CN110257425B - A PS transposon system and its mediated gene transfer method - Google Patents

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

A PS transposon system and its mediated gene transfer method

technical field

The invention relates to the establishment of a gene transfer method mediated by a PS transposon system, and also discloses a method for constructing a transgenic donor plasmid and a transposase eukaryotic expression and in vitro transcription auxiliary plasmid involved in the method, and the preparation method thereof. Transgenic animals, studies of gene function and applications in human gene therapy. The invention belongs to the field of animal genetic engineering.

Background technique

Gene transfer is an important biotechnological method at present, which can mediate the stable integration of foreign genes into the host chromosome, so it has important application value in the fields of studying gene function, preparing transgenic organisms and human gene therapy.

Transposon is a DNA sequence that can jump freely on the genome. It was first discovered in the corn chromosome by Mc.Clintock in the 1940s, and later in various organisms such as bacteria, fungi and insects. After the annotation of transposons, it is found that there are a considerable number of transposons in both prokaryotes and eukaryotes. There are great differences in the distribution of transposons among different organisms, especially in higher eukaryotes. For example, transposons are the largest component of the mammalian genome, accounting for almost half of the human genome, and almost half of the human genome. accounted for 80%. The transposition process of DNA transposons follows a "cut-and-paste" mechanism to move across the genome, and thus has been developed as an efficient gene transfer tool. In recent years, transposons have made important progress in human gene therapy, transgenic mice and zebrafish and other model organisms and functional genomics research. In addition, transposons have many advantages as a gene transfer carrier: 1) The structure is simple, and only the TIR sequences on both sides are integrated into the recipient genome together with the exogenous gene, which has little impact on the exogenous gene. The loss of large fragments and chromosomal rearrangement were not found at the site; 2) Compared with viral vectors, DNA transposon vectors have less restriction on inserted fragments; 3) Exogenous genes can be stably integrated into chromosomes, and through It can also be expressed for a long time after germ cell passage; 4) Transposase can catalyze the precise insertion of a single copy of the gene into the hand-held sequence, no longer rely on random integration and the size of the inserted fragment will not change; 5) Transposon system It can be administered in the form of naked DNA, or it can be combined with transposase in the form of DNA and RNA or protein for transposition, so its immunogenicity is low. Among them, the most widely studied DNA transposons, Sleeping Beauty (SB), PiggBac (PB) and Tol2, these three transposons not only have different origins, but also have differences in transposition activity and insertion preference. There are also differences. For example, SB prefers the insertion site to be "TA", but has no preference for genes, so SB transposons are often used in human gene therapy; PB prefers the insertion site to be "TTAA", and prefers to insert into genes, so it is often It is used for functional gene research, gene capture, etc.; Tol2 has no obvious preference for insertion sites, and the insertion positions are mostly upstream regulatory regions of genes, so it is often used to study enhancer capture and so on.

Microinjection has been widely used, and the technology is mature and stable. At the same time, scientists have further improved the method of transposon-mediated cytoplasmic microinjection by using the characteristics of transposon-mediated gene transfer. Since the transposase in the DNA transposition system contains a nuclear localization signal sequence (NLS), it can smoothly mediate the entry of the target gene into the nucleus, and the injection can be carried out directly into the cytoplasm without entering the nucleus. So this technology is receiving attention and has made encouraging progress, which may be a new way to break through the bottleneck of transgenic technology in large mammals.

The use of transposon in human gene therapy mainly includes the stages of target gene cloning, gene transfer, target cell selection and clinical trial observation. The advantages of treatment are that it is efficient and safe. To date, transposons have been called the most commonly used non-viral vectors in gene therapy. In addition, transposons can also generate mutants, carry out gene trapping, application and functional genomics research.

DNA transposon-mediated gene transfer system is a transgenic technology favored by scientists, but DNA transposons with autonomous transposition activity are rare in vertebrates. In 1996, a vertebrate transposon with natural activity, namely Tol2 transposon, was first discovered in Oryzias latipes; in 2008, the second case of Tgf2 transposon with autonomous transposition activity was found in goldfish. , and this transposon is very similar to the Qingqiang Tol2 transposon element. The inventors discovered the passer (PS) family in the research on the Tc1/Mariner transposon superfamily of vertebrates by means of bioinformatics, and the insertion age analysis showed that it may have higher activity. On the basis of phylogenetic comparative study, molecular reconstruction was carried out, and the sequences of TIR key elements and transposase of the two terminal repeats of PS transposon were obtained, and a set of gene transfer vector system was constructed. Fish and others have verified that the vector system can effectively mediate gene transfer, and has great application potential in the preparation of transgenic animals and gene therapy.

