KR101535070B1

KR101535070B1 - Recomnication expression vector of vascular growth factor and the vascular growth factor expressing stem cell line thereof

Info

Publication number: KR101535070B1
Application number: KR1020140089522A
Authority: KR
Inventors: 조제열; 조현민
Original assignee: 서울대학교산학협력단
Priority date: 2014-07-16
Filing date: 2014-07-16
Publication date: 2015-07-10

Abstract

The present invention relates to a recombinant expression vector, which comprises: a TetO- CMV promoter; a nucleotide sequence of a vascular endothelial growth factor gene; a nucleotide sequence of hEF1a gene; a nucleotide sequence of rtTA gene; a nucleotide sequence of HA-L gene; and a nucleotide sequence of HA-R gene. According to the present invention, the recombinant expression vector uses a Talen system unlike a conventional transformation using a virus so that a vascular endothelial growth factor gene is stably integrated to a safe harbor portion of a host chromosome, thereby improving the stability. Furthermore, a Tet-ON system is introduced to arbitrarily control the expression of a vascular endothelial growth factor, thus the expression of the vascular endothelial growth factor is possible when doxycycline is treated only, thereby effectively controlling the expression of the vascular endothelial growth factor.

Description

혈관성장인자 유전자의 형질전환을 위한 재조합 발현 벡터 및 이를 이용한 혈관성장인자 발현 줄기세포주 {Recomnication expression vector of vascular growth factor and the vascular growth factor expressing stem cell line thereof}[0001] The present invention relates to a recombinant expression vector for transformation of a vascular growth factor gene and a vascular growth factor expressing stem cell line using the recombinant expression vector,

본 발명은 표적 세포주에서 반영구적으로 특정 조건에만 혈관성장인자를 분비시키고 표적세포주의 염색체의 safe harbor 부위에 혈관성장인자의 유전자를 삽입하기 위한 새로운 발현벡터에 관한 기술이다. The present invention relates to a novel expression vector for secretion of vascular growth factors only under certain conditions semi-permanently in a target cell line and for inserting a gene for an angiogenic factor into the safe harbor region of the chromosome of the target cell line.

관상동맥 질환은 심장 근육에 혈관을 공급하는 동맥(관상동맥)이 프라그로 차게 되면서 혈관이 좁아지게 되는 질환으로 현재 관상동맥 질환은 전세계적으로 높은 치사율을 보이며 우리나라에서도 3대 사망원인 중 하나로 보고 되고 있다. 이러한 관상동맥 질환의 치료는 스텐트를 활용하여 관상동맥 안쪽에 쌓인 플라크 침적물을 제거하는 스텐트 삽입술에 주로 의존하고 있지만 그 후 관상동맥의 손상으로 인해 재협착이 일어나는 경우가 많다. 따라서, 관상동맥 질환을 치료할 수 있는 새로운 치료법으로 줄기세포 치료법이 시도되고 있으나 아직까지는 치료부위에서 세포생존률과 치료효과가 낮은 단점이 있다. 또한 기존의 줄기세포치료를 위한 형질전환 줄기세포주를 만드는 기술은 바이러스를 이용한 형질전환을 이용할 경우 안정성의 문제로 인해 임상적용에 어려움이 있으며,발현시키고자 하는 유전자가 너무 클 경우 바이러스가 효율적으로 만들어지지 않는 문제가 있다(Elbashir etal., 2001. Nature 411: 494-498; Trono, 2000. GeneTher. 7: 20-23). 또한 혈관성장인자의 과발현으로 인하여 혈관종(hemangioma)을 유발하는 위험이 존재하였다. 따라서 보다 안전하고 혈관성장인자의 발현조절이 가능한 줄기세포주 재조합 발현벡터 개발이 필요하며 이와 관련하여 안전한 유전자재조합을 위해 RNA-guided clustered regularly interspaced short palindromic repeats (CRISPR) 기법과 custom-designed TALE nucleases (TALENs) 기법등이 연구되고 있다. (Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. Nucleic Acids Research, 2011, Vol. 39, No. 12 e82; Programmable Sequence-Specific Transcriptional Regulation of Mammalian Genome Using Designer TAL Effectors)
Coronary artery disease is a disease in which arteries (coronary arteries) supplying blood vessels to the heart muscles are filled with plaque, resulting in narrowing of blood vessels. Coronary artery disease now has a high mortality rate in the world and is reported to be one of the three major causes of death in Korea have. The treatment of coronary artery disease depends on stent implantation to remove plaque deposition inside the coronary artery using a stent, but restenosis is often caused by coronary artery injury. Therefore, stem cell therapy has been attempted as a new therapeutic method for treating coronary artery disease. However, there is a disadvantage that cell survival rate and therapeutic effect are low at the treatment site. In addition, the technology for producing transgenic stem cell lines for the treatment of existing stem cells has difficulties in clinical application due to the problem of stability when the transformation using viruses is used, and when the gene to be expressed is too large, (Elbashir et al., 2001. Nature 411: 494-498; Trono, 2000. Gene Ther. 7: 20-23). There was also a risk of inducing hemangioma due to overexpression of vascular growth factors. Therefore, it is necessary to develop a stem cell line recombinant expression vector which is safer and able to regulate the expression of angiogenic growth factors. In this regard, for safe recombination, RNA-guided clustered regularly interspaced short palindromic repeats (CRISPR) technique and custom-designed TALE nucleases ) Techniques have been studied. (Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. Nucleic Acids Research, 2011, Vol. 39, No. 12 e82; Programmable Sequence-Specific Transcriptional Regulation of Mammalian Genome Using Designer TAL Effectors)

본 발명자들은 관상동맥 질환 치료를 위해 특정 조건에서 유전자의 발현이 조절되고 보다 안전한 혈관성장인자 유전자의 형질전환용 재조합 발현 벡터를 개발하고자 노력하였다. 그 결과 Talen 시스템과 Tet-ON 시스템을 이용한 재조합 발현벡터 및 이를 이용한 혈관성장인자 발현 줄기세포주를 완성하였다.The present inventors have sought to develop a recombinant expression vector for transfection of a vascular growth factor gene in which expression of a gene is regulated under certain conditions for the treatment of coronary artery disease. As a result, a recombinant expression vector using the Talen system and the Tet-ON system and a stem cell line expressing an angiogenic factor using the recombinant expression vector were completed.

상기 목적을 달성하기 위해 Tet-오퍼레이터(tet-o), CMV 프로모터, 혈관내피성장인자(Vascular endothelial growth factor, VEGF)유전자, hEF1a 프로모터, rtTA 유전자, HA-L 유전자(homology arm-L), HA-R(homology arm-R) 유전자를 포함하는 재조합 발현벡터를 제공한다.In order to accomplish the above object, the present invention provides a method for producing a recombinant vector comprising Tet-operator, CMV promoter, vascular endothelial growth factor (VEGF) gene, hEF1a promoter, rtTA gene, homolog arm- -R (homology arm-R) gene.

상기 재조합 발현벡터에서, 상기 Tet 오퍼레이터는 서열번호 3이고, CMV 프로모터는 서열번호 4이고, 혈관내피성장인자(Vascular endothelial growth factor, VEGF)유전자는 서열번호 5이고, hEF1a 프로모터는 서열번호 6이고, rtTA 유전자는 서열번호 7이고, HA-L 유전자는 서열번호 2이고, HA-R 유전자는 서열번호 8인 것인 재조합 발현벡터일 수 있다.In the recombinant expression vector, the Tet operator is SEQ ID NO: 3, the CMV promoter is SEQ ID NO: 4, the vascular endothelial growth factor (VEGF) gene is SEQ ID NO: 5, the hEF1a promoter is SEQ ID NO: the rtTA gene is SEQ ID NO: 7, the HA-L gene is SEQ ID NO: 2, and the HA-R gene is SEQ ID NO: 8.

상기 재조합 발현벡터는 서열번호 1인 것인 재조합 발현벡터일 수 있다.The recombinant expression vector may be the recombinant expression vector of SEQ ID NO:

또한 본 발명은 상기 재조합 발현벡터로 형질전환되어 혈관내피성장인자를 발현하는 세포주를 제공한다.The present invention also provides a cell line transformed with the recombinant expression vector to express a vascular endothelial growth factor.

상기 세포주는 제대혈 유래 줄기세포(hUCB-MSC cell)인 것인 세포주일 수 있다.The cell line may be a cell strain that is a cord blood derived stem cell (hUCB-MSC cell).

본 발명에 따른 재조합 발현벡터는 바이러스를 이용해야 하는 기존의 형질전환과 달리 Talen 시스템을 이용하여 숙주 염색체의 safe harbor 부위에 안정적으로 혈관성장인자 유전자를 도입하는 것이 가능하며 동시에 안정성 문제를 개선하였다. 또한 혈관성장인자의 발현을 임의로 조절하고자 Tet-ON 시스템을 도입하였으며 이로써 독시사이클린(doxycycline)이 처리되었을 경우에만 혈관성장인자의 발현이 가능하다.
The recombinant expression vector according to the present invention can stably introduce an angiogenic factor gene into the safe harbor site of the host chromosome using the Talen system, unlike the conventional transformation using the virus, and at the same time, the stability problem is improved. In addition, the Tet-ON system was introduced to arbitrarily regulate the expression of vascular growth factors, and thus it is possible to express vascular growth factors only when doxycycline is treated.

