With NruI digestion carrier pPS-PGK-NEO (such as Figure 17), recycling 3070bp PS frame (such as Figure 18) is connect with above-mentioned filling-in segment Tyr-TYR-bGHpA (its as a purpose box gene), is screened Positive colony out, upgrading grain and digestion identification (such as Figure 19,20) are sequenced after comparing correctly and save for use, be named as pPS-Tyr, Plasmid map such as Figure 21.

1 PS-TIR sequence of table

Sequence is respectively SEQ ID No.6 and SEQ ID No.7 in table 1.It is restriction enzyme site at thread-changing under in table 1.

2 PS-FAG-GFP primer sequence of table

Sequence is respectively SEQ ID No.8 and SEQ ID No.9 in table 2.

The transposon-mediated target gene of embodiment III, PS is examined in mouse cell horizontal expression

1, the recovery and cultivation of freeze-stored cell

PPS-PGK-NEO and pcDNA3.9-PSase plasmid (is purchased from OMEGA endotoxin-free plasmid extracts kit OMEGA company) it extracts, product final concentration is adjusted to 500ng/ul for cell transfecting.

It is removed from liquid nitrogen the cryopreservation tube (cell for being stored in this laboratory) equipped with human cervical carcinoma cell (Hela), immediately It is quickly shaken in the warm water of 37-40 DEG C of investment, until frozen stock solution melts completely；Rewarming is completed in 1-2min；By cell suspension Sterile centrifuge tube is moved into, 5mL culture solution is added, gently blows even；Cell suspension 800-1000rpm is centrifuged 5min, abandons supernatant； 1mL complete medium is added to the centrifuge tube containing cell precipitation, gently blows even, cell suspension is transferred to Tissue Culture Flask, is added Enter suitable complete medium to be cultivated.

2, cell transfecting and screening

Human cervical carcinoma cell Hela is divided into 4 groups, each group transfect respectively pPS-PGK-NEO:pcDNA3.9-PSase and Every group of 3 weights of pPS-PGK-NEO:pcDNA3.9, pSB-PGK-NEO:pT2-CMV-SB100X and pSB-PGK-NEO:pT2-CMV It is multiple.

Before transfection for 24 hours, 2000 μ LDMEM high sugar of each hole containing 10% fetal calf serum (being purchased from GIBCO company) into six orifice plates 5 × 10 are pressed in culture medium (being purchased from GIBCO company)⁵A cells/well is inoculated with Hela cell, and cell is made to reach about 70- before transfection 80% converges；2 kinds of plasmids are mixed to Opti-MEM (purchased from the GIBCO company) training being diluted in 100 μ L according to 1:1 mass ratio It supports in base, and leniently mixes；3 μ L FUGENE transfection reagents (being purchased from Promega company) are added to serum-free, the nothing of 100 μ L The Opti-MEM culture medium of antibiotic simultaneously mixes gently, in incubation at room temperature 5min；After 5min, respectively by the transfection reagent of 100 μ L Dilution is added in each group DNA dilution of 100 μ L, mixes gently and in being placed at room temperature for 20min；200 μ L mixtures are added Into ready hole, culture plate is gently shaken back and forth, places it in 37 DEG C, saturated humidity, 5%CO₂Incubator in cultivate, Transfection media is replaced with complete medium after 4h, after cultivating 24-48h completely, 1% cell inoculation is trained in 6 orifice plates It supports, when cell converges up to 10-20%, the G418 screening and culturing liquid that concentration is 600 μ g/mL is added, screens 10-12 days.With Giemsa dye liquor (being purchased from GIBCO company) dyeing, counts positive colony number.

3, cell positive clone identification is transfected

With pPS-PGK-NEO:pcDNA3.9-PSase (PS+/PSase+) and pSB-PGK-NEO:pT2-CMV-SB100X (SB+/SBase+) group is experimental group, with pPS-PGK-NEO:pcDNA3.9 (PS+/PSase-) and pSB-PGK-NEO:pT2- CMV (SB+/SBase-) group is control group, after the liquid of resistance culture containing G418 screens 10-12 days, Gimsa (Jim Sa) dyeing meter Number.

As a result, it has been found that: PS experimental group positive cell clone number is that 560, PS control group positive cell clone number is 18, SB reality It is 18 that test group positive cell clone number, which be 427, SB control group positive cell clone number,.The cell expression neomycin of PS experimental group is anti- The cell quantity of property gene is significantly higher than the number of cell clones (such as Figure 22,23) of SB experimental group.The result shows that PS Transposon System Neomycin resistance gene transfection integration can be efficiently mediated in Hela cell, and is higher than SB transposons activity, that is, uses resistance Gene confirms that PS Transposon System can efficiently mediate foreign gene transfer.

