CN110257425B

CN110257425B - PS transposon system and mediated gene transfer method thereof

Info

Publication number: CN110257425B
Application number: CN201910366530.0A
Authority: CN
Inventors: 宋成义; 王赛赛; 高波; 宗文成; 沈丹; 王亚丽; 产舒恒; 桑亚通
Original assignee: Yangzhou University
Current assignee: Shanghai Cell Therapy Group Co Ltd
Priority date: 2019-05-05
Filing date: 2019-05-05
Publication date: 2022-07-15
Anticipated expiration: 2039-05-05
Also published as: CN110257425A

Abstract

The invention discloses a PS transposon system and a mediated gene transfer method thereof, which comprises a transgenic donor plasmid which can be inserted into two terminal repetitive sequences of a target gene box and a transposase auxiliary plasmid which provides transposition activity. The gene of interest is introduced into the recipient genome by the PS transposon system. The gene transfer method based on the PS transposon system can efficiently mediate gene transfer through cell and embryo level verification, and can be applied to a plurality of biotechnology fields: 1) the method provided by the invention can be used for effectively inserting the target gene cassette into a host genome and improving the gene transfer efficiency; 2) the animal gene function research can be effectively carried out by combining with the gene capturing technology; 3) can mediate human gene therapy, etc.

Description

PS transposon system and mediated gene transfer method thereof

Technical Field

The invention relates to a method for establishing a PS transposon system-mediated gene transfer, and also discloses a construction method of a transgenic donor plasmid and transposase eukaryotic expression and in-vitro transcription auxiliary plasmid related to the method, and application of the method in preparing transgenic animals, researching gene functions and human gene therapy. The invention belongs to the field of animal gene engineering.

Background

Gene transfer is an important biotechnology means at present, and can mediate stable integration of exogenous genes into host chromosomes, so that the gene transfer has important application values in the fields of gene function research, transgenic organism preparation, human gene therapy and the like.

Transposons are DNA sequences that can freely jump on the genome, and are first found in maize chromosomes in the 20 th century and 40 th century by mc. After annotation of the transposons, it was found that a considerable number of transposons are present in both prokaryotes and eukaryotes. Transposons vary widely in distribution among different organisms, particularly higher in higher eukaryotes, e.g., transposons are the largest component of the mammalian genome, accounting for almost half of the human genome, and 80% in maize. The transposition process of DNA transposons follows the mechanism of "cut-and-paste" to move on the genome and is therefore being developed as an effective gene transfer tool. In recent years, transposons have made important progress in the study of human gene therapy, model organism preparation such as transgenic mice and zebrafish, and functional genomics. In addition, transposons have many advantages as vectors for gene transfer: 1) the structure is simple, only TIR sequences on two sides are integrated into a receptor genome together with the exogenous gene, the influence on the exogenous gene is small, and the phenomena of loss of large segments and chromosome rearrangement are not found at an integration site; 2) the DNA transposon vector is less restrictive to the inserted fragment than the viral vector; 3) the exogenous gene can be stably integrated into a chromosome and can be expressed for a long time after passage through germ cells; 4) transposase can catalyze the accurate insertion of single-copy genes into a portable sequence, random integration is not relied on, and the size of an inserted fragment is not changed; 5) the transposable system may be administered entirely as naked DNA, or DNA may be used in combination with an RNA or protein to provide transposase for transposition, and is therefore less immunogenic. Among the most widely studied DNA transposons, Sleeping Beauty (SB), PiggBac (PB) and Tol2, differ not only in origin but also in transposition activity and insertion preference, and thus differ in application. For example, SB preferential insertion sites are "TA", but there is no preference for genes, so SB transposons are often used for human gene therapy; the PB prefers to insert the site to be TTAA, and prefers to insert into the interior of a gene, so the PB is often used for functional gene research, gene capture and the like; tol2 has no obvious preferential insertion site, and the insertion site is mostly the upstream regulatory region of the gene, so it is commonly used to research enhancer capture and so on.