SUMMARY OF THE INVENTION

In order to overcome the above-mentioned defects, the present invention provides a PS transposon system and a gene transfer method mediated by the same. By using bioinformatics methods, the PS family in the Tc1/Mariner superfamily is found, and molecular reconstruction is performed to obtain two transposons. A terminal repeat sequence and transposase sequence were constructed, and a PS transposon donor plasmid and an auxiliary plasmid expressing PS transposase were constructed to assemble a set of gene transfer system, named PS transposon system. The purpose of the present invention is to provide a high-efficiency gene transfer method mediated by PS transposition system, improve the transgene preparation efficiency of animals such as mice and zebrafish, and can be effectively applied to cell gene transfection and integration, human gene therapy and gene capture, etc. field of study.

The PS transposon system of the present invention is mainly composed of a PS transposon donor plasmid and a PS transposase helper plasmid.

The transgenic donor plasmid (pLB-PS) of the present invention includes the terminal repeat sequences on both sides of the PS transposon and a polyclonal insertion site, and the polyclonal insertion site (NruI/NotI/EcoRI restriction site) can be inserted into the desired The target gene cassette transferred, the sequence of the donor plasmid (pLB-PS) is shown in SEQ ID No.1. The gene of interest can be inserted into the transgenic donor plasmid through any of the above-mentioned polyclonal insertion sites. Insertion of the target gene into the transgenic donor plasmid means: the target gene is inserted between the PS5'TIR and the PS3'TIR.

The terminal repeat sequences on both sides of the PS transposon of the present invention are derived from the molecular reconstruction of the PS family in the Tc1/Mariner superfamily, and the lengths of the terminal inverted repeats (TIR) on both sides are 28 nucleotide sequences ( Namely PS5'TIR and PS3'TIR), the sequences are shown in SEQ ID No. 2 and SEQ ID No. 3, respectively.

The target gene cassette described in the present invention can be a reporter gene expression cassette, or other exogenous gene expression cassettes, or a gene trapping element.

The PS transposase (i.e. PS CDS, also known as PSase CDS) sequence of the present invention is obtained by using bioinformatics analysis means to carry out molecular reconstruction of the PS transposon family to obtain the transposase sequence, which is obtained by chemical synthesis, As shown in SEQ ID No.5.

The PS transposase helper plasmid pcDNA3.9-PSase of the present invention has two forms, one is a eukaryotic expression plasmid that can be directly expressed in cells and in vivo, and the other is an in vitro transcription helper plasmid that can be transcribed in vitro The 5' capped PS transposase mRNA is formed.

The pcDNA3.9-PSase eukaryotic expression plasmid of the present invention comprises a viral promoter sequence (CMV), a PS transposase cDNA sequence and a bGH polyadenylation (PolyA); the vector framework is derived from the pcDNA3.9 vector, and the The length of the viral promoter sequence (CMV) is 584 nucleotides, which can guide the expression of downstream genes; the length of the PS transposase is 1275 nucleotides; the bGH polyadenylation (PolyA) The length is 225 nucleotides, the vector can express transposase autonomously, and the vector can be used as an in vitro transcription helper plasmid including T7 promoter, PS transposase cDNA sequence and bGHPolyA sequence, wherein the length of the T7 promoter is 19 The nucleotide sequence (bp) is the sequence of the vector, and the bGH PolyA sequence terminates transcription. The vector can use the mMESSAGE Mmachine T7kit (purchased from Invitrogen) to perform in vitro transcription of PS transposase 5'-capped mRNA. The sequence is shown in SEQ ID No.4.

The gene transfer method based on the PS transposon system provided by the present invention can efficiently mediate gene transfer after verification at the cell and embryo levels, and can be applied to a number of biotechnological fields: 1) The method provided in the present invention can effectively Insert the target gene cassette into the host genome to improve the efficiency of gene transfer; 2) Combine with gene capture technology, it can effectively carry out animal gene function research; 3) It can mediate human gene therapy and so on.

