WO2022019400A1 - Improved sleeping beauty transposon system and gene transposition method using same - Google Patents

Improved sleeping beauty transposon system and gene transposition method using same Download PDF

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WO2022019400A1
WO2022019400A1 PCT/KR2020/017709 KR2020017709W WO2022019400A1 WO 2022019400 A1 WO2022019400 A1 WO 2022019400A1 KR 2020017709 W KR2020017709 W KR 2020017709W WO 2022019400 A1 WO2022019400 A1 WO 2022019400A1
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cells
vector
transposon
cell
sleeping beauty
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박준태
이윤행
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인천대학교 산학협력단
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2800/00—Nucleic acids vectors
    • C12N2800/90—Vectors containing a transposable element

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  • the conventional SB transposon system was applied to CHO cells, and it was confirmed that the protein expression level was higher than that of a general vector.
  • the existing SB transposon vector ITRs it was modified by adding an additional pair of ITRs, and a new vector was created.
  • the new vector showed better protein expression than the existing SB transposon, and showed better stability.
  • an increase in protein expression was confirmed when DNA methylation was inhibited in CHO cells to which the SB transposon system was applied.
  • transposon mediator systems include PB (Piggy Bac), Tol2 and SB (Sleeping Beauty) transposons. These transposon systems use a cut and paste method to bind a transposase protein to a region of an inverted terminal repeat sequence (ITR), cut the gene of interest and integrate it into genomic DNA.
  • ITR inverted terminal repeat sequence
  • the SB transposon system has been used for many experiments and fabrications, but the focus has been on reducing the vector size and developing the transposase of the SB transposon system. Due to the low stability of the conventional SB transposon system, protein expression decreases over time.
  • the SB transposon system is mediated by a cut-and-paste mechanism, which does not induce rearrangement or modification of the target site and shows no preference with respect to the target site. Therefore, the vector introducing the SB transposon system has a higher protein expression level than the conventional expression vector. In order to double the length of the ITRs portion of the vector of the SB transposon system, a pair of ITRs was additionally added.
  • transcriptional regulation is related to DNA condensation, which is regulated by DNA methylation.
  • a DNA methylation inhibitor was used to release the condensation of DNA and increase transcription.
  • the present invention provides an inverse repeat/direct repeat (IR/DR) left arm that is repeated twice and an inverse repeat/direct repeat (IR/DR) right arm that is repeated twice It provides a sleeping beauty transposon system that includes
  • the IR/DR left arm and right arm consist of nucleotide sequences of SEQ ID NOs: 1 and 2, respectively;
  • the IR/DR repeated twice is preferably repeated with 100 to 300 bp nucleotides in between. If the interval between IR/DR repeated twice is out of the corresponding range, the efficiency of the transposon may decrease.
  • the two-repeated IR/DR left arm and right arm consist of SEQ ID NOs: 3 and 4 nucleotide sequences.
  • a transgene encoding a target protein may be included between the left arm and the right arm, and the transgene may further include a promoter.
  • transgene does not contain untranscribed flanking regions such as RNA transcription initiation signals, promoters or enhancers with respect to the DNA encoding the protein.
  • Another preferred transgene comprises a DNA sequence encoding a functional RNA.
  • transgene refers to a DNA that is introduced into a cell such as a mammalian host cell via transfection (which is also transduction in the context of the present invention) and which encodes a product of interest (a transgene expression product, eg a heterologous protein). Used herein when referring to a sequence.
  • the transgene may be functionally attached to a signal peptide coding sequence that encodes a signal peptide that mediates and/or facilitates secretion and/or translocation across the endoplasmic reticulum and/or cytoplasmic membrane and is removed prior to or during secretion.
  • a promoter is a nucleic acid sequence recognized by a host cell for expression of one or more nucleic acid sequences. Promoter sequences contain transcriptional control sequences that regulate expression of polynucleotides.
  • a promoter may be any nucleic acid sequence that exhibits transcriptional activity in a selected host cell comprising a mutant, truncated, hybrid promoter and may be obtained from a gene encoding an extracellular or intracellular polypeptide homologous or heterologous to the host cell. have.
  • Promoters according to the present invention include inducible and non-inducible promoters.
  • a nucleic acid sequence is under the control of a promoter and the promoter exerts its function on the nucleic acid. Cells/vectors of the invention often contain such promoters.
  • the promoter is a promoter derived from the genome of a mammalian cell or a promoter derived from a mammalian virus, and more preferably, the second promoter is a U6 promoter, an H1 promoter, a cytomegalo virus (CMV) promoter, an adenovirus late promoter, or vexinia.
  • CMV cytomegalo virus
  • viral 7.5K promoter SV40 promoter, HSV tk promoter, RSV promoter, human elongation factor 1 ⁇ (hEF1 ⁇ ) promoter, metallotionine promoter, beta-actin promoter, human IL-2 gene promoter, human IFN gene promoter, Human IL-4 gene promoter, human lymphotoxin gene promoter, human GM-CSF gene promoter, TERT promoter, PSA promoter, PSMA promoter, CEA promoter, E2F promoter AFP promoter or albumin promoter, most preferably CMV ( cytomegalo virus) promoter.
  • CMV cytomegalo virus
  • the transposon system may further include an antibiotic resistance gene.
  • Antibiotic resistance genes are used as marker genes and include antibiotic resistance genes commonly used in the art, for example, ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin and tetra There is a resistance gene to cyclin, but is not limited thereto.
  • a vector comprising the sleeping beauty transposon system.
  • the vector comprises an IR/DR left arm that is repeated twice; CMV promoter; transgene; SV40 promoter; antibiotic resistance gene; Two repeats of IR/DR right arm may be included in turn.
  • a vector is a means for efficiently suppressing the expression of a target gene by introducing DNA into a host cell, specifically, a gene construct comprising essential regulatory elements operably linked so that the double-stranded RNA of the target gene is expressed. have.
  • a cell transfected with a vector comprising the Sleeping Beauty transposon system.
  • Cells transfected with the vector will contain the vector.
  • transfection can be performed using various methods for injecting a vector into a cell.
  • the cell may be a cell co-transfected with a nucleic acid encoding the Sleeping Beauty transposase protein.
  • a method for producing a target protein comprising culturing cells transfected with the vector containing the Sleeping Beauty transposon system, that is, cells containing the vector containing the Sleeping Beauty transposon system. do.
  • the cell may be a cell co-transfected with a nucleic acid encoding the Sleeping Beauty transposase protein.
  • a target protein is encoded by the transgene, and the transgene can produce the target protein through transcription and translation in a cell.
  • a gene translocation method comprising the step of co-transfecting a cell with a vector comprising the Sleeping Beauty transposon system and a vector comprising a nucleic acid encoding the Sleeping Beauty transposase protein.
  • Said transposase is encoded by DNA, for example in a plasmid (vector), or, on the other hand, the polynucleotide encoding said transposase is RNA.
  • the transposon is recruited through a "cut and paste” mechanism whereby the transposase enzyme encoded by the transposon itself excises the transposon and re-integrates it at another site in the genome.
  • the transposase specifically recognizes the PB inverted terminal repeat (ITR) flanking the transposon; It binds to these sequences and facilitates excision of the transposon.
  • ITR inverted terminal repeat
  • the transposase then integrates the cleaved DNA sequence into the genome of the host cell in a relatively random manner.
  • the gene translocation method may further include treating the transfected cells with a methylation inhibitor or a histone deacetylase inhibitor.
  • Treatment with the methylation inhibitor inhibited DNA condensation when unwinding was maintained, thus increasing the expression level of the co-transfected transposase vector, and more stably integrating the transgene due to cut and paste inside the host genome and can be expressed.
  • the methylation inhibitor is any one selected from the group consisting of 5-azacytidine, 5-aza-2'-deoxycytidine, zebularine, L-methionine, apicidine, hydralazine and procainamide. It is preferably more than, but not limited thereto.
  • the histone deacetylase inhibitor is preferably at least one selected from the group consisting of trichostatin A (TSA), sodium butyrate (NaBu), valproic acid (VAP) and SAHA, but is not limited thereto.
  • said cell is a mammalian cell, said mammalian cell being a COS cell, a CHO cell, a VERO cell, an MDCK cell, a WI38 cell, a V79 cell, a B14AF28-G3 cell, a BHK cell, a HaK cell, a NS0 cell, SP2/0-Ag14 It may be one selected from the group consisting of cells, HeLa cells, HEK293 cells, and PER.C6 cells, but is not limited thereto.
  • the transposon system developed by the present inventors was found to have a higher protein expression level than the existing system. The same results were confirmed in a single stable cell line. Relative gene copy numbers and relative mRNA expression levels were investigated to confirm that the system was stably expressed with higher copy numbers. In addition, stability check was performed for one month in batch culture, and the conventional SB transposon vector showed an expression level similar to that of the control group at the end of the experiment, but the vector improved by the present inventors did not significantly decrease. As a result, the vector system improved by the present inventors showed the highest expression level.
  • Control vector luciferase expression was driven by the CMV promoter, and cells transfected with this vector carry the neomycin (Neo) gene.
  • SB transposon vector The expression vector of luciferase, named control vector, was bounded by a pair of ITRs, IR/DR left arm and right arm.
  • C The SB transposon vector was co-transfected with the SB100X transposase vector.
  • FIG. 2 is a diagram showing the effect of the SB transposon system for protein production.
  • B Analysis of luciferase control vector, SB vector and clonal cell lines generated with SB vector and SB100X transposase vector. Fifteen single cell lines were generated from each cell pool by limiting dilution. Luciferase productivity of each single cell was measured. Each dot on the diagram represents the luciferase productivity of one cell line. Horizontal bars represent the mean of luciferase production for each condition.
  • C Luciferase expression was confirmed by Western blot, and ⁇ -actin was used as a control.
  • the improved SB transposon vector construct of the present invention includes all conventional SB transposon vectors from left and right sequences. In addition, IR/DR sequences for the left and right arms are added to this.
  • E Volumetric protein production analysis of pools of recombinant cells over time. As shown in Figure 3C, cell lines were generated with a control vector (Ctrl), an SB transposon containing the SB100X transposase vector and an improved SB transposon containing the SB100X transposase vector (VdTp). The highest cell line from each group was selected. Cell lines were passaged every 4 days and grown without the use of antibiotic-resistant medium for 1 month.
  • FIG. 4 is a diagram showing changes in gene copy number and luciferase productivity after treatment with a methylation inhibitor.
  • CHO-DG44 cells (A110001; Thermo Fisher Scientific, MA, Waltham, USA) were used. Cells were cultured in Dulbecco's modified Eagle's medium containing 25 mM glucose supplemented with 10% fetal bovine serum with 100 U/ml penicillin and 100 ⁇ g/ml streptomycin and 10 mM sodium hypoxanthine and 1.6 mM thymidine. Cells were maintained in 10 ml medium in T75 cell culture flasks stirred by orbit by orbit in a 37° C. incubator with 5% CO 2 . Cells were passaged every 2 days. Cell density and viability were assessed by Cedex HiRes Analyzer.
  • Donor plasmids for the SB transposon system were constructed with inverted terminal repeat sequences (ITRs). ITRs for the SB transposon system were amplified from T2U-CAF using specific oligonucleotide primers shown in Table 1. In the DNA template, the inverted repeat/direct repeat (IR/DR) right arm and left arm are adjacent and can be amplified at once. The PCR product was then subcloned into a luciferase control vector, a modified version of pcDNA3_SP_Luciferase, to generate an IR/DR-luciferase vector. Similarly, ITRs for the improved SB transposon system were amplified from T2U-CAF using specific oligonucleotide primers shown in Table 1.
  • the improved SB transposon right and left arms were not contiguous, so a cloning step was added.
  • the amplified improved SB left arm and right arm were treated with T-Blunt? Side by side clones in the T-vector in the right direction by PCR Cloning kit.
  • the improved SB left arm and right arm adjacent to each other were amplified by PCR using specific oligonucleotide primers shown in [Table 1].
  • the PCR product was inserted into the NruI restriction site of the luciferase control vector and cloning was performed with the EZ-Fusion TM HT cloning kit.
  • SB100X transposase vector was co-transfected with SB or improved SB vector.
  • CHO-DG44 cells were counted with Cedex HiRes Analyzer and 3 ⁇ 10 5 cells were seeded in 6-well plates overnight. The next day, the plasmid was added prior to transfection and transfected with Lipofector-EZ reagent.
  • Cells were co-transfected as described above using 2 ⁇ g of donor plasmid and 0.66 ⁇ g of transposase vector. Control transfections were performed with a luciferase control vector without ITR. On day 1 post transfection, the medium was changed. When the cells were confluent in the 6-well, they were transferred to a T25 cell culture flask or a T75 cell culture flask. The medium was changed every 2 days for 14 days in the medium with 500 ⁇ g/mL G418, a selective antibiotic in neomycin. After not removing the selective pressure, the resulting cell pool was incubated as a clonal cell line by limiting dilution.