Microinjection is widely applied, the technology is mature and stable, and scientists further improve the transposon mediated cytoplasm microinjection method by utilizing the characteristic of transposon mediated gene transfer. Because the transposase in the DNA transposition system contains a nuclear localization signal sequence (NLS), the target gene can be smoothly mediated to enter a cell nucleus, and the injection can be directly carried out cytoplasm injection without entering the cell nucleus. This technology is gaining in value and has been an encouraging development, which may be a new approach to breaking the bottleneck of transgenic technology in large mammals.

The transposon is mainly used for human gene therapy, and comprises the stages of target gene cloning, gene transfer, target cell selection, clinical trial observation and the like, wherein the gene transfer is a key step of the gene therapy, and the DNA transposon mediated gene transfer has the advantages of high efficiency and safety in the gene therapy. To date, transposons have been referred to as the most commonly used non-viral vectors in gene therapy. In addition, the transposon can also be used for preparing mutants, carrying out gene capture, application and functional genomics research.

DNA transposon-mediated gene transfer systems are a highly favored transgenic technology by scientists, but DNA transposons with autonomous transposition activity are rare in vertebrates. In 1996, a naturally active vertebrate transposon, the Tol2 transposon, was first found in albino blue (oryziasalatipes); in 2008, a second case of Tgf2 transposon with autonomous transposition activity was found in goldfish and was very similar to the green Tol2 transposon element. The inventor utilizes a bioinformatics means to find a Passer (PS) family in a research on Tc1/Mariner transposon superfamily of vertebrates, and the insertion age analysis and the like show that the Passer (PS) family may have higher activity. Molecular reconstruction is carried out on the basis of systematic evolution comparative research to obtain TIR key elements of two terminal repetitive sequences of PS transposons and sequences of transposase, a set of gene transfer vector system is constructed, and the vector system can effectively mediate gene transfer through verification of cells, transgenic mice, zebra fishes and the like, and has great application potential in preparation of transgenic animals and gene therapy.

Disclosure of Invention

In order to overcome the defects, the invention provides a PS transposon system and a mediated gene transfer method thereof, which adopt a bioinformatics method to discover a PS family in a Tc1/Mariner superfamily, carry out molecular reconstruction to obtain two terminal repetitive sequences and transposase sequences of a transposon, construct a PS transposon donor plasmid and an auxiliary plasmid for expressing PS transposase, assemble a set of gene transfer system, and name the PS transposon system. The invention aims to provide a PS transposition system-mediated high-efficiency gene transfer method, which can improve the transgenic preparation efficiency of animals such as mice, zebra fish and the like, and can be effectively applied to the research fields of cell gene transfection and integration, human gene therapy, gene capture and the like.

The PS transposon system mainly comprises a PS transposon donor plasmid and a PS transposase auxiliary plasmid.

The transgenic donor plasmid (pLB-PS) comprises terminal repetitive sequences at two sides of a PS transposon and a polyclonal insertion site, wherein the polyclonal insertion site (NruI/NotI/EcoRI restriction site) can be inserted into a target gene box to be transferred, and the sequence of the donor plasmid (pLB-PS) is shown as SEQ ID No. 1. The gene of interest can be inserted into the transgenic donor plasmid through any of the polyclonal insertion sites described above. The insertion of the target gene into the transgenic donor plasmid means that the target gene is inserted between the PS5 'TIR and the PS 3' TIR.

The PS transposon bilateral terminal repetitive sequences are derived from molecular reconstruction of a PS family in a Tc1/Mariner superfamily, and bilateral Terminal Inverted Repetitive Sequences (TIRs) are 28 nucleotide sequences (namely PS5 'TIR and PS 3' TIR) respectively, and the sequences are respectively shown as SEQ ID No.2 and SEQ ID No. 3.

The target gene cassette of the invention can be a reporter gene expression cassette, or other exogenous gene expression cassettes, or gene capture elements.