도1(a)는 TALEN L, R의 AAVS1 부위 내에서 인식 부위를 나타낸 개념도이다.
도1(b)은 pTUC19-TetOCMV-VEGF-hEF1a-rtTA 재조합 발현벡터의 개열지도이다.
도2는 pTUC19 벡터의 개열지도이다.
도3는 hUCB-MSC Cell line에 TALEN Left 벡터, TALEN Right 벡터, pTUC-19 GFP 벡터를 형질주입하여 GFP가 발현되는지에 대한 결과를 나타내는 형광이미지이다.
도4는 Junction PCR을 이용하여 세포내로 도입된 GFP 유전자가 safe harbor 부위에 정상적으로 삽입되었는지 확인하기 위한 PCR 밴드 결과이다.
도5는 In fusion 방법을 이용하여 Tet-On/CMV-VEGF-hEF1a-rtTA 및 pTUC19-TetOCMV-VEGF-hEF1a-rtTA 재조합 발현백터를 제조하는 방법을 도시한 그림이다
도 6은 Tet-on 시스템이 도입된 세포의 혈관성장인자 발현 평가를 위한 웨스턴 블로트 분석(western blotting) 결과를 나타내는 이미지이다.
도 7은 인간 염색체19 의 AAVS1 부위에 혈관성장인자 발현 유전자가 정상적으로 삽입되었는지를 확인하기 위한 Junction PCR 결과이다.
도8은 TALEN 에 의해 삽입된 Tet-OnCMV-VEGF-hEF1a-rtTA 의 작동여부 확인을 위한 웨스턴 블로트 분석(western blotting) 결과이다.FIG. 1 (a) is a conceptual diagram showing recognition sites within the AAVS1 region of TALEN L, R. FIG.
1 (b) is a cleavage map of the recombinant expression vector pTUC19-TetOCMV-VEGF-hEF1a-rtTA.
2 is a cleavage map of the pTUC19 vector.
3 is a fluorescence image showing the results of the expression of GFP by transfection of TALEN Left vector, TALEN Right vector, and pTUC-19 GFP vector into the hUCB-MSC cell line.
FIG. 4 is a PCR band result for confirming whether a GFP gene introduced into a cell using Junction PCR was normally inserted into a safe harbor site.
Figure 5 shows the results of Tet-On / CMV-VEGF-hEF1a-rtTA and pTUC19-TetOCMV-VEGF-hEF1a-rtTA recombinant expression vector
6 is an image showing the result of western blotting for evaluation of vascular growth factor expression of cells into which the Tet-on system is introduced.
FIG. 7 shows a result of a Junction PCR for confirming whether an angiogenic factor expression gene was normally inserted into the AAVS1 region of human chromosome 19.
FIG. 8 is a result of western blotting for confirming the operation of Tet-OnCMV-VEGF-hEF1a-rtTA inserted by TALEN.

본 발명은 Tet-오퍼레이터(tet-o), CMV 프로모터, 혈관내피성장인자(Vascular endothelial growth factor, VEGF)유전자,hEF1a 프로모터,rtTA 유전자, HA-L 유전자, HA-R 유전자를 포함하는 재조합 발현벡터에 관한 것이다.The present invention relates to a recombinant expression vector comprising a Tet-operator (tet-o), a CMV promoter, a vascular endothelial growth factor (VEGF) gene, an hEF1a promoter, an rtTA gene, an HA- .

상기 TetO- CMV 프로모터는 테트라사이클린 오퍼레이터(tet-O)와 사이토메칼로바이러스(CMV)를 포함하며 이는 표적유전자의 발현유도를 위한 테트라사이클린/독시사이클린 전사 유도 시스템(TET-on 시스템)을 구성한다. 유전자 발현을 조절하기 위한 시스템인 TET-on 시스템은 항생제인 테트라사이클린 또는 그의 유사체인 독시사이클린 존재하에서 가역적으로 목적유전자의 전사를 조절하여 유전자 발현을 조절하는 방법이다. rtTA 단백질은 테트라사이클린과 결합한 경우에만 테트라사이클린 오퍼레이터(tet-O)에 결합한다. 따라서 테트라사이클린이 존재하는 경우에만 목적유전자의 전사가 유도된다. 테트라사이클린/독시사이클린 전사 유도 시스템은 생체내에서 가장 광범위하게 사용되고 있으며 우려할만한 부작용도 없다. 테트라사이클린과 독시사이클린은 경구투여가 가능한 매우 저렴한 물질이므로 경제적 관점에서도 다른 것에 비해 뛰어나다.The TetO-CMV promoter comprises a tetracycline operator (tet-O) and cytomegalovirus (CMV), which constitute a tetracycline / doxycycline transcription induction system (TET-on system) for induction of expression of a target gene. The TET-on system, a system for regulating gene expression, is a method for regulating gene expression by regulating transcription of a target gene reversibly in the presence of tetracycline, an antibiotic, or its analogue, a doxycycline. The rtTA protein binds to the tetracycline operator (tet-O) only when bound to tetracycline. Therefore, transcription of the target gene is induced only when tetracycline is present. Tetracycline / doxycycline transcription induction systems are the most widely used in vivo and have no worrying side effects. Because tetracycline and doxycycline are very cheap substances that can be administered orally, they are economically superior to others.

테트라사이클린/독시사이클린- 전사 유도 시스템을 구성하는 또 다른 구성요소인 rtTA의 경우, rtTA 발현 프로모터는 CMV 프로모터이며 rtTA는 테트라사이클린 또는 독시사이클린과 결합한 상태에서 인핸서에 작용하여 인핸싱 기능을 할 수 있다. rtTA는 테트라사이클린 또는 독시사이클린과 결합체를 형성하지 못하면 인핸서에 작용하는 기능을 상실함으로써 더 이상 인핸싱 기능을 행사할 수 없다. 따라서, 테트라사이클린/독시사이클린-유도성 시스템에서 rtTA은 테트라사이클린 또는 독시사이클린의 존재하에서만 표적 유전자를 발현시키는 테트라사이클린/독시사이클린- 전사 유도 시스템(Tet-On 시스템)을 구성한다. In the case of rtTA, another component of the tetracycline / doxycycline-transcription induction system, the rtTA expression promoter is the CMV promoter and rtTA acts on the enhancer in combination with tetracycline or doxycycline to enhance the function. If rtTA does not form a complex with tetracycline or doxycycline, it will no longer be able to exercise its enhancing function by losing its function of acting on the enhancer. Thus, in a tetracycline / doxycycline-inducible system, rtTA constitutes a tetracycline / doxycycline-transcription induction system (Tet-On system) that expresses the target gene only in the presence of tetracycline or doxycycline.

hEF1a (Human elongation factor-1 alpha )프로모터의 경우 인간유래의 프로모터이며 in vivo 또는 in vitro 조건에서 다양한 유전자 발현을 위해 사용된다. 본 발명에서는 CMV 프로모터와 함께 목적유전자의 발현에 기여한다. The human elongation factor-1 alpha (hEF1a) promoter is a human-derived promoter and is used for gene expression in vivo or in vitro. In the present invention, it contributes to the expression of the target gene together with the CMV promoter.

혈관성장인자는 세포의 증식, 성장, 유지 생존을 조절하는 중요한 기능을 갖는 단백질을 의미하며 VEGF(Vascular endothelial growth factor), GDNF(Glial cell-derived neurotrophic factor), HGF(hepatocyte growth factor) 또는 bFGF(basic fibroblast growth factor)일 수 있으나, 바람직하게는 VEGF일 수 있다. Vascular growth factor (VEGF), a glial cell-derived neurotrophic factor (GDNF), hepatocyte growth factor (HGF), or bFGF (vascular endothelial growth factor) basic fibroblast growth factor, but may preferably be VEGF.

TALEN(Transcription activator-like effector nuclease)시스템에 의한 유전자 삽입 유전자인 HA-L 유전자와 HA-R 유전자는 Talen 시스템에서 목적유전자가 삽입되기를 원하는 특정위치의 유전자와 상동성을 갖고 있어서 목적유전자의 상동성재조합을 유도하고 상기 상동성재조합을 통해 목적유전자를 원하는 위치에 삽입시킨다. The HA-L gene and the HA-R gene, which are gene insertion genes by the transcription activator-like effector nuclease (TALEN) system, are homologous to the gene of the specific position desired to be inserted in the Talen system, Induce recombination and insert the desired gene at the desired position through the homologous recombination.

상기 TALEN (Transcription activator-like effector nuclease)시스템은 혈관성장인자의 영구적 발현을 위해 필요한 안전한 유전자 변환기술(Safe-harbor system)로서 이 TALEN이라는 특이적 DNA-결합 효소를 통해 표적세포주의 유전자 DNA의 특정한 부위에직접 유전자 결실, 또는 유전자 삽입이 가능하여 표적세포주의 유전자 편집(gene edition)이 가능하다. 본 발명에서 TALEN L, R 벡터를 세포에 형질주입해주면 인간 염색체 19 AAVS1 부위 안에 있는 특정 인식부위들을 인식하여 절단한다. 이때 Left TALEN의 경우 인간 염색체 19 AAVS1 부위중 염기서열번호 9의 염기서열을 인식하며 Right TALEN의 경우 인간 염색체 19 AAVS1 부위중 염기서열번호 10의 염기서열을 인식한다. The transcriptional activator-like effector nuclease (TALEN) system is a safe-harbor system for the permanent expression of a vascular growth factor. Through the specific DNA-binding enzyme called TALEN, On the site Direct gene deletion, or gene insertion, is possible and gene edition of the target cell line is possible. In the present invention, when a TALEN L, R vector is transfected into a cell, specific recognition sites within the human chromosome 19 AAVS1 region are recognized and cleaved. At this time, in the case of Left TALEN, it recognizes the base sequence of base sequence No. 9 in the human chromosome 19 AAVS1 region and in the case of Right TALEN, it recognizes the base sequence of base sequence No. 10 in the human chromosome 19 AAVS1 region.