  • the cells were resuspended at a density of 0.8 cells per 150 ⁇ l medium. These diluted cells were transferred to each well of a 96-well plate to give an average of 0.8 cells per well with 500 ⁇ g/mL G418. After 15-20 days of culture, the cells were observed under a fluorescence microscope and selected to have only single colonies in one well. Individual colonies of cells were transferred to 6-well plates. After passage of one cell in 6-well plates, cells were seeded into T25 or T75 flasks. The luciferase levels of these clonal cell lines were then measured with a luciferase assay system.
  • VICTOR Multilabel Plate Reader with luminescence.
  • Genomic DNA was prepared from a stable recombinant CHO cell line; 2 ⁇ 10 6 cells were harvested and washed with PBS. The gDNA was isolated and purified using Solg TM Genomic DNA Prep Kit. Total RNA was isolated from 2 ⁇ 10 6 cells using the RNase Mini Kit. Purified RNA was stored in RNAse-free water at -80°C. Total RNA was reverse transcribed using Phusion RT-PCR kit. Purity and concentration of gDNA and cDNA were measured with a DS-11 spectrophotometer.
  • Real-time PCR (or qRT-PCR) was performed with CFX Connect TM Real-Time PCR Detection System (Bio-Rad) using the SYBR method. Primers were designed to amplify a 200 bp fragment in the luciferase region and a 200 bp fragment in the housekeeping gene GAPDH and normalize the data using specific oligonucleotide primers shown in Table 2.
  • Real-time PCR was performed with 20 ng of gDNA, and qRT-PCR was performed using 1 ⁇ l of cDNA containing non-template control and negative control, denatured at 95° C. for 5 minutes, then 94° C. for 30 seconds, 57° C. for 30 seconds. Second, 40 cycles of 10 seconds were performed at 70°C.
  • Transfected CHO cells were treated with 5-aza-2'-deoxycytidine at 5 ⁇ M/ml daily for 4 days, and treated with 20 mM/ml trichostatin A 24 hours before the experiment. After methylation inhibitor treatment, the luciferase protein concentration was analyzed with a luciferase assay system.
  • a 1kb DNA probe containing one ITR on the left side of the existing SB transposon vector was constructed, and a 1kb DNA probe containing two ITRs on the left side of the developed SB transposon vector was constructed.
  • the primers used to make the probes are shown in Table 3.
  • SB100X 6-His-tagged protein was used as the protein interacting with ITR, which was transformed into the CHO DG44 cell line and G418 selection for 2 weeks followed by the CapturemTM His-Tagged Purification Miniprep kit (635710; Takara, CA, USA). ) was obtained with The obtained probe and protein extracts were subjected to in vitro binding reactions by LightShiftTM Chemiluminescent EMSA Kit (20148; Thermo Fisher Scientific).
  • the present inventors designed a transposon vector by reverse repeat/direct repeat (IR/DR) left and right ITR comprising a CMV promoter-driven luciferase gene and an SV40 promoter-driven neomycin resistance gene (Fig. 1B). ).
  • IR/DR reverse repeat/direct repeat
  • a non-transposon vector containing the gastric vector CMV promoter-driven luciferase gene and the SV40 promoter-driven neomycin resistance gene was designed (Fig. 1A).
  • the SB transposon system requires SB100X transposase for insertion into the genome (FIG. 1C).
  • the SB transposase can be co-transfected as a helper plasmid encoding the SB transposase protein.
  • the use of transposon systems in mammalian cells can lead to retransition due to the instability of the transposon vector.
  • the transposon system is a precise process in which a defined DNA segment is moved to another part of the genome to increase the transfer efficiency of a transgene into the host genome. Therefore, to confirm that the SB transposon system promotes transgene integration, luciferase expressed in cells was measured (Fig. 2A).
  • the SB transposons with the SB100X transposase group expressed significantly more luciferase than the control and SB transposons without the SB100X transposase group ( FIG. 2A ).
  • the vector integration ability of the SB transposon system has been confirmed by many previous studies and experiments.
  • the early SB transposase went through several engineering processes with the current SB100X transposase. Our experiments also used the most advanced systems.
  • studies to date are related to the transposase protein of the SB transposon.
  • the potential for modification of the transposon vector was considered higher than that of the transposase. Therefore, a new vector was prepared by modifying the left and right IR/DR regions of the SB transposon.
  • the IR/DR of the conventional SB transposon is about 380 bp in the left and right arms. By adding pairs, IR/DR left and right arms of about 990 bp were obtained. IR/DR dual arms were designed on each side of the control vector to generate a new vector. This was named an improved SB transposon vector (Fig. 3A).
  • DNA methylation results in the formation of heterochromatin and consequently transcriptional repression. It is closely related to transposons because it evolved to protect the eukaryotic genome from transposons.
  • DNA silencing was partially suppressed after gene silencing was observed. Consequently, we investigated the effect of DNA methylation inhibitors on SB transposon-mediated expression.
  • the present inventors used 5-azacytidine to inhibit DNA methylation and treated for 4 days.
  • tricostatin A was treated in the 5-azacytidine treatment group 24 hours before the experiment for further inhibition of methylation. Trichostatin A induced DNA loosening by inhibiting histone deacetylase, which acts on DNA histone H4.
  • the group treated with the methylation inhibitor had higher protein expression than the group treated with DMSO (Fig. 4A).
  • transposase in vector form when transposase in vector form is co-transfected, the transposase protein is continuously expressed because the transposase vector is also integrated into the genome. This may result in cleavage of transposons integrated into the genome over generations, resulting in poor stability.
  • transgene re-transposition in addition to transgene silencing.
  • the gene copy number of the cell line treated with the methylation inhibitor was determined. Quantitative PCR was performed using gDNA. As a result, the copy number was reduced compared to the truncated and DMSO-treated group.
  • the copy number of the SB transposon was reduced by more than 60%, whereas our improved SB transposon was reduced by only 20%. This shows that the improved SB transposon has a relatively high incision rate and integration into the genome (Fig. 4B).
  • the transposon system developed by the present inventors was found to have a higher expression level when comparing the protein expression level compared to the existing system. The same results were confirmed in a single stable cell line. Relative gene copy number and relative mRNA expression level were investigated to confirm that the system was stably expressed with a longer copy number in the host genome. As a result, a relatively large number of copies were displayed. In addition, stability check was performed for one month in batch culture, and the vector system improved by the present inventors showed the highest expression level. The conventional SB transposon vector exhibited an expression level similar to that of the control group at the end of the experiment, but the vector improved by the present inventors did not significantly decrease.

Abstract

In the present invention, an existing SB transposon system was applied to a CHO cell, and higher protein expression than a normal vector was confirmed. A modification was made by further adding a pair of ITRs to existing SB transposon vector ITRs to create a new vector. The new vector showed an improved protein expression profile and resulted in higher stability compared to the existing SB transposon. Also, an increase in the protein expression was confirmed when DNA methylation was inhibited in the CHO cell to which the SB transposon system has been applied.

Description

개선된 슬리핑 뷰티 트랜스포존 시스템 및 이를 이용한 유전자 전위 방법Improved sleeping beauty transposon system and gene translocation method using same
본 발명은 기존의 SB 트랜스포존 시스템(transposon system)을 CHO 세포에 적용하였고, 일반 벡터보다 단백질 발현량이 높은 것을 확인하였다. 기존의 SB 트랜스포존 벡터 ITRs에 추가적으로 ITRs 한 쌍을 더 추가하여 변형을 했고, 새로운 벡터를 만들었다. 새로운 벡터는 기존의 SB 트랜스포존보다 더 나은 단백질 발현 양상을 보여줬고, 안정성도 더 좋은 결과를 보여주었다. 더해서 SB 트랜스포존 시스템을 적용한 CHO cell에서 DNA 메틸화(methylation)를 억제하였을 때의 단백질 발현 증가를 확인하였다.In the present invention, the conventional SB transposon system was applied to CHO cells, and it was confirmed that the protein expression level was higher than that of a general vector. In addition to the existing SB transposon vector ITRs, it was modified by adding an additional pair of ITRs, and a new vector was created. The new vector showed better protein expression than the existing SB transposon, and showed better stability. In addition, an increase in protein expression was confirmed when DNA methylation was inhibited in CHO cells to which the SB transposon system was applied.
진핵 생물 게놈에는 많은 반복 DNA가 포함되어 있으며, 그 중 일부는 이동성을 보여준다. 대표적인 트랜스포존 매개체 시스템은 PB(Piggy Bac), Tol2 및 SB(Sleeping Beauty) 트랜스포존을 포함한다. 이들 트랜스포존 시스템은 컷 및 페이스트 방법을 사용하여 트랜스포사제 단백질을 역 말단 반복 서열 (ITR)의 영역에 결합시키고, 관심있는 유전자를 절단하고 이를 게놈 DNA에 통합시킨다.The eukaryotic genome contains many repeating DNA, some of which show mobility. Representative transposon mediator systems include PB (Piggy Bac), Tol2 and SB (Sleeping Beauty) transposons. These transposon systems use a cut and paste method to bind a transposase protein to a region of an inverted terminal repeat sequence (ITR), cut the gene of interest and integrate it into genomic DNA.
배양된 포유동물 세포는 인간 및 번역 후 변형에 대한 그의 유사한 능력으로 인해 치료용 항체의 생산을 위한 주요 숙주로서 선택되었다. 이러한 이유로 중국 햄스터 난소(CHO) 세포는 일반적으로 재조합 항체를 생산할 때 사용되었다. 최근, 바이오 의약품 산업에서 치료용 항체의 연구 및 사용이 폭발적으로 증가함에 따라, 최근에는 바이오 의약품 시장에서 가장 큰 문제는 단백질 생산의 낮은 생산성이다. 일반적으로, 바이오 의약품 시장에서 사용되는 벡터는 세포 게놈에 간단히 통합될 수 있다는 장점이 있지만, 효율이 훨씬 떨어진다는 단점이 있다. 이러한 결함은 바이오 제약 시장에서 항체 생산의 경쟁력을 떨어뜨릴 수 있다. 항체 생산의 경쟁력을 확보하기 위하여 고효율 발현 벡터를 생성하기 위한 많은 연구가 진행되고 있다. 현재 발현 벡터의 전치 능력의 제한이 있었기 때문에 벡터의 크기의 제한이 있었다. 따라서 지금까지 발현 벡터의 크기를 줄여서 발현되는 유전자의 크기를 늘리기 위한 많은 연구가 진행되고 있다. 이 연구에서는 동일한 크기의 벡터에서 발현되는 유전자 양이 더 증가한다. 또한 DNA 메틸화는 전사를 억제하는 잘 알려진 과정이다. 따라서, DNA 메틸화를 억제하면 단백질의 발현 수준이 높아질 것으로 생각되며, DNA 메틸화를 억제할 수 있는 약물 처리 후에 단백질 발현 패턴을 조사하였다.Cultured mammalian cells have been chosen as primary hosts for the production of therapeutic antibodies due to their similar ability to human and post-translational modifications. For this reason, Chinese Hamster Ovary (CHO) cells have been commonly used to produce recombinant antibodies. Recently, as the research and use of therapeutic antibodies in the biopharmaceutical industry has exploded, the biggest problem in the biopharmaceutical market in recent years is the low productivity of protein production. In general, vectors used in the biopharmaceutical market have the advantage that they can be simply integrated into the cell genome, but have a disadvantage in that their efficiency is much lower. These deficiencies could make antibody production less competitive in the biopharmaceutical market. In order to secure the competitiveness of antibody production, many studies are being conducted to generate highly efficient expression vectors. Currently, there is a limitation in the size of the vector because there is a limitation in the translocation ability of the expression vector. Therefore, many studies have been conducted so far to increase the size of the expressed gene by reducing the size of the expression vector. In this study, the amount of genes expressed in vectors of the same size is further increased. Also, DNA methylation is a well-known process that inhibits transcription. Therefore, inhibition of DNA methylation is thought to increase the protein expression level, and the protein expression pattern was investigated after treatment with a drug capable of inhibiting DNA methylation.
지금까지 SB 트랜스포존 시스템은 많은 실험과 제작에 사용되었지만 벡터 크기를 줄이고 SB 트랜스포존 시스템의 트랜스포사제를 개발하는 데 중점을 두었다. 기존의 SB 트랜스포존 시스템은 안정성이 낮기 때문에 시간이 지남에 따라 단백질 발현이 감소한다. So far, the SB transposon system has been used for many experiments and fabrications, but the focus has been on reducing the vector size and developing the transposase of the SB transposon system. Due to the low stability of the conventional SB transposon system, protein expression decreases over time.
많은 연구로 인해 SB 트랜스포존 시스템(transposon system)에 대한 많은 발전이 있었지만, 다른 트랜스포존 시스템에 비해 상업적으로 쓰일만한 단백질 발현 벡터로써 높은 이점을 가지고 있지 않았고, 안정성 또한 떨어져 있었다.Although there have been many advances in the SB transposon system due to many studies, it did not have a high advantage as a commercially available protein expression vector compared to other transposon systems, and its stability was also poor.