The PS transposase (PS CDS, also called PSase CDS) sequence is obtained by molecular reconstruction of a PS transposon family by using a bioinformatics analysis means to obtain a transposase sequence and chemical synthesis, and is shown as SEQ ID No. 5.

The PS transposase auxiliary plasmid pcDNA3.9-PSase has two forms, one is eukaryotic expression plasmid capable of being directly expressed in cells and in vivo, and the other is in vitro transcription auxiliary plasmid capable of being transcribed in vitro to form mRNA of 5' capped PS transposase.

The pcDNA3.9-PSase eukaryotic expression plasmid comprises a virus promoter sequence (CMV), a PS transposase cDNA sequence and bGH poly A (PolyA); the vector framework is from a pcDNA3.9 vector, and the length of the virus promoter sequence (CMV) is 584 nucleotide sequences and can guide the expression of downstream genes; the PS transposase is 1275 nucleotide sequences in length; the bGH polyadenylation (PolyA) has a length of 225 nucleotide sequences, the vector can autonomously express transposase, meanwhile, the vector can be used as an in vitro transcription auxiliary plasmid and comprises a T7promoter, a PS transposase cDNA sequence and a bGH PolyA sequence, wherein the length of the T7promoter is 19 nucleotide sequences (bp) of the sequence of the vector, the bGH PolyA sequence terminates transcription, the vector can carry out in vitro transcription of PS transposase 5' capped mRNA by using an mMESSAGE Mmachine T7kit (purchased from Invitrogen company), and the sequence of the vector is shown as SEQ ID No. 4.

The gene transfer method based on the PS transposon system can efficiently mediate gene transfer through cell and embryo level verification, and can be applied to a plurality of biotechnology fields: 1) the method provided by the invention can be used for effectively inserting the target gene cassette into a host genome and improving the gene transfer efficiency; 2) the animal gene function research can be effectively carried out by combining with the gene capturing technology; 3) can mediate human gene therapy, etc.

Drawings

FIG. 1: pLB-PSase plasmid electrophoretogram;

FIG. 2 is a schematic diagram: pcDNA3.9-PSase plasmid electrophoretogram;

FIG. 3: pcDNA3.9-PSase plasmid map;

FIG. 4: pLB-PS plasmid schematic;

FIG. 5: (iii) electrophoretogram of PS-TIR annealed product;

FIG. 6: electrophoretogram of PS-TIR purified product;

FIG. 7: pLB-PS plasmid electrophoretogram;

FIG. 8: pLB-PS plasmid enzyme cutting product electrophoresis picture;

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

FIG. 10: electrophoresis picture of pPS-PGK-NEO plasmid restriction enzyme product;

FIG. 11: pPS-PGK-NEO plasmid map;

FIG. 12: PCR cutting back the product of PS-FAG-GFP;

FIG. 13 is an electrophoretogram of pPS-FAG-GFP plasmid;

FIG. 14 depicts pPS-FAG-GFP cleavage electrophoresis, 1: pPS-FAG-GFP enzyme digestion; 2: pPS-FAG-GFP plasmid;

FIG. 15 is a pPS-FAG-GFP plasmid map;

FIG. 16 shows the pZB-RT-bGH cleavage electrophoresis pattern;

FIG. 17 shows pPS-PGK-NEO cleavage electrophoresis;

FIG. 18 cut back the PS framework, Tyr-TYR-bGHpA fragment to electrophoretogram, 1: the PS frame is switched back to the electropherogram; 2: the Tyr-TYR-bGHpA fragment is excised back into the electrophoretogram;

FIG. 19 is an electrophoretogram of pPS-Tyr plasmid;

FIG. 20 shows pPS-Tyr cleavage electrophoresis patterns, 1-6: pPS-Tyr enzyme digestion; 7: pPS-Tyr plasmid;

FIG. 21 is a plasmid map of pPS-Tyr;

FIG. 22 is a graph showing G418-resistant clones obtained from Hela cells;

FIG. 23 is a graph comparing transposition activities of PS and SB;

FIG. 24 is a diagram of a PS transposition system for preparing a transgenic mouse;

FIG. 25 is a fluorescence image of zebra fish embryos injected in different periods;

FIG. 26 is a graph showing the comparison of the positive rates of zebra fish microinjected with PS transposons.