Gene transfer of the transposon-mediated target gene of embodiment IV, PS in mice embryonic

1, the preparation of mouse fertilized egg

In mature female mouse peritoneal after the pregnant mare serum gonadotrop(h)in (PMSG) (PMSG) of injection 5 international units (IU), about exist The human chorionic gonadotrophin (hCG) of 2.5-5.0IU is injected after 48-54h again, ovulation can be induced after about 12h.Female mouse It is mated at once with male mice after giving hCG.The vaginal plug of post-coitum female mice is clear to, and can use mating indicator instruction.Fertilized eggs A few houres before the next morning after mating i.e. microinjection in collection period.Fallopian tubal is carefully cut, is rinsed using fallopian tubal Art or fallopian tubal pot portion's otomy collect fertilized eggs.

2, cytoplasm microinjection mouse fertilized egg

PPS-Tyr and pcDNA3.9-PSase plasmid is extracted with OMEGA endotoxin-free plasmid extracts kit, with linear PcDNA3.9-PSase is template, prepares PSase-mRNA, product final concentration tune with mMessagemMachine kit kit Whole is 20ng/ μ L, pPS-CAG-GFP and PS-mRNA with 1:1 molar ratio mixing for standby use.

It is experimental group by pPS-Tyr plasmid and PSase mRNA hybrid injection, using FVB mouse as model organism, cytoplasm note Mouse fertilized egg is penetrated, the variation of later period mouse hair color is observed, such as Figure 24 can obviously observe the variation of wherein hair color.

Gene transfer of the transposon-mediated target gene of embodiment V, PS in zebrafish embryo

1, the preparation of zebra fish fertilized egg

Male and female fish is respectively raised several, in injection evening before that day, pipettes 1 tail mother fish, 1 tail public affairs fish is in breeding box, partition Culture is separated, is protected from light and is incubated overnight after light stimulus 1h, is taken away partition within second day, male and female fish, which chases, to be started to lay eggs, from oviposition Start timing, receive ovum after about 10min, the fertilized eggs of collection are placed under stereomicroscope and carry out zebrafish embryo injection experiment.

2, microinjection zebrafish embryo

PPS-FAG-GFP and pcDNA3.9-PSase plasmid is extracted with endotoxin-free plasmid extracts kit, is used It is saved backup after NanoPhotometer nucleic acid concentration analyzer measurement concentration.PcDNA3.9-PSase plasmid is same as above, system Standby PS transposase mRNA, saves backup after measuring concentration.

By pPS-FAG-GFP plasmid and PS transposase mRNA with various concentration hybrid injection zebra fish one cell stage. The end of fixed Transposon plasmid final concentration of 25ng/ μ L, PS-mRNA (by being obtained after the transcription of pcDNA3.9-PSase vector in vitro) Concentration sets 2 gradients: 5ng/ μ L and 25ng/ μ L, while being control with single Transposon plasmid group of injecting, and untreated fish group (is not infused Penetrate group) it is blank control group.Every group of 3 repetitions, each duplicate injection embryo number is at 100 pieces or more.By the embryo after injection It is cultivated in 1 × E3 culture solution, 12-24h is changed the liquid once, and dead embryo is removed while changing liquid, control group is according to same side Method culture.Respectively culture for 24 hours, after 48h and 5d in fluorescence microscopy microscopic observation green fluorescence expression, while statistics survives Embryo number and positive embryos number calculate positive rate.

3, various concentration transposase mediates destination gene expression detection

Luciferase expression situation is detected under fluorescence microscope, each group embryo in each period is after microinjection as the result is shown For 24 hours, there are luciferase expression in tri- periods of 48h and 5d, and single Transposon plasmid group of injecting also has luciferase expression, and blank control group does not have There is luciferase expression (such as Figure 25).Whether injecting PS transposase, the positive rate of zebra fish expressing green fluorescent protein is different；For 24 hours, When 48h and 5d, be significantly higher than containing transposase group (PS+ group) positive rate (87.14%, 92.77%, 92.77%) do not inject turn Seat enzyme group (PS- group) positive rate (48.73%, 61.51%, 61.51%) (such as Figure 26).The result shows that PS Transposon System is same PS swivel base subsystem can be confirmed on zebrafish embryo in the efficient green fluorescent protein gene mediated transfer of zebrafish embryo System has the function of mediating gene transfer.