Description of drawings

Figure 1: Electrophoresis of pLB-PSase plasmid;

Figure 2: Electrophoresis of pcDNA3.9-PSase plasmid;

Figure 3: pcDNA3.9-PSase plasmid map;

Figure 4: Schematic diagram of pLB-PS plasmid;

Figure 5: Electropherogram of PS-TIR annealing products;

Figure 6: Electropherogram of PS-TIR purified product;

Figure 7: Electrophoresis of pLB-PS plasmid;

Figure 8: Electrophoresis of pLB-PS plasmid digestion products;

Figure 9: electrophoresis of pPS-PGK-NEO plasmid;

Figure 10: Electrophoresis image of pPS-PGK-NEO plasmid digestion products;

Figure 11: pPS-PGK-NEO plasmid map;

Figure 12: PS-FAG-GFP PCR cut back product;

Figure 13: pPS-FAG-GFP plasmid electrophoresis;

Figure 14: electrophoresis of pPS-FAG-GFP digestion, 1: pPS-FAG-GFP digestion; 2: pPS-FAG-GFP plasmid;

Figure 15: pPS-FAG-GFP plasmid map;

Figure 16: pZB-RT-bGH digestion electropherogram;

Figure 17: pPS-PGK-NEO enzyme digestion electropherogram;

Figure 18: Electropherogram of PS frame and Tyr-TYR-bGHpA fragment cut back, 1: Electropherogram of PS frame cut back; 2: Electropherogram of Tyr-TYR-bGHpA fragment cut back;

Figure 19: pPS-Tyr plasmid electropherogram;

Figure 20: The electrophoresis of pPS-Tyr digestion, 1-6: pPS-Tyr digestion; 7: pPS-Tyr plasmid;

Figure 21: pPS-Tyr plasmid map;

Figure 22: Hela cell G418 resistance screening positive clone map;

Figure 23: PS and SB transposition activity comparison graph;

Figure 24: PS transposition system to prepare transgenic mice;

Figure 25: Fluorescence images of microinjected zebrafish embryos at different stages;

Figure 26: Comparison of positive rates of PS transposon microinjection in zebrafish.

Detailed ways

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

The experimental methods mentioned in the following examples are conventional methods unless otherwise specified.

Embodiment 1, the construction of transposase expression vector pcDNA3.9-PSase

1. Synthesis of transposase PSase vector.

After the PSase transposase sequence was chemically synthesized, it was cloned into the LB vector to construct the pLB-PSase vector, and the plasmid DNA was detected by 1% agarose gel electrophoresis (as shown in Figure 1).

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

The transposase CDS was excised from the pLB-PSase vector with the restriction enzymes BamHI and XhoI, and the 1296bp CDS sequence was recovered from agarose gel. At the same time, the pcDNA3.9 vector was digested with BamHI and XhoI, and the 3.9kb fragment was recovered by cutting the gel as the vector frame. After the transposase CDS and the pcDNA3.9 vector frame were connected, the competent cells Top10 were transformed, and the colonies were picked and cultured in liquid medium LA, and the plasmid was extracted for electrophoresis identification (as shown in Figure 2). The positive clones were named pcDNA3.9- PSase, the plasmid map is shown in Figure 3.

The sequence of pcDNA3.9-PSase vector is 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, SV40 poly(A) signal 2897-2998, Ampicillin Resistance gene 4208-5068.

Embodiment II, the construction of transposon expression vector

1. Construction of transgenic donor plasmid pLB-PS vector

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

The insertion age of PS transposons in the Tc1/Marinier family was analyzed for multiple species, and molecular reconstruction was carried out on the basis of phylogenetic comparative studies, and highly conserved 5' and 3' TIRs were identified, which were 28 bp transposons, respectively. to the complementary sequence. Add the restriction endonuclease site to this sequence and send it to Huada Gene Company for synthesis. The sequence is shown in Table 1.

1.2 Polymerase Amplification of PS Transposable Elements

The above chemically synthesized single-stranded nucleotide chain PS5't (including the inverted repeat element at the end of PS5' and an enzyme cleavage site, the enzyme cleavage site is used to insert the target gene) and PS3't (including the end of the PS3' end. Inverted repeat elements and restriction sites, which are used to insert the target gene), are first annealed and then extended with Klenow polymerase to form double strands. The reaction system was 50 μL, including 1ul PS5’t (100uM), 1ul PS3’t (100uM), 23ul ultrapure water, denatured at 95°C for 5min, then lowered to 25°C at a temperature of 0.1°C per second, and maintained for 5min. Then add 5 μL Klenow Buffer, 2 μL Klenow polymerase, 5 μL 4dNTP, 13 μL ultrapure water, 37°C for 1.5h, 80°C for 20min, then drop to room temperature to obtain the target sequence (PS TIR). Subsequently, it was purified by Qiagen PCR purification kit (as shown in Figures 5 and 6), and the concentration was determined by a nucleic acid concentration analyzer, and then stored at -20°C for later use. The schematic diagram of the plasmid is shown in Figure 4, and the gene insertion site in Figure 4 is the multiple cloning site (enzyme cleavage site).