Detailed Description

In order to better illustrate the invention and to facilitate the understanding of the technical solutions thereof, typical but non-limiting examples of the invention are as follows:

the experimental procedures mentioned in the following examples are conventional ones unless otherwise specified.

Example I construction of transposase expression vector pcDNA3.9-PSase

1. Transposase PSase vector synthesis.

The PSase transposase sequence was chemically synthesized and cloned to LB vector to construct p LB-PSase vector, and the plasmid DNA was detected by 1% agarose gel electrophoresis (see FIG. 1).

2. Construction of transposase eukaryotic expression plasmid and in vitro transcription auxiliary plasmid pcDNA3.9-PSase

The transposase CDS was excised from the pLB-PSase vector using the restriction enzymes BamHI and XhoI, and the 1296bp CDS sequence was recovered from the agarose gel. The pcDNA3.9 vector was digested simultaneously with BamHI and XhoI, and the 3.9kb fragment was recovered as a vector frame by gel-cutting. After the transposase CDS and pcDNA3.9 vector framework are connected, a competent cell Top10 is transformed, a single colony is picked up to a liquid medium LA for culture, the quality-improved granule is subjected to electrophoretic identification (as shown in figure 2), a positive clone is named as pcDNA3.9-PSase, and a plasmid map is shown in figure 3.

The sequence of the pcDNA3.9-PSase vector is shown in SEQ ID No.4, and comprises CMV Enhancer 235-818, T7 Enhancer 863-881, PSase CDS 919-2193, Sp6 Enhancer 2224-2242, bGH poly (A) signal 2268-2492, F1Origin 2538-2896, SV40poly (A) signal 2897-2998 and Ampicillin Resistance gene 4208-5068.

Example II construction of transposon expression vectors

1. Transgenic Donor plasmid pLB-PS vector construction

1.1 Synthesis of two inverted terminal repeat elements of PS (PS 3 'and 5')

The insertion age and other analyses of the PS transposons in the Tc1/Marinier family are carried out on multiple species, molecular reconstruction is carried out on the basis of systematic evolution comparison research, and highly conserved 5 'TIRs and 3' TIRs which are respectively reverse complementary sequences of 28bp are determined. The sequence is added with restriction endonuclease sites and sent to Huada Gene company for synthesis, and the sequence is shown in Table 1.

1.2 polymerase amplification of PS transposable elements

Annealing the chemically synthesized single-stranded nucleotide chain PS 5't (comprising the inverted repeat element and the enzyme cutting site at the end of PS 5', and the enzyme cutting site is used for inserting a target gene) and PS 3't (comprising the inverted repeat element and the enzyme cutting site at the end of PS 3', and the enzyme cutting site is used for inserting the target gene), and then extending by using Klenow polymerase to form a double chain. The reaction system is 50 μ L, comprises 1ul PS 5't (100uM), 1ul PS 3't (100uM) and 23ul ultrapure water, denatures for 5min at 95 ℃, and then reduces the temperature to 25 ℃ at 0.1 ℃ per second for 5 min. Then, 5. mu.L of Klenow Buffer, 2. mu.L of Klenow polymerase, 5. mu.L of 4dNTP, 13. mu.L of ultrapure water, 1.5h at 37 ℃ and 20min at 80 ℃ were added thereto, and the temperature was lowered to normal temperature to obtain the target sequence (PS TIR). Subsequently, the DNA was purified using Qiagen PCR purification kit (see FIGS. 5 and 6), and stored at-20 ℃ until the concentration was determined by a nucleic acid concentration measuring instrument. The plasmid is schematically shown in FIG. 4, and the gene insertion site in FIG. 4 is a multiple cloning site (restriction site).