또한 상기 인식되는 부위의 사이에 위치한 염기서열번호 11의 염기서열중 일부를 절단시킨다 (도1a). 이 때 잘려진 부위에 목적유전자를 효과적으로 삽입하기 위해 상기 좌우 TALEN 인식부위의 서열의 간격이 50bp가 되도록 설계하였다. In addition, a part of the nucleotide sequence of nucleotide sequence number 11 located between the recognized sites is cut (Fig. 1A). At this time, in order to insert the target gene effectively into the cut region, the distance between the sequences of the right and left TALEN recognition sites was designed to be 50 bp.

잘려진 서열을 회복 시키기 위하여 세포 내에서 상동성 재조합이 발생한다. 공여벡터에 HA-L 유전자와 HA-R 유전자가 삽입되어 있는 경우 상동성 재조합에 의해서 HA-L 유전자, HA-R 유전자, 그리고 그 둘 사이에 있는 목적유전자가 세포의 염색체상으로 삽입된다. 이렇게 삽입된 목적유전자들은 세포분열이 일어난 후에도 이미 염색체 19 AAVS1 부위에 들어가 있기 때문에 영구적으로 발현이 가능하다.Homologous recombination occurs within the cell to restore the cleaved sequence. When the HA-L gene and the HA-R gene are inserted into the donor vector, HA-L gene, HA-R gene, and the target gene between them are inserted into the cell chromosome by homologous recombination. These inserted target genes can be expressed permanently because they are already on chromosome 19 AAVS1 after cell division.

HA-L 유전자의 염기서열은 인간 19번 염색체의 AAVS1 부위에 위치한다.(염기서열번호 2) The nucleotide sequence of the HA-L gene is located in the AAVS1 region of the human chromosome 19. (SEQ ID NO: 2)

HA-R 유전자의 염기서열도 인간 19번 염색체의 AAVS1 부위에 위치한다. (염기서열번호8)The base sequence of the HA-R gene is also located in the AAVS1 region of the human chromosome 19. (Nucleotide sequence number 8)

본 발명에서 바람직하게는 재조합 발현벡터는 도1(b)의 개열지도로 표시되는 재조합 발현벡터일 수 있다.In the present invention, preferably, the recombinant expression vector may be a recombinant expression vector represented by the cleavage map of Fig. 1 (b).

본 발명은 또한 상기 재조합 발현벡터로 형질전환되어 혈관형성 유도인자등 목적유전자를 발현하는 줄기세포주를 제공한다.상기 줄기세포주를 제조하기 위해 (a)상동성 재조합을 통해 목적유전자를 삽입하려고 하는 표적 DNA 서열을 함유하는 줄기세포주를 제공하는 단계; (b)목적유전자를 삽입하려는 표적 DNA를 절단할 수 있는 엔도뉴클레아제 도메인 및 줄기세포주에서 표적 DNA 내의 특정 염기서열에 결합하는 다수의 TAL 이펙터 반복 서열을 포함하는 TAL 이펙터 도메인의 융합 도메인인 TALEN 발현벡터를 제공하는 단계; (c) 상기 TALEN이 줄기세포에서 표적 DNA 서열 내부 또는 표적 DNA 서열에 인접한 염기서열의 가닥 모두를 절단할 수 있도록 TALEN 벡터와 줄기세포내의 표적 DNA 서열을 접촉시키는 단계; (d) 상동성 재조합이 표적 DNA 서열사이에서 발생하도록 적어도 표적 DNA의 일부와 상동성인 서열을 포함하는 목적유전자 발현벡터를 제공하는 단계를 포함할 수 있다. 또한 TALEN과 표적 DNA 서열간의 접촉은 전기천공에 의해서 줄기세포로 TALEN 벡터를 도입하는 단계를 포함할 수 있다.The present invention also provides a stem cell line transformed with the recombinant expression vector to express a gene of interest such as an angiogenesis inducing factor. To produce the stem cell line, (a) a target to insert a target gene through homologous recombination Providing a stem cell line containing a DNA sequence; (b) an endonuclease domain capable of cleaving a target DNA into which a target gene is inserted, and a plurality of TAL effector repeat sequences which bind to a specific base sequence in the target DNA in a stem cell line. The fusion domain of the TAL effector, TALEN Providing an expression vector; (c) contacting the TALEN vector with a target DNA sequence in a stem cell so that the TALEN can cleave both the nucleotide sequence adjacent to the target DNA sequence or the target DNA sequence in the stem cell; (d) providing a target gene expression vector comprising a sequence at least homologous to a portion of the target DNA so that homologous recombination occurs between the target DNA sequences. The contact between TALEN and the target DNA sequence may also include introducing the TALEN vector into the stem cells by electroporation.

본 발명에서 상기 줄기세포주는 제대혈 유래 줄기세포(hUCB-MSC cell)일 수 있다.In the present invention, the stem cell line may be a cord blood-derived stem cell (hUCB-MSC cell).

이하 본 발명을 하기 실시예에 의해 보다 상세하게 설명하도록 한다. 다만 이러한 실시예에 의하여 본 발명이 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the present invention is not limited to these examples.

<실시예><Examples>

1. TALEN 시스템의 확인과 이를 위한 재조합 발현 벡터의 제작1. Identification of TALEN system and production of recombinant expression vector for this

본 발명과 관련하여 표적 줄기세포주에서 TALEN 시스템이 작동하는지의 여부를 확인하기 위해 TALEN이 인지할 수 있는 부위가 함유된 혈관성장인자 발현 벡터를 제작하였다. 최종적으로 혈관성장인자 발현 벡터가 특정 조건에서 혈관성장인자의 발현을 유도하고 이에 대한 조절이 가능한지 여부를 확인하였다.
In connection with the present invention, a vascular growth factor expression vector containing a site recognizable by TALEN was constructed to confirm whether the TALEN system works in a target stem cell line. Finally, it was confirmed whether or not the vascular growth factor expression vector induces the expression of angiogenic growth factor under specific conditions and is controllable.

a)pTUC -19 벡터 및 pTUC-19 GFP 벡터의 제조방법 a) pTUC method for producing a vector and -19 pTUC 19-GFP vector

TALEN 시스템에 의한 유전자 삽입 유전자인 HA-L 염기서열 및 HA-R 염기서열을 PCR을 통해 증폭한 다음 제한요소인 kpn1 과 sph1을 이용하여 pUC-19 벡터에 상기 HA-L(=left homology) 및 HA-R(=right homology)를 삽입하여 pTUC-19 벡터를 제작하였다(도2). HA-L 염기서열 및 HA-R 염기서열 사이에는 외부 유전자를 삽입할 수 있도록 Age1 절단부위와 Not1 절단부위를 삽입하였다. 또한 상기 절단부위를 이용하여 GFP 유전자를 삽입하였고 그 결과 pTUC-19 GFP 벡터를 제작하였다.
The HA-L base sequence and the HA-R base sequence, which are gene insertion genes by the TALEN system, are amplified by PCR, and then the HA-L (= left homology) and the HA-L are added to the pUC-19 vector using restriction enzymes kpn1 and sph1 HA-R (= right homology) was inserted to construct a pTUC-19 vector (Fig. 2). The Age1 and Not1 cleavage sites were inserted between the HA-L and HA-R sequences so that an external gene could be inserted. In addition, the GFP gene was inserted using the cleavage site and a pTUC-19 GFP vector was constructed.

b) hUCB-MSC Cell line에서 TALEN System의 작동여부에 대한 확인 b) Confirmation of operation of TALEN System in hUCB-MSC cell line

hUCB-MSC Cell line에서 TALEN이 제대로 작동하는지 여부를 확인하기 위하여 TALEN Left 벡터, TALEN Right 벡터, pTUC-19 GFP 벡터를 1:1:2 의 비율로 전기천공법(electroporation)을 통하여 형질주입(transfection)을 진행하였다. 형질주입 후 GFP의 발현이 12일 후에도 지속되는 것을 확인하고 (도3), 그 후 Junction PCR을 이용하여 세포 내로 도입된 GFP 유전자가 safe harbor 부위에 정상적으로 삽입(integration)되어 있는지를 확인한 결과 정확한 크기의 PCR 밴드가 나타났으며 이로써 GFP유전자 삽입이 정상적으로 유도되었음을 확인하였다. (도4)
The TALEN Left vector, TALEN Right vector, and pTUC-19 GFP vector were transfected by electroporation at a ratio of 1: 1: 2 in order to confirm whether TALEN functioned properly in the hUCB-MSC cell line. Respectively. It was confirmed that the expression of GFP persisted after 12 days of transfection (FIG. 3). After that, it was confirmed that the GFP gene introduced into the cell using Junction PCR was normally inserted into the safe harbor region, PCR bands were detected and it was confirmed that GFP gene insertion was normally induced. (Figure 4)

c) Tet-On system에 의해서 혈관성자인자의 발현이 조절되는Tet-0n/CMV-VEGF -hEF1a-rtTA vector 제작c) Production of Tet-0n / CMV-VEGF-hEF1a-rtTA vector in which expression of vasculature is controlled by Tet-On system

혈관성장인자의 발현 조절을 위한 Tet-on system 을 활용하기 위하여 pTUC-19 벡터에 혈관성장인자 유전자 발현 조절 카세트인 Tet-On/CMV-VEGF-hEF1a-rtTA를 삽입시켜 표적세포주의 염색체에 목적유전자를 삽입할 수 있는 공여벡터를 제작하였다 (도1).
In order to utilize the Tet-on system for the regulation of vascular growth factor expression, Tet-On / CMV-VEGF-hEF1a-rtTA, a vascular growth factor gene expression regulatory cassette, was inserted into the pTUC-19 vector, ( Fig. 1).