SB 트랜스포존 시스템은 컷 앤드 페이스트(cut and paste) 메커니즘으로 매개되는데 이는 표적부위의 재배열 또는 변형을 유발시키지 않고, 표적부위와 관련하여 선호도를 나타내지 않는다. 그래서 SB 트랜스포존 시스템을 도입한 벡터는 기존의 발현 벡터보다 단백질 발현량이 우세하다. 이러한 SB 트랜스포존 시스템의 벡터의 ITRs 부분의 길이를 2배로 늘려주기 위해 한 쌍의 ITRs를 추가로 넣어주었다.The SB transposon system is mediated by a cut-and-paste mechanism, which does not induce rearrangement or modification of the target site and shows no preference with respect to the target site. Therefore, the vector introducing the SB transposon system has a higher protein expression level than the conventional expression vector. In order to double the length of the ITRs portion of the vector of the SB transposon system, a pair of ITRs was additionally added.
　일반적으로 전사(transcription) 조절은 DNA 응축과 관련이 되어있는 것으로 알려져 있고, 이는 DNA 메틸화(methylation)에 의해 조절된다. DNA 메틸화 억제제(methylation inhibitor)를 이용하여 DNA의 응축을 풀어주어 전사를 높여줬다.In general, it is known that transcriptional regulation is related to DNA condensation, which is regulated by DNA methylation. A DNA methylation inhibitor was used to release the condensation of DNA and increase transcription.
상기 기술적 과제를 해결하기 위하여, 본 발명은 2번 반복되는 역 반복/직접 반복(IR/DR) 좌측 암(arm) 및 2번 반복되는 역 반복/직접 반복(IR/DR) 우측 암(arm)을 포함하는 슬리핑 뷰티 트랜스포존 시스템을 제공한다. In order to solve the above technical problem, the present invention provides an inverse repeat/direct repeat (IR/DR) left arm that is repeated twice and an inverse repeat/direct repeat (IR/DR) right arm that is repeated twice It provides a sleeping beauty transposon system that includes
상기 IR/DR 좌측 암 및 우측 암은 각각 서열번호 1 및 2의 염기서열로 이루어진다;The IR/DR left arm and right arm consist of nucleotide sequences of SEQ ID NOs: 1 and 2, respectively;
[서열번호 1] IR/DR left arm[SEQ ID NO: 1] IR / DR left arm
ACGCGTAAGCTTCCTTGAAATACATCCACAGGTACACCTCCAATTGACTCAAATGATGTCAATTAGTCTATCAGAAGCTTCTAAAGCCATGACATCATTTTCTGGAATTTTCCAAGCTGTTTAAAGGCACAGTCAACTTAGTGTATGTAAACTTCTGACCCACTGGAATTGTGATACAGTGAATTATAAGTGAAATAATCTGTCTGTAAACAATTGTTGGAAAAATGACTTGTGTCATGCACAAAGTAGATGTCCTAACTGACTTGCCAAAACTATTGTTTGTTAACAAGAAATTTGTGGAGTAGTTGAAAAACGAGTTTTAATGACTCCAACTTAAGTGTATGTAAACTTCCGACTTCAACTGTAACGCGTAAGCTTACGCGTAAGCTTCCTTGAAATACATCCACAGGTACACCTCCAATTGACTCAAATGATGTCAATTAGTCTATCAGAAGCTTCTAAAGCCATGACATCATTTTCTGGAATTTTCCAAGCTGTTTAAAGGCACAGTCAACTTAGTGTATGTAAACTTCTGACCCACTGGAATTGTGATACAGTGAATTATAAGTGAAATAATCTGTCTGTAAACAATTGTTGGAAAAATGACTTGTGTCATGCACAAAGTAGATGTCCTAACTGACTTGCCAAAACTATTGTTTGTTAACAAGAAATTTGTGGAGTAGTTGAAAAACGAGTTTTAATGACTCCAACTTAAGTGTATGTAAACTTCCGACTTCAACTGTAACGCGTAAGCTT
[서열번호 2] IR/DR right arm[SEQ ID NO: 2] IR / DR right arm
AAGCTTACGCGTTACAGTTGAAGTCGGAAGTTTACATACACTTAAGTTGGAGTCATTAAAACTCGTTTTTCAACTACTCCACAAATTTCTTGTTAACAAACAATAGTTTTGGCAAGTCAGTTAGGACATCTACTTTGTGCATGACACAAGTCATTTTTCCAACAATTGTTTACAGACAGATTATTTCACTTATAATTCACTGTATCACAATTCCAGTGGGTCAGAAGTTTACATACACTAAGTTGACTGTGCCTTTAAACAGCTTGGAAAATTCCAGAAAATGATGTCATGGCTTTAGAAGCTTCTGATAGACTAATTGACATCATTTGAGTCAATTGGAGGTGTACCTGTGGATGTATTTCAAGGAAGCTTACGCGT.AAGCTTACGCGTTACAGTTGAAGTCGGAAGTTTACATACACTTAAGTTGGAGTCATTAAAACTCGTTTTTCAACTACTCCACAAATTTCTTGTTAACAAACAATAGTTTTGGCAAGTCAGTTAGGACATCTACTTTGTGCATGACACAAGTCATTTTTCCAACAATTGTTTACAGACAGATTATTTCACTTATAATTCACTGTATCACAATTCCAGTGGGTCAGAAGTTTACATACACTAAGTTGACTGTGCCTTTAAACAGCTTGGAAAATTCCAGAAAATGATGTCATGGCTTTAGAAGCTTCTGATAGACTAATTGACATCATTTGAGTCAATTGGAGGTGTACCTGTGGATGTATTTCAAGGAAGCTTACGCGT.
상기 2번 반복되는 IR/DR은 100 ~ 300 bp 뉴클레오티드를 사이에 두고 반복되는 것이 바람직하다. 2번 반복되는 IR/DR 사이가 해당 범위를 벗어나는 경우에는 트랜스포존의 효율이 감소할 수 있다.The IR/DR repeated twice is preferably repeated with 100 to 300 bp nucleotides in between. If the interval between IR/DR repeated twice is out of the corresponding range, the efficiency of the transposon may decrease.
상기 2번 반복되는 IR/DR 좌측 암 및 우측 암은 서열번호 3 및 4 염기서열로 이루어진다. The two-repeated IR/DR left arm and right arm consist of SEQ ID NOs: 3 and 4 nucleotide sequences.
[서열번호 3] IR/DR dual left arm[SEQ ID NO: 3] IR / DR dual left arm
(서열번호 1)- ATCGCTGTCTTGCTGCGAAGCAGCGCAAAACGCCTAACCCTAAGCAGATTCTTCATGCAATTGTCGGTCAAGCCTTGCCTTGTTGTAGCTTAAATTTTGCTCGCGCACTACTCAGCGACCTCCAACACACAAGCAGGGAGCAGATACTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATAGGGGATCGGGAGATC-(서열번호 1).(SEQ ID NO: 1)-ATCGCTGTCTTGCTGCGAAGCAGCGCAAAACGCCTAACCCTAAGCAGATTCTTCATGCAATTGTCGGTCAAGCCTTGCCTTGTTGTAGCTTAAATTTTGCTCGCGCACTACTCAGCGACCTCCAACACACAACAAGCAGGGAGCAGATGTTCGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGA) (SEQ ID NO: 1)
[서열번호 4] IR/DR dual right arm[SEQ ID NO: 4] IR/DR dual right arm
(서열번호2)-GATCTCCCGATCCCCTATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATCTGCTCCCTGCTTGTGTGTTGGAGGTCGCTGAGTAGTGCGCGAGCAAAATTTAAGCTACAACAAGGCAAGGCTTGACCGACAATTGCATGAAGAATCTGCTTAGGGTTAGGCGTTTTGCGCTGCTTCGGCCATGGTGCTGGAATTCGCCCTTACGGATCGGGAGATCT-(서열번호2)(SEQ ID NO:2)-GATCTCCCGATCCCCTATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATCTGCTCCCTGCTTGTGTGTTGGAGGTCGCTGAGTAGTGCGCGAGCAAAATTTAAGCTACAACAAGGCAAGGCTGGCTGGCTGGTGCAATTGCATGAAGAATCTGCTTGGCATGGTTGGCGCG#SEQ ID NO:2
상기 좌측 암 및 우측 암 사이에 목적 단백질을 코딩하는 전이유전자(transgene)를 포함할 수 있으며, 상기 전이유전자는 프로모터를 더 포함할 수 있다. A transgene encoding a target protein may be included between the left arm and the right arm, and the transgene may further include a promoter.
상기 전이유전자는 단백질을 암호화하는 DNA와 관련하여 RNA 전사 개시 신호, 프로모터 또는 인핸서와 같은 전사되지 않는 플랭킹 영역을 포함하지 않는다. 다른 바람직한 전이유전자는 기능성 RNA를 암호화하는 DNA 서열을 포함한다. 따라서, 용어 전이유전자는 형질감염(이는 본 발명과 관련하여 또한 형질도입)을 통한 포유동물 숙주 세포와 같은 세포로 도입되고 목적하는 생성물(전이유전자 발현 생성물, 예를 들어 이종성 단백질)을 암호화하는 DNA 서열을 언급하는 경우 본원에 사용된다. 전이유전자는 소포체 및/또는 세포질 막에 걸친 전좌 및/또는 분비를 매개하고/하거나 촉진시키는 신호 펩타이드를 암호화하고 분비 전 또는 분비 동안에 제거되는 신호 펩타이드 암호화 서열에 기능적으로 부착될 수 있다.The transgene does not contain untranscribed flanking regions such as RNA transcription initiation signals, promoters or enhancers with respect to the DNA encoding the protein. Another preferred transgene comprises a DNA sequence encoding a functional RNA. Thus, the term transgene refers to a DNA that is introduced into a cell such as a mammalian host cell via transfection (which is also transduction in the context of the present invention) and which encodes a product of interest (a transgene expression product, eg a heterologous protein). Used herein when referring to a sequence. The transgene may be functionally attached to a signal peptide coding sequence that encodes a signal peptide that mediates and/or facilitates secretion and/or translocation across the endoplasmic reticulum and/or cytoplasmic membrane and is removed prior to or during secretion.
프로모터는 하나 이상의 핵산 서열의 발현을 위해 숙주 세포에 의해 인지되는 핵산 서열이다. 프로모터 서열은 폴리뉴클레오타이드의 발현을 조절하는 전사 제어 서열을 함유한다. 프로모터는 돌연변이체, 절단되고 하이브리드 프로모터를 포함하는 선택된 숙주 세포에서 전사 활성을 보여주는 임의의 핵산 서열일 수 있고 숙주 세포에 상동성 또는 이종성의 세포외 또는 세포내 폴리펩타이드를 암호화하는 유전자로부터 수득될 수 있다. 본 발명에 따른 프로모터는 유도성 및 비-유도성 프로모터를 포함한다. 핵산 서열은 프로모터의 제어하에 있고 프로모터는 상기 핵산에 대해 이의 기능을 발휘한다. 본 발명의 세포/벡터는 흔히 상기 프로모터를 함유한다.A promoter is a nucleic acid sequence recognized by a host cell for expression of one or more nucleic acid sequences. Promoter sequences contain transcriptional control sequences that regulate expression of polynucleotides. A promoter may be any nucleic acid sequence that exhibits transcriptional activity in a selected host cell comprising a mutant, truncated, hybrid promoter and may be obtained from a gene encoding an extracellular or intracellular polypeptide homologous or heterologous to the host cell. have. Promoters according to the present invention include inducible and non-inducible promoters. A nucleic acid sequence is under the control of a promoter and the promoter exerts its function on the nucleic acid. Cells/vectors of the invention often contain such promoters.
상기 프로모터는 포유동물 세포의 게놈으로부터 유래한 프로모터 또는 포유동물 바이러스로부터 유래한 프로모터이고, 보다 바람직하게는 제2프로모터는 U6 프로모터, H1 프로모터, CMV(cytomegalo virus) 프로모터, 아데노바이러스 후기 프로모터, 벡시니아 바이러스 7.5K 프로모터, SV40 프로모터, HSV의 tk 프로모터, RSV 프로모터, 인간 연장인자 1α(hEF1α) 프로모터, 메탈로티오닌 프로모터, 베타-액틴 프로모터, 인간 IL-2 유전자의 프로모터, 인간 IFN 유전자의 프로모터, 인간 IL-4 유전자의 프로모터, 인간 림포톡신 유전자의 프로모터, 인간 GM-CSF 유전자의 프로모터, TERT 프로모터, PSA 프로모터, PSMA 프로모터, CEA 프로모터, E2F 프로모터 AFP 프로모터 또는 알부민 프로모터이고, 가장 바람직하게는 CMV(cytomegalo virus) 프로모터이다.The promoter is a promoter derived from the genome of a mammalian cell or a promoter derived from a mammalian virus, and more preferably, the second promoter is a U6 promoter, an H1 promoter, a cytomegalo virus (CMV) promoter, an adenovirus late promoter, or vexinia. viral 7.5K promoter, SV40 promoter, HSV tk promoter, RSV promoter, human elongation factor 1α (hEF1α) promoter, metallotionine promoter, beta-actin promoter, human IL-2 gene promoter, human IFN gene promoter, Human IL-4 gene promoter, human lymphotoxin gene promoter, human GM-CSF gene promoter, TERT promoter, PSA promoter, PSMA promoter, CEA promoter, E2F promoter AFP promoter or albumin promoter, most preferably CMV ( cytomegalo virus) promoter.