1.3 p LB-PS vector construction

The p LB-PS vector structure schematic diagram is shown in FIG. 4, the purified PS transposon fragment (i.e. the target sequence PS TIR obtained in 1.2 above) is connected with the Tiangen zero background vector pLB by T4 ligase, competent cells Top10 are transformed, a single colony is picked up to a liquid culture medium LB containing Amp for culture, the upgraded granule is subjected to electrophoresis and enzyme digestion identification (as shown in FIGS. 7 and 8), and the positive clone is named pLB-PS. pLB-PS vector sequence is shown as SEQ ID No.1, and comprises T7promoter 305-323; PS 5' TIR 380-407(SEQ ID No. 2); PS 3' TIR 428-; ori 1257-; ampicillin Resistance gene 2016-. In SEQ ID No.1, the bases 408-427 are multiple cloning sites (restriction sites) for inserting the target gene.

2. Construction of transposon vector pPS-PGK-NEO

The PGK-NEO-bGHpA-TA cloning vector (the PGK-NEO-bGHpA-TA cloning vector is a storage vector of the laboratory) is cut by restriction endonuclease Nru1, and 1659bp of PGK-NEO-bGHpA expression frame (which is used as a target gene cassette) is recovered by cutting gel. The LB-PS vector is cut by Nru1 enzyme, the 3066bp vector framework is recovered, the expression frame is connected with the vector, the competent cell Top10 is transformed, a single colony is picked to the liquid culture medium LB containing Amp for culture, the quality-improved grains are subjected to electrophoresis and enzyme cutting identification, the grains with correct size and insertion direction are screened (as shown in figures 9 and 10), the Huada gene is sent for sequencing, and the plasmid map is shown in figure 11.

3. Construction of transposon vector pPS-FAG-GFP

The FAG-GFP gene fragment was amplified using pZB-FAG-GFP plasmid DNA as a template and the primers for amplifying PS-FAG-GFP listed in Table 2 (the amplified FAG-GFP gene fragment was used as the desired gene cassette). The reaction system is 50. mu.l, 10. mu.l of 5 XSF Buffer, 1. mu.l of dNTP, 2. mu.l of 10. mu.M primer PS-FAG-GFP-F, 2. mu.l of 10. mu.M primer PS-FAG-GFP-R (see primer sequence table 2), 1. mu.l of Phanta^TMSuper-Fidelity, 1. mu.l pZB-FAG-GFP plasmid DNA template, and ultra-pure water to 50. mu.l (high Fidelity enzyme from Novonoprazan). PCR amplification procedure: the reaction is cycled for 30 times at 94 ℃ for 40s, 55 ℃ for 40s and 72 ℃ for 1m30 s. The PCR amplification products were detected by electrophoresis on a 1% agarose gel. And (3) recovering agarose gel of a PCR product (shown in figure 12), connecting the agarose gel with a p LB vector, carrying out TA cloning, screening out positive clones, extracting plasmids, carrying out enzyme digestion identification (shown in figures 13 and 14), carrying out sequencing comparison to obtain a correct sequence, storing the sequence for the next experiment, and naming the sequence as pPS-FAG-GFP, wherein the plasmid map is shown in figure 15.

4. Construction of transposon vector pPS-Tyr

Digesting the vector pZB-RT-bGH (shown in figure 16) by using a restriction enzyme NheI, recovering a Tyr-TYR-bGHpA fragment (2555bp) (shown in figure 18), filling and purifying, and storing for later use; the vector pPS-PGK-NEO is cut by NruI enzyme (as figure 17), 3070bp PS framework is recovered (as figure 18), the obtained product is connected with the filling fragment Tyr-TYR-bGHpA (as a target gene box), positive clones are screened out, plasmids are extracted and enzyme digestion identification is carried out (as figures 19 and 20), the obtained product is stored for later use after the sequencing comparison is correct, and the obtained product is named as pPS-Tyr, and the plasmid map is as figure 21.