1.3 p LB-PS vector construction

The schematic diagram of the structure of the pLB-PS vector is shown in Figure 4. The purified PS transposon fragment (that is, the target sequence PS TIR obtained in 1.2 above) was ligated with the Tiangen zero background vector pLB with T4 ligase, and transformed into competent cells Top10 , pick a single colony to the Amp-containing liquid medium LB culture, extract the plasmid for electrophoresis and digestion identification (as shown in Figures 7, 8), and the positive clone is named pLB-PS. The pLB-PS vector sequence is 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, bases 408-427 are multiple cloning sites (enzyme cleavage sites) for inserting the target gene.

2. Construction of the transposon vector pPS-PGK-NEO

The PGK-NEO-bGHpA-TA cloning vector (the PGK-NEO-bGHpA-TA cloning vector is the laboratory preservation vector) was cut with restriction endonuclease Nru1, and the 1659bp PGK-NEO-bGHpA expression cassette was recovered by cutting the gel. as the target gene cassette). The LB-PS vector was digested with Nru 1, the 3066bp vector frame was recovered, the expression frame was connected to the vector, and the competent cells were transformed into Top10 cells. The colonies were picked and cultured in LB containing Amp liquid medium, and the plasmid was extracted for electrophoresis and digestion identification. Those with correct size and insertion direction (as shown in Figures 9 and 10) were sent to BGI for sequencing. The plasmid map is shown in Figure 11.

3. Construction of the transposon vector pPS-FAG-GFP

Using the pZB-FAG-GFP plasmid DNA as a template, the FAG-GFP gene fragment was amplified according to the primers listed in Table 2 for amplifying PS-FAG-GFP (the amplified FAG-GFP gene fragment was used as the target gene cassette). The reaction system was 50 μl, 10 μL 5×SFBuffer, 1 μl dNTP, 2 μL 10 μM primer PS-FAG-GFP-F, 2 μl 10 μM primer PS-FAG-GFP-R (see primer sequence table 2), 1 μl Phanta ^TM Super-Fidelity, 1 μl The pZB-FAG-GFP plasmid DNA template was added to 50 μl of ultrapure water (the high-fidelity enzyme was purchased from Novozymes). PCR amplification program: 94°C for 40s, 55°C for 40s, 72°C for 1m30s, cycle 30 times. PCR amplification products were detected by 1% agarose gel electrophoresis. The PCR product was recovered from agarose gel (as shown in Figure 12), connected to the pLB vector, cloned by TA, screened for positive clones, plasmid extraction and identification by restriction enzyme digestion (as shown in Figure 13, 14), and stored for future use after the sequencing and comparison were correct. In the next experiment, it was named pPS-FAG-GFP, and the plasmid map was shown in Figure 15.

4. Construction of the transposon vector pPS-Tyr

The vector pZB-RT-bGH was digested with restriction endonuclease NheI (as shown in Figure 16), and the Tyr-TYR-bGHpA fragment (2555bp) was recovered (as shown in Figure 18), which was filled and purified and stored for later use; the vector was digested with NruI pPS-PGK-NEO (as shown in Figure 17), recovered a 3070bp PS frame (as shown in Figure 18), connected with the above-mentioned fill-in fragment Tyr-TYR-bGHpA (which is used as a target gene cassette), screened out positive clones, extracted plasmids and digested with enzymes Identification (as shown in Figures 19 and 20), sequenced and aligned correctly, and stored for later use, named pPS-Tyr, and the plasmid map is shown in Figure 21.

Table 1 PS-TIR sequence

The sequences in Table 1 are SEQ ID No.6 and SEQ ID No.7, respectively. The underline in Table 1 is the restriction site.