상기 트랜스포존 시스템은 항생제 내성 유전자를 더 포함할 수 있다. 항생제 내성 유전자는 마커 유전자로 사용되고, 당업계에서 통상적으로 이용되는 항생제 내성 유전자를 포함하며, 예를 들어 암피실린, 겐타마이신, 카베니실린, 클로람페니콜, 스트렙토마이신, 카나마이신, 제네티신, 네오마이신 및 테트라사이클린에 대한 내성 유전자가 있으나, 이에 제한되지 않는다. The transposon system may further include an antibiotic resistance gene. Antibiotic resistance genes are used as marker genes and include antibiotic resistance genes commonly used in the art, for example, ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin and tetra There is a resistance gene to cyclin, but is not limited thereto.
본 발명의 또 다른 실시예에 따르면, 상기 슬리핑 뷰티 트랜스포존 시스템을 포함하는 벡터를 제공한다. According to another embodiment of the present invention, there is provided a vector comprising the sleeping beauty transposon system.
구체적인 실시예에서, 상기 벡터는 2번 반복되는 IR/DR 좌측 암; CMV 프로모터; 전이유전자; SV40 프로모터; 항생제 내성 유전자; 2번 반복되는 IR/DR 우측 암을 차례대로 포함할 수 있다. In a specific embodiment, the vector comprises an IR/DR left arm that is repeated twice; CMV promoter; transgene; SV40 promoter; antibiotic resistance gene; Two repeats of IR/DR right arm may be included in turn.
벡터는 숙주 세포에 DNA를 도입하여 목적 유전자의 발현을 효율적으로 억제하기 위한 수단으로서, 구체적으로 목적 유전자의 이중나선 RNA가 발현되도록 작동 가능하게 연결된 필수적인 조절 요소를 포함하는 유전자 작제물을 의미할 수 있다.A vector is a means for efficiently suppressing the expression of a target gene by introducing DNA into a host cell, specifically, a gene construct comprising essential regulatory elements operably linked so that the double-stranded RNA of the target gene is expressed. have.
본 발명의 또 다른 실시예에 따르면, 상기 슬리핑 뷰티 트랜스포존 시스템을 포함하는 벡터로 형질감염된 세포를 제공한다. 상기 벡터에 의하여 형질감염된 세포는 상기 벡터를 포함하게 된다. 형질감염은 일반적으로 세포 내에 벡터를 주입하기 위한 다양한 방법이 사용될 수 있다. 바람직하게 상기 세포는 슬리핑 뷰티 트랜스포사제 단백질을 코딩하는 핵산과 함께 공동-형질감염된 세포일 수 있다. According to another embodiment of the present invention, there is provided a cell transfected with a vector comprising the Sleeping Beauty transposon system. Cells transfected with the vector will contain the vector. In general, transfection can be performed using various methods for injecting a vector into a cell. Preferably, the cell may be a cell co-transfected with a nucleic acid encoding the Sleeping Beauty transposase protein.
본 발명의 또 다른 실시예에 따르면, 상기 슬리핑 뷰티 트랜스포존 시스템을 포함하는 벡터로 형질감염된 세포, 즉 슬리핑 뷰티 트랜스포존 시스템을 포함하는 벡터를 포함하는 세포를 배양하는 단계를 포함하는 목적 단백질 생산 방법을 제공한다. 바람직하게 상기 세포는 슬리핑 뷰티 트랜스포사제 단백질을 코딩하는 핵산과 함께 공동-형질감염된 세포일 수 있다.According to another embodiment of the present invention, there is provided a method for producing a target protein comprising culturing cells transfected with the vector containing the Sleeping Beauty transposon system, that is, cells containing the vector containing the Sleeping Beauty transposon system. do. Preferably, the cell may be a cell co-transfected with a nucleic acid encoding the Sleeping Beauty transposase protein.
목적 단백질은 상기 전이유전자에 의하여 인코딩되고, 상기 전이유전자는 세포 내에서 전사 및 번역 단계를 거쳐 목적 단백질을 생산할 수 있다. A target protein is encoded by the transgene, and the transgene can produce the target protein through transcription and translation in a cell.
본 발명의 또 다른 실시예에 따르면, 상기 슬리핑 뷰티 트랜스포존 시스템을 포함하는 벡터 및 슬리핑 뷰티 트랜스포사제 단백질을 코딩하는 핵산을 포함하는 벡터를 세포에 공동-형질감염시키는 단계를 포함하는 유전자 전위 방법을 제공한다. 상기 트랜스포사제는 예를 들어 플라스미드(벡터)에서 DNA에 의해 암호화되거나, 또는 한편으로 상기 트랜스포사제를 암호화하는 폴리뉴클레오타이드는 RNA이다.According to another embodiment of the present invention, a gene translocation method comprising the step of co-transfecting a cell with a vector comprising the Sleeping Beauty transposon system and a vector comprising a nucleic acid encoding the Sleeping Beauty transposase protein. to provide. Said transposase is encoded by DNA, for example in a plasmid (vector), or, on the other hand, the polynucleotide encoding said transposase is RNA.
상기 트랜스포존은 "컷 앤드 페이스트" 메커니즘을 통해 동원되고, 이에 의해 트랜스포존 자체에 의해 암호화되는 트랜스포사제 효소가 트랜스포존을 절제하여 게놈 내의 다른 부위에 재-통합시킨다. 트랜스포사제는 트랜스포존을 플랭킹하는 PB 역전된 말단 반복단위(ITR: inverted terminal repeat)를 특이적으로 인식하고; 이는 이들 서열에 결합하여 트랜스포존의 절제를 촉진시킨다. 그런 다음 트랜스포사제는 상대적으로 무작위 방식으로 숙주 세포의 게놈에 상기 절단된 DNA 서열을 통합시킨다.The transposon is recruited through a "cut and paste" mechanism whereby the transposase enzyme encoded by the transposon itself excises the transposon and re-integrates it at another site in the genome. The transposase specifically recognizes the PB inverted terminal repeat (ITR) flanking the transposon; It binds to these sequences and facilitates excision of the transposon. The transposase then integrates the cleaved DNA sequence into the genome of the host cell in a relatively random manner.
상기 유전자 전위 방법은 형질감염된 세포에 메틸화 억제제(methylation inhibitor) 또는 히스톤 디아세틸레이즈 억제제(histone deacetylase inhibitor)를 처리하는 단계를 더 포함할 수 있다. The gene translocation method may further include treating the transfected cells with a methylation inhibitor or a histone deacetylase inhibitor.
상기 메틸화 억제제로의 처리는 풀림이 유지될 때 DNA 축합을 억제하고, 따라서 공동-형질감염된 트랜스포사제 벡터의 발현 수준이 증가하였고, 숙주 게놈 내부의 컷 및 페이스트로 인해 전이유전자가 더욱 안정적으로 통합되고 발현될 수 있다. Treatment with the methylation inhibitor inhibited DNA condensation when unwinding was maintained, thus increasing the expression level of the co-transfected transposase vector, and more stably integrating the transgene due to cut and paste inside the host genome and can be expressed.
상기 메틸화 억제제는 5-아자시티딘(azacytidine), 5-아자-2'-데옥시시티딘, 제불라린, L-메티오닌, 아피시딘, 히드랄라진 및 프로카인아미드로 구성된 군으로부터 선택된 어느 하나 이상인 것이 바람직하지만, 이에 제한되지 않는다.The methylation inhibitor is any one selected from the group consisting of 5-azacytidine, 5-aza-2'-deoxycytidine, zebularine, L-methionine, apicidine, hydralazine and procainamide. It is preferably more than, but not limited thereto.
히스톤 디아세틸레이즈 억제제는 TSA(trichostatin A), NaBu(sodium butyrate), VAP(valproic acid) 및 SAHA으로 구성된 군으로부터 선택된 어느 하나 이상인 것이 바람직하지만, 이에 제한되지 않는다. The histone deacetylase inhibitor is preferably at least one selected from the group consisting of trichostatin A (TSA), sodium butyrate (NaBu), valproic acid (VAP) and SAHA, but is not limited thereto.
상기 세포는 포유동물 세포이며, 상기 포유동물 세포는 COS 세포, CHO 세포, VERO 세포, MDCK 세포, WI38 세포, V79 세포, B14AF28-G3 세포, BHK 세포, HaK 세포, NS0 세포, SP2/0-Ag14 세포, HeLa 세포, HEK293 세포 및 PER.C6 세포로 이루어진 군으로부터 선택되는 하나일 수 있으나, 이에 제한되지 않는다.wherein said cell is a mammalian cell, said mammalian cell being a COS cell, a CHO cell, a VERO cell, an MDCK cell, a WI38 cell, a V79 cell, a B14AF28-G3 cell, a BHK cell, a HaK cell, a NS0 cell, SP2/0-Ag14 It may be one selected from the group consisting of cells, HeLa cells, HEK293 cells, and PER.C6 cells, but is not limited thereto.
단백질 발현 수준을 비교할 때 본 발명자가 개발한 트랜스포존 시스템은 기존 시스템에 비해 단백질 발현 수준이 더 높은 것으로 밝혀졌다. 안정적인 단일 세포주에서 동일한 결과가 확인되었다. 상대적인 유전자 카피 수 및 상대적인 mRNA 발현 수준을 조사하여 더 많은 카피 수로 시스템이 안정적으로 발현되는지 확인하였다. 또한, 배치 배양으로 한 달 동안 안정성 점검을 수행하였고, 기존의 SB 트랜스포존 벡터는 실험 종료시 대조군의 발현 수준과 유사한 발현 수준을 나타내었지만, 본 발명자에 의해 개선된 벡터는 크게 감소하지 않았다. 결과적으로 본 발명자에 의해 개선된 벡터 시스템은 가장 높은 발현 수준을 나타냈다.When comparing the protein expression level, the transposon system developed by the present inventors was found to have a higher protein expression level than the existing system. The same results were confirmed in a single stable cell line. Relative gene copy numbers and relative mRNA expression levels were investigated to confirm that the system was stably expressed with higher copy numbers. In addition, stability check was performed for one month in batch culture, and the conventional SB transposon vector showed an expression level similar to that of the control group at the end of the experiment, but the vector improved by the present inventors did not significantly decrease. As a result, the vector system improved by the present inventors showed the highest expression level.
도 1은 본 발명에 사용된 벡터 시스템의 개략도를 나타내는 도면이다. (A) 대조군 벡터: 루시퍼라제 발현은 CMV 프로모터에 의해 구동되었고, 이 벡터로 형질감염된 세포는 네오마이신(Neo) 유전자를 가진다. (B) SB 트랜스포존 벡터: 제어 벡터로 명명된 루시퍼라제의 발현 벡터는 IR/DR 좌측 암 및 우측 암이라는 1쌍의 ITR에 의해 경계가 지어졌다. (C) SB 트랜스포존 벡터를 SB100X 트랜스포사제 벡터로 공동-형질감염시켰다.1 is a diagram showing a schematic diagram of a vector system used in the present invention. (A) Control vector: luciferase expression was driven by the CMV promoter, and cells transfected with this vector carry the neomycin (Neo) gene. (B) SB transposon vector: The expression vector of luciferase, named control vector, was bounded by a pair of ITRs, IR/DR left arm and right arm. (C) The SB transposon vector was co-transfected with the SB100X transposase vector.
도 2는 단백질 생산을 위한 SB 트랜스포존 시스템의 효과를 나타내는 도면이다. (A) SB100X 트랜스포사제 벡터를 SB 트랜스포존 벡터로 형질감염시키고 14일의 G418 선택 후 루시퍼라제 생산성을 측정하였다(***P < 0.001, student's t-test, Means ± S.D., N = 3). (B) 루시퍼라제 제어 벡터, SB 벡터 및 SB 벡터와 SB100X 트랜스포사제 벡터로 생성된 클론 세포주의 분석. 제한 희석(limiting dilution)에 의해 각 세포 풀에서 15개의 단일 세포주가 생성되었다. 각각의 단일 세포의 루시퍼라제 생산성을 측정하였다. 도표의 각 점은 하나의 세포주의 루시퍼라제 생산성을 나타낸다. 가로 막대는 각 조건에 대한 루시퍼라제 생산의 평균을 나타낸다. (C) 웨스턴 블롯으로 루시퍼라제 발현을 확인하고, β-액틴을 대조군으로 사용하였다. 2 is a diagram showing the effect of the SB transposon system for protein production. (A) SB100X transposase vector was transfected with SB transposon vector and luciferase productivity was measured 14 days after G418 selection (***P < 0.001, student's t-test, Means ± S.D., N = 3). (B) Analysis of luciferase control vector, SB vector and clonal cell lines generated with SB vector and SB100X transposase vector. Fifteen single cell lines were generated from each cell pool by limiting dilution. Luciferase productivity of each single cell was measured. Each dot on the diagram represents the luciferase productivity of one cell line. Horizontal bars represent the mean of luciferase production for each condition. (C) Luciferase expression was confirmed by Western blot, and β-actin was used as a control.