TABLE 1 PS-TIR sequences

The sequences in Table 1 are SEQ ID No.6 and SEQ ID No.7, respectively. The lower alternate in Table 1 is the enzyme cleavage site.

TABLE 2 PS-FAG-GFP primer sequences

The sequences in Table 2 are SEQ ID No.8 and SEQ ID No.9, respectively.

Example III test of expression of Gene of interest in mouse cells mediated by PS transposon

1. Recovery and culture of cryopreserved cells

The pPS-PGK-NEO and pcDNA3.9-PSase plasmids were extracted using an OMEGA endotoxin-free plasmid extraction kit (purchased from OMEGA), and the final concentration of the product was adjusted to 500ng/ul for cell transfection.

Taking out the cryopreservation tube (cells preserved in the laboratory) containing human cervical cancer cells (Hela) from the liquid nitrogen, immediately putting into warm water at 37-40 ℃ and rapidly shaking until the cryopreservation liquid is completely dissolved; completing rewarming within 1-2 min; transferring the cell suspension into a sterile centrifuge tube, adding 5mL of culture solution, and gently and uniformly blowing; centrifuging the cell suspension at 800-; adding 1mL of complete culture medium into a centrifuge tube containing the cell sediment, gently and uniformly blowing, transferring the cell suspension into a cell culture bottle, and adding a proper amount of complete culture medium for culture.

2. Cell transfection and selection

Human cervical carcinoma cell Hela was divided into 4 groups, each transfected with 3 replicates of each of pPS-PGK-NEO, pcDNA3.9-PSase and pPS-PGK-NEO, pcDNA3.9, pSB-PGK-NEO, pT2-CMV-SB100X and pSB-PGK-NEO, pT 2-CMV.

24h before transfection, 5X 10 aliquots of 2000. mu.L MEM high-sugar medium (from GIBCO) containing 10% fetal bovine serum (from GIBCO) per well in six-well plates⁵Seeding each cell/well with Hela cells to achieve about 70-80% confluence before transfection; and (3) mixing 2 plasmids according to the proportion of 1:1 mass ratio was mixed and diluted in 100. mu.L of Opti-MEM (purchased from GIBCO Co.) medium and gently mixed; mu.L of FUGENE transfection reagent (available from Promega) was added to 100. mu.L of serum-free, antibiotic-free reagentThe Opti-MEM medium was mixed gently and incubated at room temperature for 5 min; after 5min, 100. mu.L of the transfection reagent diluent was added to each 100. mu.L of the DNA diluent, gently mixed and left at room temperature for 20 min; add 200. mu.L of the mixture to the prepared wells, gently shake the plates back and forth, place them at 37 ℃ with saturated humidity and 5% CO₂After 4 hours, the transfection medium is replaced by the complete medium, after 24-48 hours of complete culture, 1% of cells are inoculated into a 6-well plate for culture, when the cells reach 10-20% confluence, G418 screening culture solution with the concentration of 600 mug/mL is added, and screening is carried out for 10-12 days. Positive clones were counted by staining with Giemsa stain (purchased from GIBCO).

3. Positive clone identification of transfected cells

The experimental group of pPS-PGK-NEO pcDNA3.9-PSase (PS +/PSase +) and pSB-PGK-NEO pT2-CMV-SB100X (SB +/SBase +) was used, the control group of pPS-PGK-NEO pcDNA3.9(PS +/PSase-) and pSB-PGK-NEO pT2-CMV (SB +/SBase-) was used, and after screening the G418-containing resistant culture solution for 10-12 days, Gimsa (Giemsa) staining was counted.

As a result, it was found that: the number of positive cell clones in the PS experimental group is 560, the number of positive cell clones in the PS control group is 18, the number of positive cell clones in the SB experimental group is 427, and the number of positive cell clones in the SB control group is 18. The number of cells expressing the neomycin resistance gene in the PS experimental group was significantly higher than the number of cell clones in the SB experimental group (see fig. 22, 23). The result shows that the PS transposon system can efficiently mediate the transfection and integration of the neomycin resistance gene in Hela cells and is higher than the SB transposon activity, namely the resistance gene proves that the PS transposon system can efficiently mediate the transfer of foreign genes.