<Tet-On/CMV-VEGF-hEF1a-rtTA의 제조방법><Preparation of Tet-On / CMV-VEGF-hEF1a-rtTA>

공여벡터에 삽입될 유전자인 Tet-on/CMV-VEGF-hEF1a-rtTA의 경우 In-fusion 시스템을 도입하여 제작하였다. In-fusion 시스템은 벡터의 선형화(linearization) 단계, PCR을 통한 각 유전자 단편의 증폭 및 정제 단계, In-Fusion 반응 단계를 거쳐서 진행된다 (도5) .Tet-on / CMV-VEGF-hEF1a-rtTA, a gene to be inserted into the donor vector, was prepared by introducing an In-fusion system. The in-fusion system proceeds through a linearization step of the vector, an amplification and purification step of each gene fragment through PCR, and an In-Fusion reaction step (FIG. 5).

먼저 Age1과 EcoR1 제한효소를 통해 벡터의 선형화를 해 준 다음 필요한 각각의 염기서열( Tet-O CMV, VEGF, hEF1a-rtTA )를 PCR을 통해 증폭시켰다.First, the vector was linearized with Age1 and EcoR1 restriction enzymes, and then each of the necessary nucleotide sequences (Tet-O CMV, VEGF and hEF1a-rtTA) was amplified by PCR.

PCR 과정에서 프라이머를 제작할 때는 각각의 염기서열 단편이 서로 15bp씩 겹쳐지도록 제작해야 하며 그리고 각각의 다른 벡터에 들어 있던 삽입유전자를 증폭시키기 위하여 다음과 같은 프라이머를 사용하였다. When preparing primers in the PCR, each of the base sequence fragments should be overlapped with each other by 15 bp, and the following primers were used to amplify the inserted gene in each other vector.

Tet-Ocmv의 프라이머로서 Fragment 1 FW Primer (F1_FW): TTGGGCCCGACGTCGCATGCCGCGGCCGCTTAATTAAC Tm : 124 GC : 63.2 % (염기서열번호 12)Fragment 1 FW Primer (F1_FW) as a primer of Tet-Ocmv: TTGGGCCCGACGTCGCATGCCGCGGCCGCTTAATTAAC Tm: 124 GC: 63.2% (nucleotide sequence number 12)

Fragment 1 RV Primer (F1_RV): AATTAAGCGGCCGCGGATGGCCGCCACCGCGGA Tm : 112 GC : 69.7 % (염기서열번호 13)를 사용하였고,Fragment 1 RV Primer (F1_RV): AATTAAGCGGCCGCGGATGGCCGCCACCGCGGA Tm: 112 GC: 69.7% (SEQ ID NO: 13)

VEGF 의 프라이머로서 Fragment 2 FW Primer (F2_FW) : CGCGGCCGCTTAATTAAC Tm : 56 GC : 55.6 % (염기서열번호 14)Fragment 2 FW Primer (F2_FW) as a primer of VEGF: CGCGGCCGCTTAATTAAC Tm: 56 GC: 55.6% (nucleotide sequence number 14)

Fragment 2 RV Primer (F2_RV) CGATCGCAGATCCTTGATGGCCGCCACCGCGGA Tm : 110 GC : 66.7 % (염기서열번호 15)를 사용하였으며,Fragment 2 RV Primer (F2_RV) CGATCGCAGATCCTTGATGGCCGCCACCGCGGA Tm: 110 GC: 66.7% (nucleotide sequence number 15)

hEF1a-rtTA의 프라이머로서 Fragment 3 FW Primer (F3_FW): AAGGATCTGCGATCGCTC Tm : 56 GC : 55.6 % (염기서열번호 16)Fragment 3 FW Primer (F3_FW) as a primer of hEF1a-rtTA: AAGGATCTGCGATCGCTC Tm: 56 GC: 55.6% (nucleotide sequence number 16)

Fragment 3 RV Primer (F3_RV) GGGAGCTCTCCCATATGGCGGCCGCAAAAAACCTC Tm : 112 GC : 60 % (염기서열번호 17)를 사용하였다.Fragment 3 RV Primer (F3_RV) GGGAGCTCTCCCATATGGCGGCCGCAAAAAACCTC Tm: 112 GC: 60% (nucleotide sequence number 17) was used.

마지막으로 선형화된 발현벡터와 In-Fusion 효소를 함께 클로닝 반응을 통해 pTUC19-VEGF 벡터를 제작 하였다. VEGF 삽입체 부분의 경우 직접 서열합성을 통하여 제작하였으며, Tet-O CMV, hEF1a-rtTA 삽입유전자 부분의 경우 다른 벡터에 들어가 있는 부분을 프라이머만 제작하여 단편을 증폭시켜 제작하였다. 이렇게 제작된 삽입유전자 (Tet-on-CMV-VEGF-hEF1a-rtTA)는 age1, not1 제한효소를 통해 TALEN시스템의 공여벡터인 pTUC19 Vector에 옮겨져 pTUC19-TetOCMV-VEGF-hEF1a-rtTA 재조합 발현백터를 제작 하였다.Finally, the pTUC19-VEGF vector was constructed by cloning the linearized expression vector and the In-Fusion enzyme together. VEGF inserts were constructed by direct sequence synthesis. For the Tet-O CMV and hEF1a-rtTA insert gene fragments, only the primer in the other vector was prepared and amplified. The constructed transgene (Tet-on-CMV-VEGF-hEF1a-rtTA) was transferred to the pTUC19 Vector, a donor vector of the TALEN system via age1 and not1 restriction enzymes, to construct a recombinant expression vector pTUC19-TetOCMV-VEGF-hEF1a-rtTA Respectively.

d) 제작된 Tet-on system에 의해 혈관성장인자 발현이 조절되는 벡터가 도입된 세포에서의 특정 조건에서 혈관성장인자 발현 평가d) Assessment of vascular growth factor expression under specific conditions in cells transfected with a vector whose vascular growth factor expression is regulated by the constructed Tet-on system

완성된 pTUC19-TetOCMV-VEGF-hEF1a-rtTA 재조합 발현백터가 도입된 세포에서 독시사이클린(doxycycline, Dox)에 의해 혈관형성 유도물질(VEGF)의 발현이 조절 된다는 것을 증명하기 위해 hUCB-MSC cell에 전기천공(electroporation)을 통해 상기 pTUC19-TetOCMV-VEGF-hEF1a-rtTA 재조합 발현백터를 형질주입 시킨 후, 이틀 후 단백질 수준에서 증명하기 위하여 프로테아제 억제효소(protease inhibitor)를 첨가한 RIPA 버퍼를 사용하여 단백질을 회수하였다. 이후 대조군, 형질주입시킨 군, 독시사이클린을 처리한 군등 모두 3군을 웨스턴 블로트 분석(western blot)를 통한 정량방법으로 동일한 양의 단백질을 사용하여, 베타 액틴(beta actin)의 발현을 확인하고 VEGF 의 발현 양상을 서로 비교하였다. 그 결과 독시사이클린이 처리된 군에서 VEGF의 증가된 발현이 관찰되어 Tet-on system에 의하여 혈관성장인자의 발현 조절이 가능함을 입증하였다(도6).
To demonstrate that the expression of angiogenesis inducing substance (VEGF) is regulated by doxycycline (Dox) in the cells transfected with the recombinant expression vector pTUC19-TetOCMV-VEGF-hEF1a-rtTA completed, the hUCB- After transfection of the recombinant expression vector pTUC19-TetOCMV-VEGF-hEF1a-rtTA by electroporation, the protein was recovered using a RIPA buffer supplemented with a protease inhibitor to prove it at the protein level two days later Respectively. Subsequently, expression of beta actin was confirmed using the same amount of protein by Western blotting in all three groups (control, transfected, and treated with doxycycline), and VEGF Were compared with each other. As a result, increased expression of VEGF was observed in the treated group with doxycycline, and the Tet-on system It is possible to control the expression of vascular growth factors (Fig. 6).