The basic principles, main features and advantages of the present invention have been shown and described above.The technology of the industry Personnel are it should be appreciated that the present invention is not limited to the above embodiments, and what is described in the above embodiment and the description is only in order to say Bright the principle of the present invention, various changes and improvements may be made to the invention without departing from the spirit and scope of the present invention, this A little changes and improvements all fall within the protetion scope of the claimed invention.The claimed scope of the invention is by appended claims And its equivalent defines.

Sequence table

<110>Yangzhou University

<120>a kind of PS Transposon System and its gene transfer method of mediation

<130> xhx2019050501

<141> 2019-05-05

<160> 9

<170> SIPOSequenceListing 1.0

<210> 1

<211> 3070

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 1

gcccctgcag ccgaattata ttatttttgc caaataattt ttaacaaaag ctctgaagtc 60

ttcttcattt aaattcttag atgatacttc atctggaaaa ttgtcccaat tagtagcatc 120

acgctgtgag taagttctaa accatttttt tattgttgta ttatctctaa tcttactact 180

cgatgagttt tcggtattat ctctattttt aacttggagc aggttccatt cattgttttt 240

ttcatcatag tgaataaaat caactgcttt aacacttgtg cctgaacacc atatccatcc 300

ggcgtaatac gactcactat agggagagcg gccgccagat cttccggatg gctcgagttt 360

ttcagcaaga tggatcctac cgtattttcc gcactataag gcgcacctcg cgagcggccg 420

cgaattcggt gcgccttata gtgcggaaaa tacggtagga tccccatctt tctagaagat 480

ctcctacaat attctcagct gccatggaaa atcgatgttc ttcttttatt ctctcaagat 540

tttcaggctg tatattaaaa cttatattaa gaactatgct aaccacctca tcaggaaccg 600

ttgtaggtgg cgtgggtttt cttggcaatc gactctcatg aaaactacga gctaaatatt 660

caatatgttc ctcttgacca actttattct gcattttttt tgaacgaggt ttagagcaag 720

cttcaggaaa ctgagacagg aattttatta aaaatttaaa ttttgaagaa agttcagggt 780

taatagcatc cattttttgc tttgcaagtt cctcagcatt cttaacaaaa gacgtctctt 840

ttgacatgtt taaagtttaa acctcctgtg tgaaattgtt atccgctcac aattccacac 900

attatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc 960

acattaattg cgttgcgctc actgccaatt gctttccagt cgggaaacct gtcgtgccag 1020

ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc 1080

gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct 1140

cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg 1200

tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc 1260

cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga 1320

aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct 1380

cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg 1440

gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag 1500

ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat 1560

cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac 1620

aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac 1680

tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc 1740

ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt 1800

tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc 1860

ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg 1920

agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca 1980

atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca 2040

cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag 2100

ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac 2160

ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc 2220

agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct 2280

agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc 2340

gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg 2400

cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc 2460

gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat 2520

tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag 2580

tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat 2640

aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg 2700

cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca 2760

cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga 2820

aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc 2880

ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata 2940

tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg 3000

ccacctgacg tctaagaaac cattattatc atgacattaa cctataaaaa taggcgtatc 3060

acgaggcccc 3070

<210> 2

<211> 28

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 2

ccgtattttc cgcactataa ggcgcacc 28

<210> 3

<211> 28

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 3

ggtgcgcctt atagtgcgga aaatacgg 28

<210> 4

<211> 5206

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 4

gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg 60

ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120

cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180

ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240

gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300

tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360

cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420

attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt 480

atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540

atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600

tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660

actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720

aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780

gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840

ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttggtacc 900

gagctcggat ccgccaccat ggctcctacc aaaagacacg cgtacaacgc tgagtttaaa 960

ctcaaggcga taagccacgc acaagaacac ggcaatagag cagcagcgag agaatttaat 1020

atcaatgaat caatggtgag gaagtggagg aagtatgagg atgagctccg ccaagtaaag 1080

aagacaacac agagtttccg cgggaacaaa gcgagatggc cacagttaga ggacaaagtt 1140

gaacagtggg ttgctgaaca aagagcagca agcagaagtg ttagtacagt cacaattcgt 1200

atgaaggcaa tagcgctagc tcgcgaacat aacatcagtg aattcagagg cggtccttct 1260

tggtgcttcc gttttatgaa acgacgtcat ctctccatcc gtacgcgcac tactgtgtca 1320

caacaactac cagctgatta tcaggaaaag ttggccactt tccgcacata ctgcagaaac 1380

aagataactg aaaaaaagat ccagccagag catatcatca atatggacga ggttccactc 1440

accttcgata tccctgtaaa ccgcactgtg gataaaacag gagcacgtac ggtgaatatt 1500

cgcaccacag ggaatgagaa aacgtccttc actgtagttc tcgcctgcca ggctaatggc 1560

cacaaacttc cacccatggt tattttcaag aggaagacct tgccgaaaga aaactttcca 1620

gctggcattg tcataaaagc taactcgaag ggatggatgg atgaagaaaa gatgagtgag 1680

tggttgagag aaatttatgt gaagagaccg ggtggttttt ttcacacagc tccgtcccta 1740

ttgatctatg actccatgcg cgcacatatc accgagcatg tcaaaaaaca agtgaagcac 1800

actaattcag tgctcgccgt cattccgggt ggattaacaa aagaactcca gccgctcgat 1860

gttggcgtca acagagcatt caaagctcga ctgcgaactg cgtgggagca gtggatgacc 1920

gaaggcgaac acacgttcac caagacgggg agacagcgcc ggacgacata tgctaatatc 1980

tgcaagtgga tagtaaatgc ctgggctggt atatcagtca caactgtggt ccgagctttt 2040

aggaaggcag gaattgtcac cgaactgcca gacaacagca gcgacactga ctcggttaat 2100

gatgactttg ataagacgga gccaggcgtt ttggatgccg caatagccca gctgttcaat 2160

tcggacacgg aagaagaagt tttcgaggga ttttagctcg agcatgcatc tagagggccc 2220

tattctatag tgtcacctaa atgctagagc tcgctgatca gcctcgactg tgccttctag 2280

ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac 2340

tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca 2400

ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag 2460

caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa ccagctgggg 2520

ctctaggggg tatccccacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt 2580

tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt 2640

cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc ggggcatccc 2700

tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga 2760

tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc 2820

cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt 2880

ctattctttt gatttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata 2940

aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc 3000

atgtctgtat accgtcgacc tctagctaga gcttggcgta atcatggtca tagctgtttc 3060

ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt 3120

gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc 3180

ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg 3240

ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct 3300

cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca 3360

cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga 3420

accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc 3480

acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg 3540

cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat 3600

acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcaatgctca cgctgtaggt 3660

atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc 3720

agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg 3780

acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg 3840

gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg 3900

gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg 3960

gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca 4020

gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga 4080

acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga 4140

tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt 4200

ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt 4260

catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat 4320

ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag 4380

caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct 4440

ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt 4500

tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg 4560

cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca 4620

aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt 4680

tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat 4740

gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac 4800

cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa 4860

aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt 4920

tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt 4980

tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa 5040

gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt 5100

atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa 5160

taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtctc 5206

<210> 5

<211> 1275

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 5

atggctccta ccaaaagaca cgcgtacaac gctgagttta aactcaaggc gataagccac 60

gcacaagaac acggcaatag agcagcagcg agagaattta atatcaatga atcaatggtg 120

aggaagtgga ggaagtatga ggatgagctc cgccaagtaa agaagacaac acagagtttc 180

cgcgggaaca aagcgagatg gccacagtta gaggacaaag ttgaacagtg ggttgctgaa 240

caaagagcag caagcagaag tgttagtaca gtcacaattc gtatgaaggc aatagcgcta 300

gctcgcgaac ataacatcag tgaattcaga ggcggtcctt cttggtgctt ccgttttatg 360

aaacgacgtc atctctccat ccgtacgcgc actactgtgt cacaacaact accagctgat 420