도 3은 기존의 SB 벡터와 본 발명의 SB 벡터 시스템 간의 단백질 발현 효율을 비교한 도면이다. (A) 본 발명의 개선된 SB 트랜스포존 벡터 구조물은 좌측 및 우측 서열로부터의 모든 통상적인 SB 트랜스포존 벡터를 포함한다. 또한 여기에는 좌측과 우측 암이 각각 동일한 IR/DR 서열이 추가된다. (B) 개선된 SB 트랜스포존 시스템 및 SB 트랜스포존 시스템의 루시퍼라제 발현 수준을 비교하였다. 세포 풀 공동-형질감염된 0.66 μg의 SB100X 트랜스포사제 벡터를 대조군으로 선택하였다(***P < 0.001, **P < 0.01, student's t-test, Means ± S.D., N = 3). (C) 루시퍼라제 대조군 벡터, SB100X 트랜스포사제 벡터를 포함하는 SB 트랜스포존 벡터 및 SB100X 트랜스포사제 벡터를 포함하는 개선된 SB 트랜스포존 벡터의 클로닝 세포주의 루시퍼라제 발현 수준의 비교. 제한 희석에 의해 각 세포 풀에서 20개의 단일 세포주가 생성되었다. 각각의 단일 세포의 부피 루시퍼라제 생산성을 루시퍼라제 분석에 의해 측정하였다. 도표의 각 점은 하나의 세포주의 루시퍼라제 생산성을 나타낸다. 가로 막대는 각 조건에 대한 루시퍼라제 생성 평균을 나타낸다(**P < 0.01, *P < 0.05 student's t-test, Means ± S.D., N = 3). (D) 웨스턴 블롯으로 루시퍼라제 발현을 확인하고, β-액틴을 대조군으로 사용하였다. (E) 시간 경과에 따른 재조합 세포 풀의 부피 단백질 생산 분석. 도 3C에 나타난 바와 같이, 세포주를 대조군 벡터(Ctrl), SB100X 트랜스포사제 벡터를 포함하는 SB 트랜스포존 및 SB100X 트랜스포사제 벡터를 포함하는 개선된 SB 트랜스포존(VdTp)과 함께 생성하였다. 각 그룹에서 가장 높은 세포주가 선택되었다. 세포주를 4일마다 계대하면서 1개월 동안 항생제 저항성 배지를 사용하지 않고 성장시켰다. 지시된 시간에 4일 배치 배양의 말기에 루시퍼라제 분석에 의해 각 세포주의 부피 생산성을 측정하였다(***P < 0.001, two-way ANOVA, Means ± S.D., N = 3). (F) 게놈 DNA에 통합된 상대 유전자 카피 수의 비교. 도 3C에서 가장 높은 단백질 발현을 하는 세포주를 각각의 그룹에 대해 선택하였고, 상대적인 루시퍼라제 유전자 카피 수를 비교하기 위해 세포주의 게놈 DNA로 real-time PCR을 수행하였다(***P < 0.001, two-way ANOVA, Means ± S.D., N = 3). (G) 루시퍼라제 mRNA 발현 수준의 비교. 도 3C에서 가장 높은 단백질 발현을 하는 세포주를 각 그룹에 대해 선택하였고, 상대적인 루시퍼라제 발현 수준을 비교하기 위해 세포주의 cDNA로 real-time PCR을 수행하였다(***P < 0.001, two-way ANOVA, Means ± S.D., N = 3). (H) 전기 영동 이동성 이동 분석 (EMSA)을 사용하여 기존 SB 트랜스포존 벡터 및 개발된 SB 트랜스포존 벡터와 SB100X 트랜스포사제 및 6- 히스티딘 항체의 결합을 확인.3 is a diagram comparing the protein expression efficiency between the conventional SB vector and the SB vector system of the present invention. (A) The improved SB transposon vector construct of the present invention includes all conventional SB transposon vectors from left and right sequences. In addition, IR/DR sequences for the left and right arms are added to this. (B) The luciferase expression level of the improved SB transposon system and the SB transposon system was compared. Cell pool co-transfected 0.66 μg of SB100X transposase vector was selected as a control (***P < 0.001, **P < 0.01, student's t-test, Means ± S.D., N = 3). (C) Comparison of luciferase expression levels of cloning cell lines of luciferase control vector, SB transposon vector containing SB100X transposase vector, and improved SB transposon vector containing SB100X transposase vector. Limiting dilution resulted in 20 single cell lines from each cell pool. Volumetric luciferase productivity of each single cell was determined by luciferase assay. Each dot on the diagram represents the luciferase productivity of one cell line. Horizontal bars represent the mean of luciferase production for each condition (**P < 0.01, *P < 0.05 student's t-test, Means ± S.D., N = 3). (D) Luciferase expression was confirmed by Western blot, and β-actin was used as a control. (E) Volumetric protein production analysis of pools of recombinant cells over time. As shown in Figure 3C, cell lines were generated with a control vector (Ctrl), an SB transposon containing the SB100X transposase vector and an improved SB transposon containing the SB100X transposase vector (VdTp). The highest cell line from each group was selected. Cell lines were passaged every 4 days and grown without the use of antibiotic-resistant medium for 1 month. Volumetric productivity of each cell line was determined by luciferase assay at the indicated times at the end of the 4-day batch culture (***P < 0.001, two-way ANOVA, Means ± S.D., N = 3). (F) Comparison of relative gene copy numbers integrated into genomic DNA. In Figure 3C, the cell line with the highest protein expression was selected for each group, and real-time PCR was performed with the genomic DNA of the cell line to compare the relative luciferase gene copy number (***P < 0.001, two -way ANOVA, Means ± SD, N = 3). (G) Comparison of luciferase mRNA expression levels. The cell line with the highest protein expression in FIG. 3C was selected for each group, and real-time PCR was performed with cDNA of the cell line to compare the relative luciferase expression level (***P < 0.001, two-way ANOVA) , Means ± SD, N = 3). (H) Confirmation of binding of SB100X transposase and 6-histidine antibody to the existing SB transposon vector and the developed SB transposon vector using electrophoretic mobility shift assay (EMSA).
도 4는 메틸화 억제제 처리 후 유전자 카피 수 및 루시퍼라제 생산성의 변화를 나타낸 도면이다. (A) 도 3C에서 가장 높은 단백질 발현을 하는 세포주를 선택하고, 각 그룹에 대해 5 μM/ml의 5-아자-2'-데옥시시티딘(5-aza-2'-deoxycytidine), 20 mM/ml의 트리코스타틴 A(trichostatin A)를 처리하고 루시퍼라제 발현 수준을 비교하였다(***P < 0.001, student's t-test, Means ± S.D., N = 3). (B) 실시간 PCR을 사용한 상대 유전자 카피 수의 비교(***P < 0.001, student's t-test, Means ± S.D., N = 3)4 is a diagram showing changes in gene copy number and luciferase productivity after treatment with a methylation inhibitor. (A) Selecting the cell line with the highest protein expression in Figure 3C, 5 μM/ml of 5-aza-2'-deoxycytidine, 20 mM for each group Treatment with /ml of trichostatin A (trichostatin A) and comparing the expression level of luciferase (***P < 0.001, student's t-test, Means ± SD, N = 3). (B) Comparison of relative gene copy number using real-time PCR (***P < 0.001, student's t-test, Means ± S.D., N = 3)
이하, 실시예를 통해 본 발명을 보다 구체적으로 설명한다. 그러나 이들 예는 본 발명의 이해를 돕기 위한 것일 뿐 어떠한 의미로든 본 발명의 범위가 이들 예로 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail through examples. However, these examples are only for helping the understanding of the present invention, and the scope of the present invention is not limited to these examples in any sense.
실험예Experimental example
1. 세포 배양1. Cell Culture
CHO-DG44 세포(A1100001; Thermo Fisher Scientific, MA, Waltham, USA)가 사용되었다. 세포를 100U/ml 페니실린 및 100μg/ml 스트렙토마이신 및 10mM 나트륨 하이포산틴(sodium hypoxanthine) 및 1.6mM 티미딘과 함께 10% 태아 소 혈청이 첨가된 25mM 포도당을 함유하는 Dulbecco's modified Eagle's 배지에서 배양하였다. 세포를 5% CO 2로 37℃ 인큐베이터에서 궤도에 의해 오비탈에 의해 교반되는 T75 세포 배양 플라스크에서 10ml 배지에 유지시켰다. 세포는 2일마다 계대되었다. 세포 밀도 및 생존력은 Cedex HiRes Analyzer에 의해 평가되었다.CHO-DG44 cells (A110001; Thermo Fisher Scientific, MA, Waltham, USA) were used. Cells were cultured in Dulbecco's modified Eagle's medium containing 25 mM glucose supplemented with 10% fetal bovine serum with 100 U/ml penicillin and 100 μg/ml streptomycin and 10 mM sodium hypoxanthine and 1.6 mM thymidine. Cells were maintained in 10 ml medium in T75 cell culture flasks stirred by orbit by orbit in a 37° C. incubator with 5% CO 2 . Cells were passaged every 2 days. Cell density and viability were assessed by Cedex HiRes Analyzer.
2. 플라스미드2. Plasmids
SB 트랜스포존 시스템에 대한 공여 플라스미드를 역 말단 반복 서열(inverted terminal repeat sequences, ITRs)로 구축하였다. SB 트랜스포존 시스템에 대한 ITR을 [표 1]에 나타낸 특정 올리고 뉴클레오티드 프라이머를 사용하여 T2U-CAF로부터 증폭시켰다. DNA 주형에서, 역 반복(inverted repeat)/직접 반복(direct repeat) (IR/DR) 우측 암과 좌측 암은 인접하여 한 번에 증폭될 수 있었다. 이어서 PCR 생성물을 pcDNA3_SP_Luciferase의 변형된 버전인 루시퍼라제 컨트롤 벡터로 서브 클로닝하여 IR/DR-루시퍼라제 벡터를 생성하였다. 유사하게, 개선된 SB 트랜스포존 시스템에 대한 ITR을 [표 1]에 나타낸 특정 올리고 뉴클레오티드 프라이머를 사용하여 T2U-CAF로부터 증폭시켰다.Donor plasmids for the SB transposon system were constructed with inverted terminal repeat sequences (ITRs). ITRs for the SB transposon system were amplified from T2U-CAF using specific oligonucleotide primers shown in Table 1. In the DNA template, the inverted repeat/direct repeat (IR/DR) right arm and left arm are adjacent and can be amplified at once. The PCR product was then subcloned into a luciferase control vector, a modified version of pcDNA3_SP_Luciferase, to generate an IR/DR-luciferase vector. Similarly, ITRs for the improved SB transposon system were amplified from T2U-CAF using specific oligonucleotide primers shown in Table 1.
Figure PCTKR2020017709-appb-img-000001
Figure PCTKR2020017709-appb-img-000001
위와는 달리, 개선된 SB 트랜스포존 우측 및 좌측 암은 인접하지 않았으므로 복제 단계가 추가되었다. 증폭된 개선된 SB 좌측 암 및 우측 암을 T-Blunt? PCR Cloning 키트에 의해 T-벡터에서 오른쪽 방향으로 나란히 복제하였다. 클로닝된 T-벡터에서, 서로 인접한 개선된 SB 좌측 암 및 우측 암은 [표 1]에 나타낸 특정 올리고 뉴클레오티드 프라이머를 사용하여 PCR에 의해 증폭되었다. IR/DR 클로닝 및 SB 트랜스포존 개발시, PCR 생성물을 루시퍼라제 제어 벡터의 NruI 제한 부위에 삽입하고 EZ-Fusion TM HT 클로닝 키트로 클로닝을 수행하였다. 전이(transposition)의 경우, SB100X 트랜스포사제 벡터를 SB 또는 개선된 SB 벡터와 함께 공동-형질감염시켰다.Contrary to the above, the improved SB transposon right and left arms were not contiguous, so a cloning step was added. The amplified improved SB left arm and right arm were treated with T-Blunt? Side by side clones in the T-vector in the right direction by PCR Cloning kit. In the cloned T-vector, the improved SB left arm and right arm adjacent to each other were amplified by PCR using specific oligonucleotide primers shown in [Table 1]. During IR/DR cloning and SB transposon development, the PCR product was inserted into the NruI restriction site of the luciferase control vector and cloning was performed with the EZ-Fusion TM HT cloning kit. For transposition, SB100X transposase vector was co-transfected with SB or improved SB vector.