<실험예><Experimental Example>

1. Inducible VEGF Secreting Cell line 제작1. Manufacture of Inducible VEGF Secreting Cell line

본 발명과 관련하여 표적세포주의 염색체에 삽입된 혈관성장인자 발현벡터에 대한 직접적 검증 실험을 진행하였다.
In accordance with the present invention, a direct validation experiment of an angiogenic factor expression vector inserted into the chromosome of the target cell line was carried out.

a) 표적세포 염색체의 safe harbor 부위 (AAVS1) 로의 삽입a) Insertion of the target cell chromosome into the safe harbor site (AAVS1)

hUCB-MSC Cell line 100 mm Plate ( 1x 10⁶ 개의 hUCB-MSC ) 에 TALEN L 벡터,TALEN R 벡터,Ptuc19-TetOCMV-VEGF-hEF1a-rtTA 벡터(농도: 1ug/ul)를 3ug : 3ug : 6ug 의 비율로 Buffer R 200ul 에 넣어주었다.이때 각각의 벡터는 모두 모두 500ng/ul 이상의 농도가 되어야 한다. 상기 벡터들은 NEON 형질주입 시스템을 사용하여 전기천공(electroporation)(조건: Pulse Voltage : 1300, Pulse Width : 20, Pulse No :2)을 통해 동반 형질주입(co-Transfection)을 진행하였고 이후에 100 mm 플레이트 2개에 나누어 분주하였다. 14일 뒤 safe harbor 부위인 인간염색체19 의 AAVS1 부위에 혈관성장인자 발현 카세트가 정상적으로 삽입되었는지를 확인하였다. 이를 위해, cell line으로부터 genomic DNA를 추출 한 뒤 Junction PCR 을 진행하였다. (PCR에 사용된 프라이머의 경우 AAVS1 F , VEGF R 혹은 rtTA F, AAVS1 R 부위로부터 설계하였다.(도 7)
3 ug: 3 ug: 6 ug of TALEN L vector, TALEN R vector, Ptuc19-TetOCMV-VEGF-hEF1a-rtTA vector (concentration: 1 ug / ul) was added to hUCB-MSC Cell line 100 mm plate (1x10 ⁶ hUCB- In Buffer R 200ul at a concentration of 500ng / ul or higher. These vectors were subjected to co-transfection using electroporation (condition: pulse voltage: 1300, pulse width: 20, pulse no. 2) using NEON transfection system, The plates were divided into two. After 14 days, it was confirmed whether the cassette for the expression of an angiogenic factor was normally inserted into the AAVS1 region of the human chromosome 19, which is the safe harbor site. For this purpose, genomic DNA was extracted from the cell line and then subjected to Junction PCR. (Primers used for PCR were designed from AAVS1 F, VEGF R or rtTA F and AAVS1 R sites (Figure 7)

b) TALEN 에 의해 integration 된 Tet-OnCMV-VEGF-hEF1a-rtTA 의 작동여부 확인 b) Confirmation of whether Tet-OnCMV-VEGF-hEF1a-rtTA integrated by TALEN works

AAVS1 부위에 삽입된 Tet-OnCMV-VEGF-hEF1a-rtTA에 의해서 VEGF의 발현이 일시적 발현이 아닌 영구적 발현여부를 확인하기 위해 위와 동일한 방법으로 동반 형질주입(co-Transfection)시켜 준 후 14일후 RIPA 버퍼를 활용하여 Protein을 회수 한 후 브래드포드 분석(bradford assay)를 통한 정량방법으로 동일한 양의 단백질을 사용하여 독시사이클린 처리를 통해 VEGF의 분비를 조절할 수 있음을 증명하였다. (도 8)
The expression of VEGF was transiently expressed by Tet-OnCMV-VEGF-hEF1a-rtTA inserted in the AAVS1 region, and co-transfection was carried out in the same manner as described above to confirm the expression of VEGF, , We demonstrated that the secretion of VEGF can be regulated through the use of the same amount of protein by the quantification method using the bradford assay after recovery of the protein using doxycycline. (Fig. 8)

c) Tet-OnCMV-VEGF-hEF1a-rtTA vector가 삽입된 줄기세포에서 세포 외로 분비된 단백질의 양적 확인 c) quantitative confirmation of extracellular secreted proteins from stem cells with Tet-OnCMV-VEGF-hEF1a-rtTA vector inserted

혈관형성유도물질인 VEGF의 치료 효과를 최대화 하면서 반대로 혈관종(hemangioma) 문제는 해결하기 위하여 유도 단백질의 양적, 농도 및 양적 확인을 위하여 ELISA 실험을 수행하였다. VEGF 유전자가 삽입된 표적세포에 독시사이클린을 처리할때 발현되는 VEGF의 양을 비교분석 하였다(도9). 그 결과, 벡터가 삽입된 세포에서 독시사이클린이 처리되었을 경우 VEGF의 발현이 다른 대조군들에 비해 증가하는 것을 확인하였다. 이 결과를 바탕으로 Tet-On 시스템이 삽입된 후에도 VEGF 유전자가 확실하게 작동한다는 것을 검증하였으며, VEGF 농도가 일차적으로 형질주입(transfection)시켰을 때의 농도와 크게 차이가 없고 최적화된 독시사이클린의 처리를 통해 혈관성장인자인 VEGF의 발현을 조절할 수 있음을 확인하였다.
In order to solve the hemangioma problem, an ELISA experiment was conducted to confirm the quantitative, concentration and quantitative determination of the inducible protein while maximizing the therapeutic effect of VEGF, an angiogenesis inducing substance. The amount of VEGF expressed when treating the target cell into which the VEGF gene was inserted was analyzed (Fig. 9). As a result, it was confirmed that the expression of VEGF was increased when the vector was treated with doxycycline in comparison with the other control groups. Based on these results, it was verified that the VEGF gene works well after the insertion of the Tet-On system, and the VEGF concentration is not significantly different from the concentration when the transfection is performed first, and the optimized treatment with doxycycline It is possible to control the expression of VEGF, an angiogenic factor.