Example IV PS transposon mediated Gene transfer of Gene of interest in mouse embryos

1. Preparation of mouse fertilized eggs

After 5 International Units (IU) of pregnant horse serum gonadotropin (PMSG) is injected into the abdominal cavity of a mature female mouse, 2.5-5.0IU of human chorionic gonadotropin (hCG) is injected after about 48-54 h, and ovulation can be induced after about 12 h. The female mice were caged with the male mice immediately after administration of hCG. Vaginal plugs were readily visible in female mice after mating and were indicated by mating indicators. The fertilized egg collection period is the next morning after caging, i.e., several hours prior to microinjection. The fallopian tubes are carefully dissected and fertilized eggs are collected by salpingectomy or tubal ampullation.

2. Fertilized egg of mouse injected with cytoplasm

pPS-Tyr and pcDNA3.9-PSase plasmids are extracted by an OMEGA endotoxin-free plasmid extraction kit, linear pcDNA3.9-PSase is used as a template, a mMessageminal kit is used for preparing PSase-mRNA, the final concentration of a product is adjusted to be 20 ng/mu L, and pPS-CAG-GFP and PS-mRNA are mixed for later use according to the molar ratio of 1: 1.

pPS-Tyr plasmid and PSase mRNA were mixed and injected into the experimental group, FVB mouse was used as model organism, mouse fertilized egg was injected into cytoplasm, and the change of the hair color of the mouse in the later stage was observed, as shown in FIG. 24, the change of the hair color was observed.

Example V, PS transposon mediated Gene transfer of Gene of interest in Zebra Fish embryos

1. Preparation of fertilized zebra fish egg

Breeding a plurality of male and female fishes respectively, transferring 1 female fish one night before injection, placing 1 male fish in a breeding box, carrying out partition culture by using a partition plate, carrying out light-shading overnight culture after 1h of light stimulation, taking the partition plate away the next day, starting spawning when the male and female fishes chase after chasing, timing when spawning is started, collecting the spawns after about 10min, and placing the collected fertilized ova under a stereomicroscope to carry out zebra fish embryo injection experiment.

2. Microinjection of zebrafish embryos

pPS-FAG-GFP and pcDNA3.9-PSase plasmids are extracted by an endotoxin-free plasmid extraction kit, and are stored for later use after the concentration is determined by a NanoPhotometer nucleic acid concentration determinator. The pcDNA3.9-PSase plasmid was used to prepare PS transposase mRNA as described above, and the concentration was determined and stored for future use.

The pPS-FAG-GFP plasmid and the PS transposase mRNA were mixed at different concentrations and injected into zebrafish single-cell stage embryos. The final concentration of the immobilized transposon plasmid was 25 ng/. mu.L, and the final concentration of the PS-mRNA (obtained after in vitro transcription from the pcDNA3.9-PSase vector) was graded by 2: 5 ng/. mu.L and 25 ng/. mu.L, while the single-injection transposon plasmid group was used as a control, and the untreated group (i.e., non-injection group) was used as a blank control. Each group had 3 replicates, and the number of embryos injected per replicate was over 100. Culturing the injected embryo in 1 × E3 culture medium, changing the culture medium once for 12-24h, removing dead embryo while changing the culture medium, and culturing the control group according to the same method. And observing the green fluorescence expression condition under a fluorescence microscope after culturing for 24h, 48h and 5d respectively, counting the number of surviving embryos and the number of positive embryos, and calculating the positive rate.