3. 형질감염(Transfections)3. Transfections
CHO-DG44 세포를 Cedex HiRes Analyzer로 계수하고 3 x 10 5 세포를 6-웰 플레이트에 밤새 시딩하였다. 다음날, 형질감염 전에 플라스미드를 첨가하고 Lipofector-EZ 시약으로 형질감염시켰다.CHO-DG44 cells were counted with Cedex HiRes Analyzer and 3×10 5 cells were seeded in 6-well plates overnight. The next day, the plasmid was added prior to transfection and transfected with Lipofector-EZ reagent.
4. 세포주 개발4. Cell Line Development
공여체 플라스미드 2μg 및 트랜스포사제 벡터 0.66μg를 사용하여 상기 기재된 바와 같이 세포를 공동-형질감염시켰다. ITR이 없는 루시퍼라제 대조군 벡터에 의해 대조군 형질감염을 수행하였다. 형질감염 후 1일차에, 배지를 교체하였다. 세포가 6-웰에 합류(confluent)될 때, 이들 세포를 T25 세포 배양 플라스크 또는 T75 세포 배양 플라스크로 옮겼다. 네오마이신에서 선별 항생제인 500 ㎍/mL G418을 갖는 배지에서 14일 동안 2일마다 배지를 교체하였다. 선택압(selective pressure)을 제거하지 않은 후, 생성된 세포 풀을 제한 희석에 의해 클론 세포주로 배양하였다. 간략하게, 상기 세포를 150㎕ 배지 당 0.8 세포의 밀도로 재현탁시켰다. 이들 희석된 세포를 96-웰 플레이트의 각 웰로 옮기고 500 μg/mL G418와 함께 웰당 평균 0.8 세포를 제공하였다. 배양 15-20일 후, 세포를 형광 현미경으로 관찰하고 하나의 웰에 단일 콜로니만을 갖도록 선택하였다. 세포의 개별 콜로니를 6-웰 플레이트로 옮겼다. 6-웰 플레이트에서 하나의 세포 계대 후, 세포를 T25 또는 T75 플라스크에 시딩하였다. 이어서, 이들 클론 세포주의 루시퍼라제 수준을 루시퍼라제 분석 시스템으로 측정하였다.Cells were co-transfected as described above using 2 μg of donor plasmid and 0.66 μg of transposase vector. Control transfections were performed with a luciferase control vector without ITR. On day 1 post transfection, the medium was changed. When the cells were confluent in the 6-well, they were transferred to a T25 cell culture flask or a T75 cell culture flask. The medium was changed every 2 days for 14 days in the medium with 500 μg/mL G418, a selective antibiotic in neomycin. After not removing the selective pressure, the resulting cell pool was incubated as a clonal cell line by limiting dilution. Briefly, the cells were resuspended at a density of 0.8 cells per 150 μl medium. These diluted cells were transferred to each well of a 96-well plate to give an average of 0.8 cells per well with 500 μg/mL G418. After 15-20 days of culture, the cells were observed under a fluorescence microscope and selected to have only single colonies in one well. Individual colonies of cells were transferred to 6-well plates. After passage of one cell in 6-well plates, cells were seeded into T25 or T75 flasks. The luciferase levels of these clonal cell lines were then measured with a luciferase assay system.
5. 단백질 정량5. Protein Quantification
세포 수는 Cedex HiRes Analyzer를 사용하여 측정하였고, 루시퍼라제 단백질 농도는 루시퍼라제 분석 시스템에 의해 분석되었다; 1 Х 10 6 세포를 2,000 rpm에서 2분 동안 원심 분리하였다. 이들 세포를 PBS로 2회 세척하였다. 그런 다음, 이들 세포의 펠릿을 100 μl의 1 Х 10 6 세포 배양 용해 시약에서 볼텍싱한다. 이들 용해된 샘플을 100 ㎕의 백색 96-웰의 각 웰에 옮기고, 100 ㎕의 루시퍼라제 분석 시약 (LARII)을 각 웰에 첨가 하였다(용해된 샘플:LARII=1:1). 그 후, 플레이트는 발광성을 갖는 VICTOR Multilabel Plate Reader로 측정되었다.Cell number was determined using Cedex HiRes Analyzer, and luciferase protein concentration was analyzed by luciferase assay system; 1 Х 10 6 cells were centrifuged at 2,000 rpm for 2 minutes. These cells were washed twice with PBS. Then, the pellet of these cells is vortexed in 100 μl of 1 Х 10 6 cell culture lysis reagent. These lysed samples were transferred to each well of 100 μl white 96-well, and 100 μl luciferase assay reagent (LARII) was added to each well (lysed sample: LARII=1:1). The plates were then measured with a VICTOR Multilabel Plate Reader with luminescence.
6. gDNA 및 cDNA 준비6. gDNA and cDNA Preparation
게놈 DNA (gDNA)는 안정한 재조합 CHO 세포주로부터 제조되었다; 2 Х 10 6 세포를 수확하고 PBS로 세척하였다. Solg TM Genomic DNA Prep Kit를 사용하여 gDNA를 분리하고 정제했다. RNase Mini Kit를 사용하여 2 Х 10 6 세포에서 총 RNA를 분리했다. 정제된 RNA를 RNAse-free water에 -80℃에서 저장하였다. Phusion RT-PCR 키트를 사용하여 총 RNA를 역전사시켰다. gDNA 및 cDNA의 순도 및 농도는 DS-11 분광 광도계로 측정되었다.Genomic DNA (gDNA) was prepared from a stable recombinant CHO cell line; 2 Х 10 6 cells were harvested and washed with PBS. The gDNA was isolated and purified using Solg TM Genomic DNA Prep Kit. Total RNA was isolated from 2 Х 10 6 cells using the RNase Mini Kit. Purified RNA was stored in RNAse-free water at -80°C. Total RNA was reverse transcribed using Phusion RT-PCR kit. Purity and concentration of gDNA and cDNA were measured with a DS-11 spectrophotometer.
7. Real-time PCR 및 qRT-PCR7. Real-time PCR and qRT-PCR
Real-time PCR(또는 qRT-PCR)은 SYBR 방법을 이용하여 CFX Connect TM Real-Time PCR Detection System (Bio-Rad)으로 수행되었다. 프라이머는 [표 2]에 나타낸 특정 올리고 뉴클레오티드 프라이머를 사용하여 루시퍼라제 영역에서 200 bp 단편, 하우스 키핑 유전자 GAPDH에서 200 bp 단편을 증폭시키고 데이터를 정규화하도록 설계되었다.Real-time PCR (or qRT-PCR) was performed with CFX Connect ™ Real-Time PCR Detection System (Bio-Rad) using the SYBR method. Primers were designed to amplify a 200 bp fragment in the luciferase region and a 200 bp fragment in the housekeeping gene GAPDH and normalize the data using specific oligonucleotide primers shown in Table 2.
Figure PCTKR2020017709-appb-img-000002
Figure PCTKR2020017709-appb-img-000002
Real-time PCR은 20ng의 gDNA로 수행되었고, qRT-PCR은 비템플릿 대조군 및 음성 대조군을 포함하는 1μl의 cDNA를 사용하여 95℃에서 5분 동안 변성시킨 후 94℃에서 30초, 57℃에서 30초, 70℃에서 10초 40사이클을 수행하였다. Real-time PCR was performed with 20 ng of gDNA, and qRT-PCR was performed using 1 μl of cDNA containing non-template control and negative control, denatured at 95° C. for 5 minutes, then 94° C. for 30 seconds, 57° C. for 30 seconds. Second, 40 cycles of 10 seconds were performed at 70°C.
8. 웨스턴 블롯 분석8. Western Blot Analysis
세포를 5% β-머캅토 에탄올을 함유하는 Laemmli 샘플 완충액에 용해시키고 95℃에서 5분 동안 가열하였다. 이어서 단백질 용해물을 8% 구배 트리스-글리신 미니 단백질 겔에서 분리하고 반 건조 장치(Bio-Rad)를 사용하여 폴리비닐리덴 디플루오라이드 막으로 옮겼다. 0.1% 트윈 20을 갖는 트리스-버퍼 식염수 중 5% 무지방 분유로 막을 차단하였다. HRP-접합된 일차 항체와 함께 배양되었다. 단백질은 Image lab이 있는 Chemidoc XRS+ 시스템을 사용하여 강화된 super signal west pico chemiluminescence 용액으로 검출되었다. 본 발명에 사용된 일차 항체는 HRP-접합된 항-루시퍼라제 항체, 및 로딩 제어에 사용되는 하우스 키핑 유전자의 HRP-접합된 β-액틴을 포함하였다. Cells were lysed in Laemmli sample buffer containing 5% β-mercapto ethanol and heated at 95° C. for 5 min. The protein lysate was then separated on an 8% gradient Tris-Glycine mini protein gel and transferred to a polyvinylidene difluoride membrane using a semi-drying apparatus (Bio-Rad). The membrane was blocked with 5% nonfat dry milk in Tris-buffered saline with 0.1% Tween 20. Incubated with HRP-conjugated primary antibody. Proteins were detected with enhanced super signal west pico chemiluminescence solution using Chemidoc XRS+ system with Image lab. The primary antibody used in the present invention included an HRP-conjugated anti-luciferase antibody, and HRP-conjugated β-actin of the housekeeping gene used for loading control.
9. 메틸화 억제제 처리9. Methylation Inhibitor Treatment
형질감염된 CHO 세포를 매일 5 μM/ml의 5-아자-2'-데옥시시티딘으로 4일 동안 처리하고, 실험 24시간 전에 20 mM/ml의 트리코스타틴 A(trichostatin A)를 처리하였다. 메틸화 억제제 처리 후, 루시퍼라제 단백질 농도를 루시퍼라제 분석 시스템으로 분석하였다.Transfected CHO cells were treated with 5-aza-2'-deoxycytidine at 5 μM/ml daily for 4 days, and treated with 20 mM/ml trichostatin A 24 hours before the experiment. After methylation inhibitor treatment, the luciferase protein concentration was analyzed with a luciferase assay system.
10. 전기영동 이동성 분석(EMSA)10. Electrophoretic Mobility Analysis (EMSA)
기존 SB 트랜스포존 벡터의 좌측 ITR 1 개를 포함하는 1kb DNA 프로브를 구축하고, 개발된 SB 트랜스포존 벡터의 좌측 ITR 2 개를 포함하는 1kb DNA 프로브를 구축 하였다. 프로브를 만드는데 사용된 프라이머는 표 3에 표시하였다. SB100X 6-His 태그가 달린 단백질을 ITR과 상호 작용하는 단백질로 사용했으며, 이는 2주 동안 CHO DG44 세포주 및 G418 선택으로 형질 전환한 후 Capturem ™ His-Tagged Purification Miniprep 키트 (635710; Takara, CA, USA)로 얻었다. 얻어진 프로브 및 단백질 추출물을 LightShift ™ Chemiluminescent EMSA Kit (20148; Thermo Fisher Scientific)에 의해 시험관 내 결합 반응에 적용하였다. 이 과정에서 His tag 항체 (sc-8036 HRP; Santa cruz)가 함께 반응하여 단백질 이동을 일으켰다. 이 EMSA 반응 용액을 2.5 % 0.5X TBE 아가로스 겔에 놓고 Image lab이 있는 Chemidoc XRS + 시스템으로 사진을 촬영하였다.A 1kb DNA probe containing one ITR on the left side of the existing SB transposon vector was constructed, and a 1kb DNA probe containing two ITRs on the left side of the developed SB transposon vector was constructed. The primers used to make the probes are shown in Table 3. SB100X 6-His-tagged protein was used as the protein interacting with ITR, which was transformed into the CHO DG44 cell line and G418 selection for 2 weeks followed by the Capturem™ His-Tagged Purification Miniprep kit (635710; Takara, CA, USA). ) was obtained with The obtained probe and protein extracts were subjected to in vitro binding reactions by LightShift™ Chemiluminescent EMSA Kit (20148; Thermo Fisher Scientific). In this process, His tag antibody (sc-8036 HRP; Santa cruz) reacted together to cause protein movement. This EMSA reaction solution was placed on a 2.5% 0.5X TBE agarose gel, and pictures were taken with Chemidoc XRS + system with Image lab.
Figure PCTKR2020017709-appb-img-000003
Figure PCTKR2020017709-appb-img-000003
11. 통계 분석11. Statistical Analysis
차이가 유의한지 여부를 확인하기 위해 Student 's t-test, two-way ANOVA에 이어 Bonferroni의 사후 테스트 또는 Bonferroni의 다중 비교 테스트를 통한 one-way ANOVA을 사용했다.To determine whether the differences were significant, Student's t-test, two-way ANOVA followed by one-way ANOVA with Bonferroni's post hoc test or Bonferroni's multiple comparison test was used.