<110> seoul national university <120> Recomnication expression vector of vascular growth factor <130> 14P1128 <160> 17 <170> KopatentIn 2.0 <210> 1 <211> 7078 <212> DNA <213> Artificial Sequence <220> <223> artificial sequence <400> 1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgagctcggt accctcttcc 420 ccttgctctc tgctgtgttg ctgcccaagg atgctctttc cggagcactt ccttctcggc 480 gctgcaccac gtgatgtcct ctgagcggat cctccccgtg tctgggtcct ctccgggcat 540 ctctcctccc tcacccaacc ccatgccgtc ttcactcgct gggttccctt ttccttctcc 600 ttctggggcc tgtgccatct ctcgtttctt aggatggcct tctccgacgg atgtctccct 660 tgcgtcccgc ctccccttct tgtaggcctg catcatcacc gtttttctgg acaaccccaa 720 agtaccccgt ctccctggct ttagccacct ctccatcctc ttgctttctt tgcctggaca 780 ccccgttctc ctgtggattc gggtcacctc tcactccttt catttgggca gctcccctac 840 cccccttacc tctctagtct gtgctagctc ttccagcccc ctgtcatggc atcttccagg 900 ggtccgagag ctcagctagt cttcttcctc caacccgggc ccctatgtcc acttcaggac 960 agcatgtttg ctgcctccag ggatcctgtg tccccgagct gggaccacct tatattccca 1020 gggccggtta atgtggctct ggttctgggt acttttatct gtcccctcca ccccacagtg 1080 gggccactag ggacaggatt ggtgacagaa aagccccatc cttaggcctc ctccttccta 1140 gtctcctgat attgggtcta acccccacct cctgttaggc agattcctta tctggtgaca 1200 cacccccatt tccaccggtt taattaactc gagggatcca agctggccgc acgtctccct 1260 atcagtgata gagaagtcga cacgtctcga gctccctatc agtgatagag aaggtacgtc 1320 tagaacgtct ccctatcagt gatagagaag tcgacacgtc tcgagctccc tatcagtgat 1380 agagaaggta cgtctagaac gtctccctat cagtgataga gaagtcgaca cgtctcgagc 1440 tccctatcag tgatagagaa ggtacgtcta gaacgtctcc ctatcagtga tagagaagtc 1500 gacacgtctc gagctcccta tcagtgatag agaaggtacc ccctatataa gcagagctcg 1560 tttagtgaac cgtcagatcg cctggagacg ccatccacgc tgttttgacc tccatagaag 1620 acaccgggac cgatccagcc tccgcggtgg cggccatcac catgaacttt ctgctgtctt 1680 gggtgcattg gagccttgcc ttgctgctct acctccacca tgccaagtgg tcccaggctg 1740 cacccatggc agaaggagga gggcagaatc atcacgaagt ggtgaagttc atggatgtct 1800 atcagcgcag ctactgccat ccaatcgaga ccctggtgga catcttccag gagtaccctg 1860 atgagatcga gtacatcttc aagccatcct gtgtgcccct gatgcgatgc gggggctgct 1920 gcaatgacga gggcctggag tgtgtgccca ctgaggagtc caacatcacc atgcagatta 1980 tgcggatcaa acctcaccaa ggccagcaca taggagagat gagcttccta cagcacaaca 2040 aatgtgaatg cagaccaaag aaagatagag caagacaaga aaatccctgt gggccttgct 2100 cagagcggag aaagcatttg tttgtacaag atccgcagac gtgtaaatgt tcctgcaaaa 2160 acacagactc gcgttgcaag gcgaggcagc ttgagttaaa cgaacgtact tgcagatgtg 2220 acaagccgag gcgggactac aaagacgatg acgacaagtg actgtgcctt ctagttgcca 2280 gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac 2340 tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat 2400 tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca 2460 tgctggggat gcggtgggct ctatggaagg atctgcgatc gctccggtgc ccgtcagtgg 2520 gcagagcgca catcgcccac agtccccgag aagttggggg gaggggtcgg caattgaacg 2580 ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg atgtcgtgta ctggctccgc 2640 ctttttcccg agggtggggg agaaccgtat ataagtgcag tagtcgccgt gaacgttctt 2700 tttcgcaacg ggtttgccgc cagaacacag ctgaagcttc gaggggctcg catctctcct 2760 tcacgcgccc gccgccctac ctgaggccgc catccacgcc ggttgagtcg cgttctgccg 2820 cctcccgcct gtggtgcctc ctgaactgcg tccgccgtct aggtaagttt aaagctcagg 2880 tcgagaccgg gcctttgtcc ggcgctccct tggagcctac ctagactcag ccggctctcc 2940 acgctttgcc tgaccctgct tgctcaactc tacgtctttg tttcgttttc tgttctgcgc 3000 cgttacagat ccaagctgtg accggcgcct acatgtctag actggacaag agcaaagtca 3060 taaacggcgc tctggaatta ctcaatggag tcggtatcga aggcctgacg acaaggaaac 3120 tcgctcaaaa gctgggagtt gagcagccta ccctgtactg gcacgtgaag aacaagcggg 3180 ccctgctcga tgccctgcca atcgagatgc tggacaggca tcatacccac ttctgccccc 3240 tggaaggcga gtcatggcaa gactttctgc ggaacaacgc caagtcattc cgctgtgctc 3300 tcctctcaca tcgcgacggg gctaaagtgc atctcggcac ccgcccaaca gagaaacagt 3360 acgaaaccct ggaaaatcag ctcgcgttcc tgtgtcagca aggcttctcc ctggagaacg 3420 cactgtacgc tctgtccgcc gtgggccact ttacactggg ctgcgtattg gaggaacagg 3480 agcatcaagt agcaaaagag gaaagagaga cacctaccac cgattctatg cccccacttc 3540 tgagacaagc aattgagctg ttcgaccggc agggagccga acctgccttc cttttcggcc 3600 tggaactaat catatgtggc ctggagaaac agctaaagtg cgaaagcggc gggccggccg 3660 acgcccttga cgattttgac ttagacatgc tcccagccga tgcccttgac gactttgacc 3720 ttgatatgct gcctgctgac gctcttgacg attttgacct tgacatgctc cccgggtaac 3780 taagtaagga tccagacatg ataagataca ttgatgagtt tggacaaacc acaactagaa 3840 tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta tttgtaacca 3900 ttataagctg caataaacaa gttaacaaca acaattgcat tcattttatg tttcaggttc 3960 agggggaggt gtgggaggtt ttttgaattc tcatgatatc ttccgtcgac gctctctaga 4020 gctagcggcc gcgccagaga ggatcctggg agggagagct tggcaggggg tgggagggaa 4080 gggggggatg cgtgacctgc ccggttctca gtggccaccc tgcgctaccc tctcccagaa 4140 cctgagctgc tctgacgcgg ccgtctggtg cgtttcactg atcctggtgc tgcagcttcc 4200 ttacacttcc caagaggaga agcagtttgg aaaaacaaaa tcagaataag ttggtcctga 4260 gttctaactt tggctcttca cctttctagt ccccaattta tattgttcct ccgtgcgtca 4320 gttttacctg tgagataagg ccagtagcca gccccgtcct ggcagggctg tggtgaggag 4380 gggggtgtcc gtgtggaaaa ctccctttgt gagaatggtg cgtcctaggt gttcaccagg 4440 tcgtggccgc ctctactccc tttctctttc tccatccttc tttccttaaa gagtccccag 4500 tgctatctgg gacatattcc tccgcccaga gcagggtccc gcttccctaa ggccctgctc 4560 tgggcttctg ggtttgagtc cttggcaagc ccaggagagg cgctcaggct tccctgtccc 4620 ccttcctcgt ccaccatctc atgcccctgg ctctcctgcc ccttccctac aggggttcct 4680 ggctctgctc ttcagactga gccccgttcc cctgcatccc cgttcccctg catccccctt 4740 cccctgcatc ccccagaggc cccaggccac ctacttggcc tggaccccac gagaggccac 4800 cccagccctg tctaccaggc tgccttttgg gtgcatgcaa gcttggcgta atcatggtca 4860 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga 4920 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg 4980 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc 5040 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac 5100 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata 5160 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa 5220 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct 5280 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa 5340 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg 5400 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca 5460 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa 5520 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg 5580 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg 5640 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg 5700 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc 5760 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag 5820 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac 5880 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc 5940 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag 6000 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt 6060 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag 6120 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca 6180 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact 6240 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca 6300 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg 6360 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc 6420 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg 6480 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca 6540 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt 6600 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc 6660 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc 6720 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca 6780 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa 6840 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat 6900 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa 6960 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtctaagaa 7020 accattatta tcatgacatt aacctataaa aataggcgta tcacgaggcc ctttcgtc 7078 <210> 2 <211> 800 <212> DNA <213> Artificial Sequence <220> <223> TALEN left Homology <400> 2 ctcttcccct tgctctctgc tgtgttgctg cccaaggatg ctctttccgg agcacttcct 60 tctcggcgct gcaccacgtg atgtcctctg agcggatcct ccccgtgtct gggtcctctc 120 cgggcatctc tcctccctca cccaacccca tgccgtcttc actcgctggg ttcccttttc 180 cttctccttc tggggcctgt gccatctctc gtttcttagg atggccttct ccgacggatg 240 tctcccttgc gtcccgcctc cccttcttgt aggcctgcat catcaccgtt tttctggaca 300 accccaaagt accccgtctc cctggcttta gccacctctc catcctcttg ctttctttgc 360 ctggacaccc cgttctcctg tggattcggg tcacctctca ctcctttcat ttgggcagct 420 cccctacccc ccttacctct ctagtctgtg ctagctcttc cagccccctg tcatggcatc 480 ttccaggggt ccgagagctc agctagtctt cttcctccaa cccgggcccc tatgtccact 