3. Transposase-mediated target gene expression detection at different concentrations

The fluorescence expression was detected under a fluorescence microscope, and the results showed that the embryos of each group in each period had fluorescence expression at three time periods of 24h, 48h and 5d after microinjection, the single injected transposon plasmid group also had fluorescence expression, and the blank control group had no fluorescence expression (see fig. 25). If PS transposase is not injected, the positive rates of the zebra fish expressing green fluorescent protein are different; at 24h, 48h and 5d, the positive rate (87.14%, 92.77%, 92.77%) of the transposase-containing group (PS + group) was significantly higher than the positive rate (48.73%, 61.51%, 61.51%) of the non-injected transposase group (PS-group) (see fig. 26). The result shows that the PS transposon system can also efficiently mediate the gene transfer of the green fluorescent protein in the zebra fish embryo, namely the PS transposon system is proved to have the function of mediating the gene transfer on the zebra fish embryo.

The foregoing shows and describes the general principles, principal features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification for the purpose of illustrating the principles of the present invention, but that various changes and modifications may be made without departing from the spirit and scope of the invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.

Sequence listing

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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

1. A PS transposon system comprising a transgenic donor plasmid insertable into the two terminal repeats of a gene cassette of interest and a transposase helper plasmid providing transposition activity; the target gene cassette is a reporter gene expression cassette or other exogenous gene expression cassettes or a gene capture element; two terminal repetitive sequences are respectively shown as SEQ ID No.2 and SEQ ID No. 3; the transposase auxiliary plasmid contains PS CDS, the PS CDS is a transposase sequence of 1275bp, 425 amino acids are coded, and the transposase sequence is shown as SEQ ID No. 5.

2. A PS transposon system as in claim 1, wherein the transgenic donor plasmid comprises: PS transposon terminal repetitive sequence and target gene expression box; the transposase auxiliary plasmid is a transposase eukaryotic expression plasmid pcDNA3.9-PSase, and can also be used as an in vitro transcription vector; the sequence of the transgenic donor plasmid is shown as SEQ ID No. 1; the sequence of pcDNA3.9-PSase is shown in SEQ ID No. 4.

3. A PS transposon system as claimed in claim 1, wherein the two terminal repeats of the transgenic donor plasmid are obtained by molecular reconstruction of the PS transposon family in the Tc1/Mariner superfamily.

4. The PS transposon system of claim 1, wherein the transposase sequence is obtained by molecular reconstruction of the PS transposon family in the Tc1/Mariner superfamily; the construction steps of transposase eukaryotic expression plasmid pcDNA3.9-PSase are as follows: connecting the DNA sequence of PS transposase with pcDNA3.9 vector to construct auxiliary plasmid for expressing transposase, and the transposase auxiliary plasmid can also be used as in vitro transcription transposase auxiliary plasmid because pcDNA3.9 contains T7promoter and T7 transcription termination sequence; the sequence of the transposase helper plasmid is shown as SEQ ID No. 4.

5. A PS transposable system according to claim 4, characterized in that the in vitro transcription transposase helper plasmid pcDNA3.9-PSase vector is capable of performing in vitro transcription of PS transposase 5' capped mRNA using the mMessagemachine T7 kit.

6. A method of gene transfer based on a PS transposon system, comprising introducing a gene of interest into a recipient genome by gene cloning using the PS transposon system of any one of claims 1 to 5, wherein the PS transposon system comprises: a transgenic donor plasmid into which the two terminal repeats of the gene cassette of interest can be inserted and a transposase helper plasmid that provides transposition activity.

7. A method of making a PS transposon system as claimed in any one of claims 1 to 5, wherein (1) the transgenic donor plasmid is constructed: respectively cloning elements required by the target gene expression cassette by using a PCR technology; constructing a target gene expression cassette; cloning the target gene expression cassette between PS5 'TIR and PS 3' TIR of the PS transposon by a gene cloning technology to construct a transgenic donor plasmid; (2) construction of transgenic helper plasmids: chemically synthesizing a transposase sequence Psase CDS, connecting the PSase CDS with a pcDNA3.9 vector, and constructing an auxiliary plasmid capable of expressing or transcribing the transposase in vitro; the PSase CDS is the PS CDS.