실시예Example
1. 고 단백질 발현을 위해 SB 트랜스포존 시스템을 도입한 벡터의 효과1. Effect of vector introduced with SB transposon system for high protein expression
SB 트랜스포존 시스템을 확인하기 위해, 본 발명자들은 CMV 프로모터 구동 루시퍼라제 유전자 및 SV40 프로모터 구동 네오마이신 내성 유전자를 포함하는 역 반복/직접 반복(IR/DR) 좌우 ITR에 의한 트랜스포존 벡터를 설계하였다(도 1B). 위 트랜스포존 시스템과 비교하기 위해, 위 벡터 CMV 프로모터 구동 루시퍼라제 유전자 및 SV40 프로모터 구동 네오마이신 내성 유전자를 포함하는 비-트랜스포존 벡터를 설계 하였다(도 1A). 상기 SB 트랜스포존 시스템에는 게놈에 삽입하기 위해 필요한 SB100X 트랜스포사제(transposase)가 필요하다(도 1C). SB 트랜스포사제는 SB 트랜스포사제 단백질을 코딩하는 헬퍼 플라스미드로서 공동-형질감염(co-transfected)될 수 있다. 그러나 포유동물 세포에서 트랜스포존 시스템의 사용은 트랜스포존 벡터의 불안정성으로 인해 재전이(retransition)를 초래할 수 있다.To confirm the SB transposon system, the present inventors designed a transposon vector by reverse repeat/direct repeat (IR/DR) left and right ITR comprising a CMV promoter-driven luciferase gene and an SV40 promoter-driven neomycin resistance gene (Fig. 1B). ). To compare with the gastric transposon system, a non-transposon vector containing the gastric vector CMV promoter-driven luciferase gene and the SV40 promoter-driven neomycin resistance gene was designed (Fig. 1A). The SB transposon system requires SB100X transposase for insertion into the genome (FIG. 1C). The SB transposase can be co-transfected as a helper plasmid encoding the SB transposase protein. However, the use of transposon systems in mammalian cells can lead to retransition due to the instability of the transposon vector.
트랜스포존 시스템은 정의된 DNA 세그먼트가 게놈의 다른 부분으로 이동하여 숙주 게놈으로의 전이 유전자(transgene)의 전이 효율을 증가시키는 정확한 과정이다. 따라서, SB 트랜스포존 시스템이 트랜스진 통합을 촉진한다는 것을 확인하기 위해, 세포에서 발현된 루시퍼라제가 측정되었다(도 2A). SB100X 트랜스포사제 그룹을 갖는 SB 트랜스포존은 대조군 및 SB100X 트랜스포사제 그룹이 없는 SB 트랜스포존보다 현저히 더 많은 루시퍼라제를 발현하였다(도 2A).The transposon system is a precise process in which a defined DNA segment is moved to another part of the genome to increase the transfer efficiency of a transgene into the host genome. Therefore, to confirm that the SB transposon system promotes transgene integration, luciferase expressed in cells was measured (Fig. 2A). The SB transposons with the SB100X transposase group expressed significantly more luciferase than the control and SB transposons without the SB100X transposase group ( FIG. 2A ).
루시퍼라제 발현이 확인된 세포주를 사용하여, 안정한 단일 세포주를 수득하고, 루시퍼라제 발현 수준을 각 그룹에서 15개의 단일 세포주에 대해 측정하였다(도 2B). SB100X 트랜스포사제 그룹을 갖는 SB 트랜스포존은 가장 높은 발현 수준을 나타냈다(도 2B). 각 그룹에 대해 두 번째로 높은 측정된 세포주를 사용하여 웨스턴 블롯을 수행하였고, 이는 다시 확인되었다(도 2C). 이러한 결과에 기초하여, SB 트랜스포존 시스템에서 비-트랜스포존 시스템보다 전이 유전자의 유전자 발현이 증가되었다.Using the cell line with confirmed luciferase expression, a stable single cell line was obtained, and the luciferase expression level was measured for 15 single cell lines in each group (Fig. 2B). The SB transposon with the SB100X transposase group showed the highest expression level (Fig. 2B). Western blot was performed using the second highest measured cell line for each group, which was confirmed again (Fig. 2C). Based on these results, gene expression of transgenes was increased in the SB transposon system than in the non-transposon system.
2. 기존의 SB 트랜스포존과 본 발명의 SB 트랜스포존 간의 루시퍼라제 발현 수준 비교2. Comparison of luciferase expression levels between the conventional SB transposon and the SB transposon of the present invention
SB 트랜스포존 시스템의 벡터 통합 능력은 이전의 많은 연구와 실험에 의해 확인되었다. 초기 SB 트랜스포사제는 현재 SB100X 트랜스포사제로 여러 공학적 프로세스를 거쳤다. 우리의 실험은 또한 가장 진보된 시스템을 사용했다. 그러나 현재까지의 연구는 SB 트랜스포존의 트랜스포사제 단백질과 관련이 있다. 트랜스포존 벡터의 변형 가능성은 트랜스포사제 변형 가능성보다 높은 것으로 간주하였다. 따라서 SB 트랜스포존의 왼쪽 및 오른쪽 IR/DR 영역을 수정하여 새로운 벡터를 제조하였다. 종래의 SB 트랜스포존의 IR/DR은 좌우 암에서 약 380bp이다. 쌍을 추가함으로써, 약 990 bp의 IR/DR 좌우 암이 얻어졌다. IR/DR 이중 암은 새로운 벡터를 생성하기 위해 제어 벡터의 각 측면에 설계되었다. 이는 개선된 SB 트랜스포존 벡터로 명명되었다(도 3A).The vector integration ability of the SB transposon system has been confirmed by many previous studies and experiments. The early SB transposase went through several engineering processes with the current SB100X transposase. Our experiments also used the most advanced systems. However, studies to date are related to the transposase protein of the SB transposon. The potential for modification of the transposon vector was considered higher than that of the transposase. Therefore, a new vector was prepared by modifying the left and right IR/DR regions of the SB transposon. The IR/DR of the conventional SB transposon is about 380 bp in the left and right arms. By adding pairs, IR/DR left and right arms of about 990 bp were obtained. IR/DR dual arms were designed on each side of the control vector to generate a new vector. This was named an improved SB transposon vector (Fig. 3A).
기존의 SB 트랜스포존과 개선된 SB 트랜스포존 간의 단백질 발현 수준을 비교하기 위해, 형질감염 후 500μg/mL에서 14일 G418 선택 후 일시적 루시퍼라제 발현을 측정하였다. 결과적으로, 개선된 SB 트랜스포존은 기존의 SB 트랜스포존보다 단백질 발현이 증가한 것으로 밝혀졌다. 이전 실험에서와 같이, 본 발명자는 안정적인 세포주를 얻고 단일 세포 배양을 수행했다. 루시퍼라제는 각 그룹에서 20개의 단일 세포주 각각에서 측정되었다. 도 3B에 도시된 바와 같이, 개선된 SB 트랜스포존은 비교적 많은 양의 루시퍼라제를 발현하였다. 또한, 그룹당 최고를 나타내는 세포주를 선택하고 실험을 수행하였다(도 3C). 본 발명자는 이것을 다시 한번 확인하기 위해 웨스턴 블롯을 수행했고 이는 이전 결과와 같은 결과를 보여주었다(도 3D). To compare the protein expression level between the conventional SB transposon and the improved SB transposon, transient luciferase expression was measured after G418 selection 14 days after transfection at 500 μg/mL. As a result, the improved SB transposon was found to have increased protein expression than the conventional SB transposon. As in previous experiments, we obtained stable cell lines and performed single cell cultures. Luciferase was measured in each of 20 single cell lines in each group. As shown in Figure 3B, the improved SB transposon expressed a relatively high amount of luciferase. In addition, the cell line showing the best per group was selected and the experiment was performed (Fig. 3C). To confirm this once again, the present inventors performed western blot, which showed the same results as the previous results (Fig. 3D).
개선된 SB 트랜스포존이 각 세포주의 장기 안정성에 미치는 영향을 조사하기 위해 배치 배양(batch culture)을 한 달 동안 수행하였다. 각 세포주의 루시퍼라제 발현을 4일 주기로 측정하였다. 배치 배양 기간 동안, 개선된 SB 트랜스포존의 발현 패턴은 종래의 SB 트랜스포존의 발현 패턴보다 꾸준히 높았으며, 20일 후에, 종래의 SB 트랜스포존은 대조군과 유사한 발현 패턴을 나타냈다. 결과적으로, 개선된 SB 트랜스포존은 가장 진보된 형태인 종래의 SB 트랜스포존보다 더 안정적이고 높은 수준으로 발현되었다(도 3E).To investigate the effect of the improved SB transposon on the long-term stability of each cell line, batch culture was performed for one month. Luciferase expression in each cell line was measured at a 4-day cycle. During the batch culture period, the expression pattern of the improved SB transposon was consistently higher than that of the conventional SB transposon, and after 20 days, the conventional SB transposon showed an expression pattern similar to that of the control. As a result, the improved SB transposon was more stable and expressed at a higher level than the most advanced form, the conventional SB transposon (Fig. 3E).
단백질 발현 수준을 확인한 후, 게놈 DNA (gDNA)를 사용하여 SB 트랜스포존과 개선된 SB 트랜스포존 사이의 게놈 DNA 통합을 비교하기 위해 정량적 PCR을 수행하였다. 이것은 개선된 SB 트랜스포존의 보다 안정적인 단백질 발현의 원인을 찾기 위해 수행되었다. 개선된 SB 트랜스포존은 SB 트랜스포존보다 약 2배 더 많은 유전자 카피를 나타내었다. 또한 cDNA를 이용한 정량적 PCR로 mRNA 발현을 확인하였다. mRNA 발현도 2배 이상 차이가 났다(도 3G). EMSA 결과는 SB100X 트랜스포사제가 기존 SB벡터의 단일 ITR보다 개발된 SB벡터의 이중 ITR에 더 많은 결합을 한다는 것을 확인하였다(도 3H). 이러한 결과는 개선된 SB 트랜스포존이 원래의 SB 트랜스포존보다 유전자 통합에 더 효과적이며, 이는 센트럴 도그마(central dogma)에 의해 mRNA 및 단백질 수준이 증가함을 시사한다. 또한, 이러한 유전자 통합의 증가는 안정한 단백질 발현의 증가로 이어졌다.After confirming the protein expression level, quantitative PCR was performed to compare the genomic DNA integration between the SB transposon and the improved SB transposon using genomic DNA (gDNA). This was done to find the cause of the more stable protein expression of the improved SB transposon. The improved SB transposon exhibited approximately two-fold more gene copies than the SB transposon. In addition, mRNA expression was confirmed by quantitative PCR using cDNA. There was also a difference of more than two-fold in mRNA expression (FIG. 3G). The EMSA results confirmed that the SB100X transposase binds more to the double ITR of the developed SB vector than the single ITR of the existing SB vector (FIG. 3H). These results suggest that the improved SB transposon is more effective for gene integration than the original SB transposon, suggesting that the mRNA and protein levels are increased by the central dogma. In addition, this increase in gene integration led to an increase in stable protein expression.
3. DNA 메틸화 억제를 통한 전이유전자 침묵 및 재배치 확인3. Confirmation of transgene silencing and rearrangement through DNA methylation inhibition
DNA 메틸화는 헤테로 염색질의 형성 및 결과적으로 전사 억제를 초래한다. 그것은 진핵 생물 게놈을 트랜스포존으로부터 보호하기 위해 진화했기 때문에 트랜스포존과 밀접한 관련이 있다. SB 트랜스포존이 인간 세포에서 사용될 때, 유전자 침묵(gene silencing)이 관찰된 후 DNA 침묵이 부분적으로 억제되었다. 결과적으로, 본 발명자들은 SB 트랜스포존-매개 발현에 대한 DNA 메틸화 억제제의 효과를 조사하였다. DNA 메틸화 억제제로서, 본 발명자는 DNA 메틸화를 억제하기 위해 5-아자시티딘(5-azacytidine)을 사용하고 4일 동안 처리하였다. 또한 트리코스타틴 A를 추가 메틸화 억제에 대한 실험 24시간 전에 5-아자시티딘 처리 그룹에서 처리 하였다. 트리코스타틴 A(Trichostatin A)는 DNA 히스톤 H4에 작용하는 히스톤 탈아세틸화효소(Histone deacetylase)를 억제함으로써 DNA 이완(DNA loosening)을 유발했다. 결과적으로, 메틸화 억제제로 처리된 그룹은 DMSO로 처리된 그룹보다 단백질의 발현이 더 높았다(도 4A).DNA methylation results in the formation of heterochromatin and consequently transcriptional repression. It is closely related to transposons because it evolved to protect the eukaryotic genome from transposons. When the SB transposon was used in human cells, DNA silencing was partially suppressed after gene silencing was observed. Consequently, we investigated the effect of DNA methylation inhibitors on SB transposon-mediated expression. As a DNA methylation inhibitor, the present inventors used 5-azacytidine to inhibit DNA methylation and treated for 4 days. In addition, tricostatin A was treated in the 5-azacytidine treatment group 24 hours before the experiment for further inhibition of methylation. Trichostatin A induced DNA loosening by inhibiting histone deacetylase, which acts on DNA histone H4. As a result, the group treated with the methylation inhibitor had higher protein expression than the group treated with DMSO (Fig. 4A).