540 tcaggacagc atgtttgctg cctccaggga tcctgtgtcc ccgagctggg accaccttat 600 attcccaggg ccggttaatg tggctctggt tctgggtact tttatctgtc ccctccaccc 660 cacagtgggg ccactaggga caggattggt gacagaaaag ccccatcctt aggcctcctc 720 cttcctagtc tcctgatatt gggtctaacc cccacctcct gttaggcaga ttccttatct 780 ggtgacacac ccccatttcc 800 <210> 3 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> tet operator <400> 3 tccctatcag tgatagaga 19 <210> 4 <211> 106 <212> DNA <213> Artificial Sequence <220> <223> CMV promotor <400> 4 cctatataag cagagctcgt ttagtgaacc gtcagatcgc ctggagacgc catccacgct 60 gttttgacct ccatagaaga caccgggacc gatccagcct ccgcgg 106 <210> 5 <211> 576 <212> DNA <213> Artificial Sequence <220> <223> vegf <400> 5 accatgaact ttctgctgtc ttgggtgcat tggagccttg ccttgctgct ctacctccac 60 catgccaagt ggtcccaggc tgcacccatg gcagaaggag gagggcagaa tcatcacgaa 120 gtggtgaagt tcatggatgt ctatcagcgc agctactgcc atccaatcga gaccctggtg 180 gacatcttcc aggagtaccc tgatgagatc gagtacatct tcaagccatc ctgtgtgccc 240 ctgatgcgat gcgggggctg ctgcaatgac gagggcctgg agtgtgtgcc cactgaggag 300 tccaacatca ccatgcagat tatgcggatc aaacctcacc aaggccagca cataggagag 360 atgagcttcc tacagcacaa caaatgtgaa tgcagaccaa agaaagatag agcaagacaa 420 gaaaatccct gtgggccttg ctcagagcgg agaaagcatt tgtttgtaca agatccgcag 480 acgtgtaaat gttcctgcaa aaacacagac tcgcgttgca aggcgaggca gcttgagtta 540 aacgaacgta cttgcagatg tgacaagccg aggcgg 576 <210> 6 <211> 546 <212> DNA <213> Artificial Sequence <220> <223> Human elongation factor-1 alpha <400> 6 aaggatctgc gatcgctccg gtgcccgtca gtgggcagag cgcacatcgc ccacagtccc 60 cgagaagttg gggggagggg tcggcaattg aacgggtgcc tagagaaggt ggcgcggggt 120 aaactgggaa agtgatgtcg tgtactggct ccgccttttt cccgagggtg ggggagaacc 180 gtatataagt gcagtagtcg ccgtgaacgt tctttttcgc aacgggtttg ccgccagaac 240 acagctgaag cttcgagggg ctcgcatctc tccttcacgc gcccgccgcc ctacctgagg 300 ccgccatcca cgccggttga gtcgcgttct gccgcctccc gcctgtggtg cctcctgaac 360 tgcgtccgcc gtctaggtaa gtttaaagct caggtcgaga ccgggccttt gtccggcgct 420 cccttggagc ctacctagac tcagccggct ctccacgctt tgcctgaccc tgcttgctca 480 actctacgtc tttgtttcgt tttctgttct gcgccgttac agatccaagc tgtgaccggc 540 gcctac 546 <210> 7 <211> 747 <212> DNA <213> Artificial Sequence <220> <223> rtTA <400> 7 atgtctagac tggacaagag caaagtcata aacggcgctc tggaattact caatggagtc 60 ggtatcgaag gcctgacgac aaggaaactc gctcaaaagc tgggagttga gcagcctacc 120 ctgtactggc acgtgaagaa caagcgggcc ctgctcgatg ccctgccaat cgagatgctg 180 gacaggcatc atacccactt ctgccccctg gaaggcgagt catggcaaga ctttctgcgg 240 aacaacgcca agtcattccg ctgtgctctc ctctcacatc gcgacggggc taaagtgcat 300 ctcggcaccc gcccaacaga gaaacagtac gaaaccctgg aaaatcagct cgcgttcctg 360 tgtcagcaag gcttctccct ggagaacgca ctgtacgctc tgtccgccgt gggccacttt 420 acactgggct gcgtattgga ggaacaggag catcaagtag caaaagagga aagagagaca 480 cctaccaccg attctatgcc cccacttctg agacaagcaa ttgagctgtt cgaccggcag 540 ggagccgaac ctgccttcct tttcggcctg gaactaatca tatgtggcct ggagaaacag 600 ctaaagtgcg aaagcggcgg gccggccgac gcccttgacg attttgactt agacatgctc 660 ccagccgatg cccttgacga ctttgacctt gatatgctgc ctgctgacgc tcttgacgat 720 tttgaccttg acatgctccc cgggtaa 747 <210> 8 <211> 800 <212> DNA <213> Artificial Sequence <220> <223> TALEN right Homology <400> 8 gccagagagg atcctgggag ggagagcttg gcagggggtg ggagggaagg gggggatgcg 60 tgacctgccc ggttctcagt ggccaccctg cgctaccctc tcccagaacc tgagctgctc 120 tgacgcggcc gtctggtgcg tttcactgat cctggtgctg cagcttcctt acacttccca 180 agaggagaag cagtttggaa aaacaaaatc agaataagtt ggtcctgagt tctaactttg 240 gctcttcacc tttctagtcc ccaatttata ttgttcctcc gtgcgtcagt tttacctgtg 300 agataaggcc agtagccagc cccgtcctgg cagggctgtg gtgaggaggg gggtgtccgt 360 gtggaaaact ccctttgtga gaatggtgcg tcctaggtgt tcaccaggtc gtggccgcct 420 ctactccctt tctctttctc catccttctt tccttaaaga gtccccagtg ctatctggga 480 catattcctc cgcccagagc agggtcccgc ttccctaagg ccctgctctg ggcttctggg 540 tttgagtcct tggcaagccc aggagaggcg ctcaggcttc cctgtccccc ttcctcgtcc 600 accatctcat gcccctggct ctcctgcccc ttccctacag gggttcctgg ctctgctctt 660 cagactgagc cccgttcccc tgcatccccg ttcccctgca tcccccttcc cctgcatccc 720 ccagaggccc caggccacct acttggcctg gaccccacga gaggccaccc cagccctgtc 780 taccaggctg ccttttgggt 800 <210> 9 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> left recognition site <400> 9 atctggtgac acacccccat ttcc 24 <210> 10 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> right recognition site <400> 10 gccagagagg atcctgggag gg 22 <210> 11 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> talen cleavage site <400> 11 tggagccatc tctctccttg ccagaacctc taaggtttgc ttacgatgga 50 <210> 12 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> Fragment 1 FW Primer <400> 12 ttgggcccga cgtcgcatgc cgcggccgct taattaac 38 <210> 13 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Fragment 1 RV Primer <400> 13 aattaagcgg ccgcggatgg ccgccaccgc gga 33 <210> 14 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Fragment 2 FW Primer <400> 14 cgcggccgct taattaac 18 <210> 15 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Fragment 2 RV Primer <400> 15 cgatcgcaga tccttgatgg ccgccaccgc gga 33 <210> 16 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Fragment 3 FW Primer <400> 16 aaggatctgc gatcgctc 18 <210> 17 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Fragment 3 RV Primer <400> 17 gggagctctc ccatatggcg gccgcaaaaa acctc 35 <110> seoul national university <120> Recomnication expression vector of vascular growth factor <130> 14P1128 <160> 17 <170> Kopatentin 2.0 <210> 1 <211> 7078 <212> DNA <213> Artificial Sequence <220> Artificial sequence <400> 1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgagctcggt accctcttcc 420 ccttgctctc tgctgtgttg ctgcccaagg atgctctttc cggagcactt ccttctcggc 480 gctgcaccac gtgatgtcct ctgagcggat cctccccgtg tctgggtcct ctccgggcat 540 ctctcctccc tcacccaacc ccatgccgtc ttcactcgct gggttccctt ttccttctcc 600 ttctggggcc tgtgccatct ctcgtttctt aggatggcct tctccgacgg atgtctccct 660 tgcgtcccgc ctccccttct tgtaggcctg catcatcacc gtttttctgg acaaccccaa 720 agtaccccgt ctccctggct ttagccacct ctccatcctc ttgctttctt tgcctggaca 780 ccccgttctc ctgtggattc gggtcacctc tcactccttt catttgggca gctcccctac 840 cccccttacc tctctagtct gtgctagctc ttccagcccc ctgtcatggc atcttccagg 900 ggtccgagag ctcagctagt cttcttcctc caacccgggc ccctatgtcc acttcaggac 960 agcatgtttg ctgcctccag ggatcctgtg tccccgagct gggaccacct tatattccca 1020 gggccggtta atgtggctct ggttctgggt acttttatct gtcccctcca ccccacagtg 1080 gggccactag ggacaggatt ggtgacagaa aagccccatc cttaggcctc ctccttccta 1140 gtctcctgat attgggtcta acccccacct cctgttaggc agattcctta tctggtgaca 1200 cacccccatt tccaccggtt taattaactc gagggatcca agctggccgc acgtctccct 1260 atcagtgata gagaagtcga cacgtctcga gctccctatc agtgatagag aaggtacgtc 1320 tagaacgtct ccctatcagt gatagagaag tcgacacgtc tcgagctccc tatcagtgat 1380 agagaaggta cgtctagaac gtctccctat cagtgataga gaagtcgaca cgtctcgagc 1440 tccctatcag tgatagagaa ggtacgtcta gaacgtctcc ctatcagtga tagagaagtc 1500 gacacgtctc gagctcccta tcagtgatag agaaggtacc ccctatataa gcagagctcg 1560 tttagtgaac cgtcagatcg cctggagacg ccatccacgc tgttttgacc tccatagaag 1620 acaccgggac cgatccagcc tccgcggtgg cggccatcac catgaacttt ctgctgtctt 1680 gggtgcattg gagccttgcc ttgctgctct acctccacca tgccaagtgg tcccaggctg 1740 cacccatggc agaaggagga gggcagaatc atcacgaagt ggtgaagttc atggatgtct 1800 atcagcgcag ctactgccat ccaatcgaga ccctggtgga catcttccag gagtaccctg 1860 atgagatcga gtacatcttc aagccatcct gtgtgcccct gatgcgatgc gggggctgct 1920 gcaatgacga gggcctggag tgtgtgccca ctgaggagtc caacatcacc atgcagatta 1980 tgcggatcaa acctcaccaa ggccagcaca taggagagat gagcttccta cagcacaaca 2040 aatgtgaatg cagaccaaag aaagatagag caagacaaga aaatccctgt gggccttgct 2100 cagagcggag aaagcatttg tttgtacaag atccgcagac gtgtaaatgt tcctgcaaaa 2160 acacagactc gcgttgcaag gcgaggcagc ttgagttaaa cgaacgtact tgcagatgtg 2220 acaagccgag gcgggactac aaagacgatg acgacaagtg actgtgcctt ctagttgcca 2280 gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac 2340 tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat 2400 tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca 2460 tgctggggat gcggtgggct ctatggaagg atctgcgatc gctccggtgc ccgtcagtgg 2520 gcagagcgca catcgcccac agtccccgag aagttggggg gaggggtcgg caattgaacg 2580 ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg atgtcgtgta ctggctccgc 2640 ctttttcccg agggtggggg agaaccgtat ataagtgcag tagtcgccgt gaacgttctt 2700 tttcgcaacg ggtttgccgc cagaacacag ctgaagcttc gaggggctcg catctctcct 2760 tcacgcgccc gccgccctac ctgaggccgc catccacgcc ggttgagtcg cgttctgccg 2820 cctcccgcct gtggtgcctc ctgaactgcg tccgccgtct aggtaagttt aaagctcagg 2880 tcgagaccgg gcctttgtcc ggcgctccct tggagcctac