Table 2 PS-FAG-GFP primer sequences

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

Embodiment III, PS transposon mediates target gene expression test at mouse cell level

1. Recovery and culture of cryopreserved cells

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

Take out the cryopreservation tube containing human cervical cancer cells (Hela) from liquid nitrogen (cells stored in our laboratory), and immediately put it into warm water at 37-40°C and shake quickly until the cryopreservation solution is completely thawed; after 1-2min Complete rewarming within 10 minutes; transfer the cell suspension to a sterile centrifuge tube, add 5 mL of culture medium, and blow gently; centrifuge the cell suspension at 800-1000 rpm for 5 min, discard the supernatant; add 1 mL of complete cell pellet to the centrifuge tube containing the cell pellet The medium, gently blow evenly, transfer the cell suspension into a cell culture flask, and add an appropriate amount of complete medium for cultivation.

2. Cell transfection and screening

Human cervical cancer cells Hela were divided into 4 groups, each group was transfected with pPS-PGK-NEO:pcDNA3.9-PSase and pPS-PGK-NEO:pcDNA3.9, pSB-PGK-NEO:pT2-CMV-SB100X and pSB respectively -PGK-NEO:pT2-CMV with 3 replicates per group.

24h before transfection, HeLa cells were seeded at 5×10 ⁵ cells/well in 2000 μL DMEM high-glucose medium (purchased from GIBCO company) containing 10% fetal bovine serum (purchased from GIBCO company) in each well of a six-well plate, Make the cells about 70-80% confluent before transfection; mix and dilute the two plasmids in a 1:1 mass ratio in 100 μL of Opti-MEM (purchased from GIBCO) medium, and mix gently; Add 3 μL of FUGENE transfection reagent (purchased from Promega) to 100 μL of serum-free, antibiotic-free Opti-MEM medium, mix gently, and incubate at room temperature for 5 min; after 5 min, add 100 μL of transfection reagent dilution solution respectively 100 μL of each group of DNA dilutions, mix gently and leave at room temperature for 20 min; add 200 μL of the mixture to the prepared wells, gently shake the culture plate back and forth, and place it at 37 °C, saturated humidity, 5% CO ₂ in an incubator, replace the transfection medium with complete medium after 4 hours, and inoculate 1% of the cells in a 6-well plate after 24-48 hours of complete culture. When the cells reach 10-20% confluence, Add G418 screening medium at a concentration of 600 μg/mL, and screen for 10-12 days. The cells were stained with Giemsa staining solution (purchased from GIBCO), and the number of positive clones was counted.

3. Identification of positive clones of transfected cells

The pPS-PGK-NEO:pcDNA3.9-PSase(PS+/PSase+) and pSB-PGK-NEO:pT2-CMV-SB100X(SB+/SBase+) groups were used as the experimental groups, and the pPS-PGK-NEO:pcDNA3.9( PS+/PSase-) and pSB-PGK-NEO:pT2-CMV (SB+/SBase-) groups were used as control groups. After 10-12 days of selection in G418-resistant culture medium, Gimsa (Giemsa) staining was counted.

The results showed that the number of positive cell clones in the PS experimental group was 560, the number of positive cell clones in the PS control group was 18, the number of positive cell clones in the SB experimental group was 427, and the number of positive cell clones in the SB control group was 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 (as shown in Figures 22 and 23). The results show that the PS transposon system can efficiently mediate the transfection and integration of the neomycin resistance gene in Hela cells, and the activity is higher than that of the SB transposon. Induce foreign gene transfer.

Embodiment IV, PS transposon mediates the gene transfer of target gene in mouse embryo

1. Preparation of mouse fertilized eggs

Mature female mice were intraperitoneally injected with 5 International Units (IU) of pregnant horse serum gonadotropin (PMSG), and then injected with 2.5-5.0 IU of human chorionic gonadotropin (hCG) about 48-54 hours later for about 12 hours Ovulation can then be induced. Female mice were caged with male mice immediately after administration of hCG. Vaginal plugs of female mice after mating are easy to see and can be indicated by mating indicators. The zygote collection period was the next morning after co-cage, a few hours before microinjection. The fallopian tubes are carefully incised, and fertilized eggs are collected by tubal irrigation or salpingoampulotomy.

2. Cytoplasmic microinjection of mouse fertilized eggs

The pPS-Tyr and pcDNA3.9-PSase plasmids were extracted with the OMEGA endotoxin-free plasmid extraction kit, and the linear pcDNA3.9-PSase was used as the template to prepare PSase-mRNA with the mMessagemMachine kit. The final concentration of the product was adjusted to 20ng/μL, pPS-CAG-GFP and PS-mRNA were mixed in a 1:1 molar ratio for use.

Mixed injection of pPS-Tyr plasmid and PSase mRNA was used as the experimental group. FVB mice were used as model organisms. The fertilized eggs of mice were injected into the cytoplasm, and the changes in the coat color of the mice were observed in the later period, as shown in Figure 24. The changes in the coat color were clearly observed.