일반적으로, 벡터 형태의 트랜스포사제를 공동-형질감염시키는 경우, 트랜스포사제 벡터도 게놈에 통합되기 때문에 트랜스포사제 단백질이 연속적으로 발현된다. 이로 인해 게놈에 통합된 트랜스포존이 세대 동안 절단되어 안정성이 떨어질 수 있다. 그래서, 본 발명자는 또한 전이 유전자 침묵(transgene silencing) 외에 전이 유전자 재배치(transgene re-transposition)를 조사했다. 메틸화 억제제로 처리된 세포주의 유전자 카피 수를 측정하였다. gDNA를 사용하여 정량적 PCR을 수행하였다. 결과적으로, 카피 수는 절단되고 DMSO로 처리된 그룹에 비해 감소되었다. SB 트랜스포존의 카피 수는 60% 이상 감소한 반면, 본 발명자의 개선된 SB 트랜스포존은 20%만 감소했다. 이것은 개선된 SB 트랜스포존이 비교적 높은 절개 비율(incision rate)과 게놈으로의 통합을 가지고 있음을 보여준다(도 4B).In general, when transposase in vector form is co-transfected, the transposase protein is continuously expressed because the transposase vector is also integrated into the genome. This may result in cleavage of transposons integrated into the genome over generations, resulting in poor stability. So, we also investigated transgene re-transposition in addition to transgene silencing. The gene copy number of the cell line treated with the methylation inhibitor was determined. Quantitative PCR was performed using gDNA. As a result, the copy number was reduced compared to the truncated and DMSO-treated group. The copy number of the SB transposon was reduced by more than 60%, whereas our improved SB transposon was reduced by only 20%. This shows that the improved SB transposon has a relatively high incision rate and integration into the genome (Fig. 4B).
종합하면, 이 연구 데이터는 DNA 메틸화의 억제가 SB 트랜스포존 시스템에서 전이 유전자 활성화에 매우 효과적임을 시사한다. 또한, 개선된 SB 트랜스포존은 재전이가 발생하더라도 높은 재통합 비율을 갖는다. Taken together, these study data suggest that inhibition of DNA methylation is highly effective for transgene activation in the SB transposon system. In addition, the improved SB transposon has a high rate of reintegration even if re-metastasis occurs.
결과 검토Review the results
본 발명자 트랜스포존 벡터를 개선하는 방법을 연구했다. 트랜스포존의 IR/DR 면적은 이미 충분히 작았지만, 본 발명자는 생산량을 늘리기 위해 약간의 증가가 필요할 것이라고 생각했다. IR/DR 영역이 이전보다 두 배가 되었을 때, 본 발명자는 기질의 양이 효소 기반의 복잡한 이론 에서처럼 두 배가 될 것이라고 생각하여 특정 서열을 인식하는 더 많은 효소가 존재할 것이라고 생각했다. 결합량이 증가함에 따라 컷 및 페이스트(cut & paste)가 자연적으로 증가하여 숙주 게놈으로의 통합량 및 단백질 발현량이 증가할 것으로 예상되었다. 위의 아이디어를 기반으로, 본 발명자는 복제를 통해 제어 벡터의 양쪽 끝에 두 쌍의 IR/DR 영역을 삽입하여 IR/DR 이중 암을 포함하는 개선된 SB 트랜스포존을 개발했다.The inventors studied a method of improving transposon vectors. Although the IR/DR area of the transposon was already small enough, we thought that a slight increase would be necessary to increase the production. When the IR/DR region was doubled than before, we thought that the amount of substrate would double as in the complex theory based on enzymes, so there would be more enzymes recognizing specific sequences. As the amount of binding increased, cut and paste naturally increased, and it was expected that the amount of integration into the host genome and the amount of protein expression would increase. Based on the above idea, the present inventors have developed an improved SB transposon containing an IR/DR double arm by inserting two pairs of IR/DR regions at both ends of the control vector via cloning.
본 발명자가 개발한 트랜스포존 시스템은 기존 시스템에 비해 단백질 발현 수준을 비교할 때 발현 수준이 더 높은 것으로 밝혀졌다. 안정적인 단일 세포주에서 동일한 결과가 확인되었다. 상대 유전자 카피 수 및 상대 mRNA 발현 수준을 조사하여 숙주 게놈에서 더 이상 카피 수로 시스템이 안정적으로 발현되는지 확인하였다. 결과적으로 비교적 많은 수의 카피로 표시되었다. 또한, 배치 배양으로 한 달 동안 안정성 점검을 수행하였고, 본 발명자에 의해 개선된 벡터 시스템은 가장 높은 발현 수준을 나타냈다. 기존의 SB 트랜스포존 벡터는 실험 종료시 대조군의 발현 수준과 유사한 발현 수준을 나타내었지만, 본 발명자에 의해 개선된 벡터는 크게 감소하지 않았다. The transposon system developed by the present inventors was found to have a higher expression level when comparing the protein expression level compared to the existing system. The same results were confirmed in a single stable cell line. Relative gene copy number and relative mRNA expression level were investigated to confirm that the system was stably expressed with a longer copy number in the host genome. As a result, a relatively large number of copies were displayed. In addition, stability check was performed for one month in batch culture, and the vector system improved by the present inventors showed the highest expression level. The conventional SB transposon vector exhibited an expression level similar to that of the control group at the end of the experiment, but the vector improved by the present inventors did not significantly decrease.
염색질이 응축될 때 mRNA 발현이 잘 일어나지 않으며, 반대로, 염색질이 플레어되면(flared) 발현이 잘 발생한다고 생각할 수 있다. 이러한 사실을 가정하면, 메틸화 억제제로의 처리는 풀림이 유지될 때 DNA 축합을 억제하고 단백질 발현을 증가시킬 것으로 생각되었다. 그러나 메틸화 억제제 처리를 수행할 때, 공동-형질감염된 트랜스포사제 벡터의 발현 수준이 증가하였고, 숙주 게놈 내부의 컷 및 페이스트로 인해 재배치가 일어날 것으로 생각되었다. 이를 확인하기 위해 메틸화 억제제 처리 후의 상대적 유전자 카피 수를 확인하였다. 카피 수의 감소가 확인되었지만 본 발명의 개선된 벡터의 사본 감소는 적었다. 이것은 새로 개선된 벡터가 재배치가 발생하더라도 더욱 안정적으로 통합되고 발현된다고 해석할 수 있다.It can be considered that mRNA expression does not occur well when chromatin is condensed, and on the contrary, expression occurs well when chromatin is flared. Given this fact, it was thought that treatment with a methylation inhibitor would inhibit DNA condensation and increase protein expression when unwinding was maintained. However, when the methylation inhibitor treatment was performed, the expression level of the co-transfected transposase vector was increased, and it was thought that the rearrangement would occur due to the cut and paste inside the host genome. To confirm this, the relative gene copy number after methylation inhibitor treatment was confirmed. A decrease in the number of copies was observed, but the decrease in the number of copies of the improved vector of the present invention was small. This can be interpreted that the newly improved vector is more stably integrated and expressed even if rearrangement occurs.

Claims (15)

  1. 2번 반복되는 역 반복/직접 반복(IR/DR) 좌측 암(arm) 및 2번 반복되는 역 반복/직접 반복(IR/DR) 우측 암(arm)을 포함하는 슬리핑 뷰티 트랜스포 시스템. A Sleeping Beauty Transform system comprising a double repeat inverse repeat/direct repeat (IR/DR) left arm and a two repeat inverse repeat/direct repeat (IR/DR) right arm.
  2. 제1항에 있어서, According to claim 1,
    상기 IR/DR 좌측 암 및 우측 암은 각각 서열번호 1 및 서열번호 2의 염기서열로 이루어진 슬리핑 뷰티 트랜스포존 시스템.The IR / DR left arm and right arm are sleeping beauty transposon system consisting of nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 2, respectively.
  3. 제1항에 있어서, According to claim 1,
    상기 2번 반복되는 IR/DR은 100 ~ 300 bp 뉴클레오티드를 사이에 두고 반복되는 슬리핑 뷰티 트랜스포존 시스템.The double-repeated IR/DR is a sleeping beauty transposon system that is repeated with 100 to 300 bp nucleotides in between.
  4. 제1항에 있어서, According to claim 1,
    상기 2번 반복되는 IR/DR 좌측 암 및 우측 암은 서열번호 3 및 서열번호 4의 염기서열로 이루어진 슬리핑 뷰티 트랜스포존 시스템.The sleeping beauty transposon system consisting of the nucleotide sequences of SEQ ID NO: 3 and SEQ ID NO: 4 in the left arm and right arm of the IR/DR repeated twice.
  5. 제1항에 있어서, According to claim 1,
    상기 좌측 암 및 우측 암 사이에 목적 단백질을 코딩하는 전이유전자(transgene)을 포함하는 슬리핑 뷰티 트랜스포존 시스템.A sleeping beauty transposon system comprising a transgene encoding a target protein between the left arm and the right arm.
  6. 제5항에 있어서, 6. The method of claim 5,
    상기 전이유전자는 프로모터를 더 포함하는 슬리핑 뷰티 트랜스포존 시스템.The transgene is a sleeping beauty transposon system further comprising a promoter.
  7. 제1항에 있어서,According to claim 1,
    상기 좌측 암 및 우측 암 사이에 항생제 내성 유전자를 포함하는 슬리핑 뷰티 트랜스포존 시스템.A sleeping beauty transposon system comprising an antibiotic resistance gene between the left arm and the right arm.
  8. 제1항 내지 제7항 중 어느 한 항의 슬리핑 뷰티 트랜스포존 시스템을 포함하는 벡터.A vector comprising the Sleeping Beauty transposon system according to any one of claims 1 to 7.
  9. 제8항의 벡터를 포함하는 세포. A cell comprising the vector of claim 8 .
  10. 제9항의 세포를 배양하는 단계를 포함하는 목적 단백질 생산 방법.10. A method for producing a target protein comprising the step of culturing the cell of claim 9.
  11. 제8항의 벡터 및 슬리핑 뷰티 트랜스포사제 단백질을 코딩하는 핵산을 포함하는 벡터를 세포에 공동-형질감염시키는 단계를 포함하는 유전자 전위 방법. A gene translocation method comprising the step of co-transfecting a cell with the vector of claim 8 and a vector comprising a nucleic acid encoding a sleeping beauty transposase protein.
  12. 제11항에 있어서, 12. The method of claim 11,
    형질감염된 세포에 메틸화 억제제(methylation inhibitor) 또는 히스톤 디아세틸레이즈 억제제(histone deacetylase inhibitor)를 처리하는 단계를 더 포함하는 유전자 전위 방법.A gene translocation method further comprising treating the transfected cells with a methylation inhibitor or a histone deacetylase inhibitor.
  13. 제12항에 있어서, 13. The method of claim 12,
    상기 메틸화 억제제는 5-아자시티딘(azacytidine)이고 히스톤 디아세틸레이즈 억제제는 트리코스타틴 A(Trichostatin A)인 유전자 전위 방법.The gene translocation method wherein the methylation inhibitor is 5-azacytidine and the histone deacetylase inhibitor is Trichostatin A.
  14. 제11항에 있어서, 12. The method of claim 11,
    상기 세포는 포유동물 세포인 유전자 전위 방법.wherein the cell is a mammalian cell.
  15. 제14항에 있어서, 15. The method of claim 14,
    상기 포유동물 세포는 COS 세포, CHO 세포, VERO 세포, MDCK 세포, WI38 세포, V79 세포, B14AF28-G3 세포, BHK 세포, HaK 세포, NS0 세포, SP2/0-Ag14 세포, HeLa 세포, HEK293 세포 및 PER.C6 세포로 이루어진 군으로부터 선택되는 하나인 유전자 전위 방법.Said mammalian cells include COS cells, CHO cells, VERO cells, MDCK cells, WI38 cells, V79 cells, B14AF28-G3 cells, BHK cells, HaK cells, NS0 cells, SP2/0-Ag14 cells, HeLa cells, HEK293 cells and A gene translocation method which is one selected from the group consisting of PER.C6 cells.
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US20060205081A1 (en) * 2005-02-18 2006-09-14 Linhong Li Use of methyltransferase inhibitors to enhance transgene expression
US20180135032A1 (en) * 2007-07-04 2018-05-17 Max-Delbrück-Centrum für Molekulare Medizin Hyperactive Variants of the Transposase Protein of the Transposon System Sleeping Beauty
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