tatcaggaaa agttggccac tttccgcaca tactgcagaa acaagataac tgaaaaaaag 480

atccagccag agcatatcat caatatggac gaggttccac tcaccttcga tatccctgta 540

aaccgcactg tggataaaac aggagcacgt acggtgaata ttcgcaccac agggaatgag 600

aaaacgtcct tcactgtagt tctcgcctgc caggctaatg gccacaaact tccacccatg 660

gttattttca agaggaagac cttgccgaaa gaaaactttc cagctggcat tgtcataaaa 720

gctaactcga agggatggat ggatgaagaa aagatgagtg agtggttgag agaaatttat 780

gtgaagagac cgggtggttt ttttcacaca gctccgtccc tattgatcta tgactccatg 840

cgcgcacata tcaccgagca tgtcaaaaaa caagtgaagc acactaattc agtgctcgcc 900

gtcattccgg gtggattaac aaaagaactc cagccgctcg atgttggcgt caacagagca 960

ttcaaagctc gactgcgaac tgcgtgggag cagtggatga ccgaaggcga acacacgttc 1020

accaagacgg ggagacagcg ccggacgaca tatgctaata tctgcaagtg gatagtaaat 1080

gcctgggctg gtatatcagt cacaactgtg gtccgagctt ttaggaaggc aggaattgtc 1140

accgaactgc cagacaacag cagcgacact gactcggtta atgatgactt tgataagacg 1200

gagccaggcg ttttggatgc cgcaatagcc cagctgttca attcggacac ggaagaagaa 1260

gttttcgagg gattt 1275

<210> 6

<211> 56

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 6

ggatcctacc gtattttccg cactataagg cgcacctcgc gagcggccgc gaattc 56

<210> 7

<211> 56

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 7

ggatcctacc gtattttccg cactataagg cgcaccgaat tcgcggccgc tcgcga 56

<210> 8

<211> 58

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 8

ccgtattttc cgcactataa ggcgcaccac tagtgctttt agaccttctt acttttgg 58

<210> 9

<211> 56

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 9

ccgtattttc cgcactataa ggcgcacctt aattaaagct tgggctgcag gtcgag 56

Claims (9)

1. a kind of PS Transposon System, which is characterized in that including containing two terminal repeats that can be inserted into target gene box Transgenic donor plasmid and provide transposition activity transposase helper plasmid；Target gene box can for reporter gene expression box or Other exogenous gene expression boxes or gene trap element；Two terminal repeats are respectively such as SEQ ID No.2 and SEQ ID Shown in No.3.

2. Transposon System according to claim 1, which is characterized in that two terminal repeats of PS transposons are distinguished It is respectively the inverted terminal repeat sequence of 28bp for PS5 ' TIR and PS3 ' TIR, PS5 ' TIR and PS3 ' TIR；Transposase assists matter Contain PS CDS in grain, PS CDS is the transposase sequence of 1275bp, encodes 425 amino acid.

3. PS Transposon System according to claim 1, which is characterized in that the transgenic donor plasmid includes: PS Transposon ends repetitive sequence, destination gene expression box；The transposase helper plasmid is transposase eukaryon expression plasmid PcDNA3.9-PSase, transposase helper plasmid can also be used as that carrier is transcribed in vitro；The transgenic donor plasmid sequence is such as Shown in SEQ ID No.1；The sequence of pcDNA3.9-PSase is as shown in SEQ ID No.4.

4. PS Transposon System according to claim 1, which is characterized in that two ends of the transgenic donor plasmid Repetitive sequence is to carry out obtained by Molecular remodeling to PS swivel base sub-family in Tc1/Mariner superfamily.

5. PS Transposon System according to claim 3, which is characterized in that the target gene box can be report Expression casette perhaps other exogenous gene expression boxes or gene trap element.

6. PS Transposon System according to claim 2, which is characterized in that the transposase sequence is to Tc1/ PS swivel base sub-family carries out Molecular remodeling acquisition in Mariner superfamily；Transposase eukaryon expression plasmid pcDNA3.9-PSase Construction step are as follows: PS transposase dna sequence is connect with pcDNA3.9 carrier, building expression transposase helper plasmid, because Contain T7 promoter and T7 transcription terminator on pcDNA3.9, therefore transposase helper plasmid is alternatively arranged as that transposase is transcribed in vitro Helper plasmid；The transposase helper plasmid sequence is as shown in SEQ ID No.4.

7. PS Transposon System according to claim 6, which is characterized in that transposase helper plasmid is transcribed in vitro It is capped that pcDNA3.9-PSase carrier can carry out PS transposase 5 ' using mMessagemMachine T7 kit kit The in-vitro transcription of mRNA.

8. a kind of gene transfer method based on PS Transposon System, which is characterized in that be by claim 1-7 any one Target gene is imported acceptor gene group, the PS Transposon System in the way of gene cloning by the PS Transposon System It include: the transgenic donor plasmid containing two terminal repeats that can be inserted into target gene box and turn that transposition activity is provided Seat enzyme helper plasmid.

9. a kind of preparation method of the PS Transposon System as described in claim 1-7 any one, which is characterized in that (1) turn The building of genetic donor plasmid: element needed for cloning destination gene expression box respectively with round pcr；Construct destination gene expression Box；The destination gene expression box is cloned between PS5 ' TIR and PS3 ' TIR of PS transposons by gene clone technology, Construct transgenic donor plasmid；(2) building of transgenosis helper plasmid: chemical synthesis transposase sequence Psase CDS makes PSase CDS is connect with pcDNA3.9 carrier, and the helper plasmid of transposase can be expressed or be transcribed in vitro to building；PSase CDS For PS CDS.