Embodiment V, PS transposon mediates the gene transfer of target gene in zebrafish embryo

1. Preparation of zebrafish zygotes

A number of male and female fish were raised each. One night before the injection, one female fish was removed, and one male fish was placed in a breeding box. The partitions were separated and cultured. After 1 h of light stimulation, they were cultured overnight in the dark. Removed, the male and female fish started to lay eggs after chasing, and the time from the start of spawning, about 10 minutes later, the eggs were collected, and the collected fertilized eggs were placed under a stereo microscope for zebrafish embryo injection experiments.

2. Microinjection of Zebrafish Embryos

The pPS-FAG-GFP and pcDNA3.9-PSase plasmids were extracted with an endotoxin-free plasmid extraction kit, and the concentrations were measured with a NanoPhotometer nucleic acid concentration analyzer and stored for later use. The pcDNA3.9-PSase plasmid was as described above, and PS transposase mRNA was prepared, and the concentration was determined and stored for later use.

The pPS-FAG-GFP plasmid and PS transposase mRNA were mixed and injected into zebrafish one-cell stage embryos at different concentrations. The final concentration of the immobilized transposon plasmid was 25ng/μL, and the final concentration of PS-mRNA (obtained by in vitro transcription of the pcDNA3.9-PSase vector) was set to 2 gradients: 5ng/μL and 25ng/μL. The daughter plasmid group was the control, and the untreated group (ie, the uninjected group) was the blank control group. There were 3 replicates in each group, and the number of embryos injected in each replicate was more than 100. The injected embryos were cultured in 1×E3 medium, and the medium was changed every 12-24 h, and the dead embryos were removed while changing the medium. The control group was cultured in the same way. After culturing for 24h, 48h and 5d, the expression of green fluorescence was observed under a fluorescence microscope, and the number of viable embryos and positive embryos were counted to calculate the positive rate.

3. Detection of target gene expression mediated by different concentrations of transposase

Fluorescence expression was detected under a fluorescence microscope. The results showed that the embryos of each stage had fluorescence expression at 24h, 48h and 5d after microinjection, and the single-injection transposon plasmid group also had fluorescence expression, while the blank control group had fluorescence expression. There was no fluorescent expression (Figure 25). The positive rate of zebrafish expressing green fluorescent protein was different whether PS transposase was injected or not; at 24h, 48h and 5d, the positive rate of transposase containing group (PS+ group) was significantly higher (87.14%, 92.77%, 92.77%) In the non-injected transposase group (PS-group), the positive rate (48.73%, 61.51%, 61.51%) (Fig. 26). The results show that the PS transposon system can also efficiently mediate GFP gene transfer in zebrafish embryos, that is, it is confirmed that the PS transposon system has the function of mediating gene transfer in zebrafish embryos.

The foregoing has shown and described the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above-mentioned embodiments, and the descriptions in the above-mentioned embodiments and the description are only to illustrate the principle of the present invention, and the present invention will also have Various changes and modifications fall within the scope of the claimed invention. The claimed scope of the present invention is defined by the appended claims and their equivalents.

sequence listing

<110> Yangzhou University

<120> A PS transposon system and its mediated gene transfer method

<130> xhx2019050501

<141> 2019-05-05

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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

<400> 2

ccgtattttc cgcactataa ggcgcacc 28

<210> 3

<211> 28

<212> DNA

<213> Artificial Sequence

<400> 3

ggtgcgcctt atagtgcgga aaatacgg 28

<210> 4

<211> 5206

<212> DNA

<213> 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 ggtggtttttt 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 gacggtttttt 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

<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

<400> 6

ggatcctacc gtattttccg cactataagg cgcacctcgc gagcggccgc gaattc 56

<210> 7

<211> 56

<212> DNA

<213> Artificial Sequence

<400> 7

ggatcctacc gtattttccg cactataagg cgcaccgaat tcgcggccgc tcgcga 56

<210> 8

<211> 58

<212> DNA

<213> Artificial Sequence

<400> 8

ccgtattttc cgcactataa ggcgcaccac tagtgctttt agaccttctt acttttgg 58

<210> 9

<211> 56

<212> DNA

<213> Artificial Sequence

<400> 9

ccgtattttc cgcactataa ggcgcacctt aattaaagct tgggctgcag gtcgag 56

Claims (7)

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.

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