ctagactcag ccggctctcc 2940 acgctttgcc tgaccctgct tgctcaactc tacgtctttg tttcgttttc tgttctgcgc 3000 cgttacagat ccaagctgtg accggcgcct acatgtctag actggacaag agcaaagtca 3060 taaacggcgc tctggaatta ctcaatggag tcggtatcga aggcctgacg acaaggaaac 3120 tcgctcaaaa gctgggagtt gagcagccta ccctgtactg gcacgtgaag aacaagcggg 3180 ccctgctcga tgccctgcca atcgagatgc tggacaggca tcatacccac ttctgccccc 3240 tggaaggcga gtcatggcaa gactttctgc ggaacaacgc caagtcattc cgctgtgctc 3300 tcctctcaca tcgcgacggg gctaaagtgc atctcggcac ccgcccaaca gagaaacagt 3360 acgaaaccct ggaaaatcag ctcgcgttcc tgtgtcagca aggcttctcc ctggagaacg 3420 cactgtacgc tctgtccgcc gtgggccact ttacactggg ctgcgtattg gaggaacagg 3480 agcatcaagt agcaaaagag gaaagagaga cacctaccac cgattctatg cccccacttc 3540 tgagacaagc aattgagctg ttcgaccggc agggagccga acctgccttc cttttcggcc 3600 tggaactaat catatgtggc ctggagaaac agctaaagtg cgaaagcggc gggccggccg 3660 acgcccttga cgattttgac ttagacatgc tcccagccga tgcccttgac gactttgacc 3720 ttgatatgct gcctgctgac gctcttgacg attttgacct tgacatgctc cccgggtaac 3780 taagtaagga tccagacatg ataagataca ttgatgagtt tggacaaacc acaactagaa 3840 tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta tttgtaacca 3900 ttataagctg caataaacaa gttaacaaca acaattgcat tcattttatg tttcaggttc 3960 agggggaggt gtgggaggtt ttttgaattc tcatgatatc ttccgtcgac gctctctaga 4020 gctagcggcc gcgccagaga ggatcctggg agggagagct tggcaggggg tgggagggaa 4080 gggggggatg cgtgacctgc ccggttctca gtggccaccc tgcgctaccc tctcccagaa 4140 cctgagctgc tctgacgcgg ccgtctggtg cgtttcactg atcctggtgc tgcagcttcc 4200 ttacacttcc caagaggaga agcagtttgg aaaaacaaaa tcagaataag ttggtcctga 4260 gttctaactt tggctcttca cctttctagt ccccaattta tattgttcct ccgtgcgtca 4320 gttttacctg tgagataagg ccagtagcca gccccgtcct ggcagggctg tggtgaggag 4380 gggggtgtcc gtgtggaaaa ctccctttgt gagaatggtg cgtcctaggt gttcaccagg 4440 tcgtggccgc ctctactccc tttctctttc tccatccttc tttccttaaa gagtccccag 4500 tgctatctgg gacatattcc tccgcccaga gcagggtccc gcttccctaa ggccctgctc 4560 tgggcttctg ggtttgagtc cttggcaagc ccaggagagg cgctcaggct tccctgtccc 4620 ccttcctcgt ccaccatctc atgcccctgg ctctcctgcc ccttccctac aggggttcct 4680 ggctctgctc ttcagactga gccccgttcc cctgcatccc cgttcccctg catccccctt 4740 cccctgcatc ccccagaggc cccaggccac ctacttggcc tggaccccac gagaggccac 4800 cccagccctg tctaccaggc tgccttttgg gtgcatgcaa gcttggcgta atcatggtca 4860 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga 4920 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg 4980 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc 5040 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac 5100 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata 5160 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa 5220 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct 5280 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa 5340 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg 5400 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca 5460 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa 5520 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg 5580 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg 5640 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg 5700 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc 5760 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag 5820 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac 5880 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc 5940 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag 6000 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt 6060 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag 6120 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca 6180 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact 6240 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca 6300 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg 6360 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc 6420 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg 6480 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca 6540 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt 6600 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc 6660 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc 6720 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca 6780 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa 6840 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat 6900 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa 6960 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtctaagaa 7020 accattatta tcatgacatt aacctataaa aataggcgta tcacgaggcc ctttcgtc 7078 <210> 2 <211> 800 <212> DNA <213> Artificial Sequence <220> <223> TALEN left Homology <400> 2 ctcttcccct tgctctctgc tgtgttgctg cccaaggatg ctctttccgg agcacttcct 60 tctcggcgct gcaccacgtg atgtcctctg agcggatcct ccccgtgtct gggtcctctc 120 cgggcatctc tcctccctca cccaacccca tgccgtcttc actcgctggg ttcccttttc 180 cttctccttc tggggcctgt gccatctctc gtttcttagg atggccttct ccgacggatg 240 tctcccttgc gtcccgcctc cccttcttgt aggcctgcat catcaccgtt tttctggaca 300 accccaaagt accccgtctc cctggcttta gccacctctc catcctcttg ctttctttgc 360 ctggacaccc cgttctcctg tggattcggg tcacctctca ctcctttcat ttgggcagct 420 cccctacccc ccttacctct ctagtctgtg ctagctcttc cagccccctg tcatggcatc 480 ttccaggggt ccgagagctc agctagtctt cttcctccaa cccgggcccc tatgtccact 540 tcaggacagc atgtttgctg cctccaggga tcctgtgtcc ccgagctggg accaccttat 600 attcccaggg ccggttaatg tggctctggt tctgggtact tttatctgtc ccctccaccc 660 cacagtgggg ccactaggga caggattggt gacagaaaag ccccatcctt aggcctcctc 720 cttcctagtc tcctgatatt gggtctaacc cccacctcct gttaggcaga ttccttatct 780 ggtgacacac ccccatttcc 800 <210> 3 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> tet operator <400> 3 tccctatcag tgatagaga 19 <210> 4 <211> 106 <212> DNA <213> Artificial Sequence <220> <223> CMV promotor <400> 4 cctatataag cagagctcgt ttagtgaacc gtcagatcgc ctggagacgc catccacgct 60 gttttgacct ccatagaaga caccgggacc gatccagcct ccgcgg 106 <210> 5 <211> 576 <212> DNA <213> Artificial Sequence <220> <223> VEGF <400> 5 ctacctccac 60 catgccaagt ggtcccaggc tgcacccatg gcagaaggag gagggcagaa tcatcacgaa 120 gtggtgaagt tcatggatgt ctatcagcgc agctactgcc atccaatcga gaccctggtg 180 gacatcttcc aggagtaccc tgatgagatc gagtacatct tcaagccatc ctgtgtgccc 240 ctgatgcgat gcgggggctg ctgcaatgac gagggcctgg agtgtgtgcc cactgaggag 300 tccaacatca ccatgcagat tatgcggatc aaacctcacc aaggccagca cataggagag 360 atgagcttcc tacagcacaa caaatgtgaa tgcagaccaa agaaagatag agcaagacaa 420 gaaaatccct gtgggccttg ctcagagcgg agaaagcatt tgtttgtaca agatccgcag 480 acgtgtaaat gttcctgcaa aaacacagac tcgcgttgca aggcgaggca gcttgagtta 540 aacgaacgta cttgcagatg tgacaagccg aggcgg 576 <210> 6 <211> 546 <212> DNA <213> Artificial Sequence <220> <223> Human elongation factor-1 alpha <400> 6 aaggatctgc gatcgctccg gtgcccgtca gtgggcagag cgcacatcgc ccacagtccc 60 cgagaagttg gggggagggg tcggcaattg aacgggtgcc tagagaaggt ggcgcggggt 120 aaactgggaa agtgatgtcg tgtactggct ccgccttttt cccgagggtg ggggagaacc 180 gtatataagt gcagtagtcg ccgtgaacgt tctttttcgc aacgggtttg ccgccagaac 240 acagctgaag cttcgagggg ctcgcatctc tccttcacgc gcccgccgcc ctacctgagg 300 ccgccatcca cgccggttga gtcgcgttct gccgcctccc gcctgtggtg cctcctgaac 360 tgcgtccgcc gtctaggtaa gtttaaagct caggtcgaga ccgggccttt gtccggcgct 420 cccttggagc ctacctagac tcagccggct ctccacgctt tgcctgaccc tgcttgctca 480 actctacgtc tttgtttcgt tttctgttct gcgccgttac agatccaagc tgtgaccggc 540 gcctac 546 <210> 7 <211> 747 <212> DNA <213> Artificial Sequence <220> <223> rtTA <400> 7 atgtctagac tggacaagag caaagtcata aacggcgctc tggaattact caatggagtc 60 ggtatcgaag gcctgacgac aaggaaactc gctcaaaagc tgggagttga gcagcctacc 120 ctgtactggc acgtgaagaa caagcgggcc ctgctcgatg ccctgccaat cgagatgctg 180 gacaggcatc atacccactt ctgccccctg gaaggcgagt catggcaaga ctttctgcgg 240 aacaacgcca agtcattccg ctgtgctctc ctctcacatc gcgacggggc taaagtgcat 300 ctcggcaccc gcccaacaga gaaacagtac gaaaccctgg aaaatcagct cgcgttcctg 360 tgtcagcaag gcttctccct ggagaacgca ctgtacgctc tgtccgccgt gggccacttt 420 acactgggct gcgtattgga ggaacaggag catcaagtag caaaagagga aagagagaca 480 cctaccaccg attctatgcc cccacttctg agacaagcaa ttgagctgtt cgaccggcag 540 ggagccgaac ctgccttcct tttcggcctg gaactaatca tatgtggcct ggagaaacag 600 ctaaagtgcg aaagcggcgg gccggccgac gcccttgacg attttgactt agacatgctc 660 ccagccgatg cccttgacga ctttgacctt gatatgctgc ctgctgacgc tcttgacgat 720 tttgaccttg acatgctccc cgggtaa 747 <210> 8 <211> 800 <212> DNA <213> Artificial Sequence <220> <223> TALEN right Homology <400> 8 gccagagagg atcctgggag ggagagcttg gcagggggtg ggagggaagg gggggatgcg 60 tgacctgccc ggttctcagt ggccaccctg cgctaccctc tcccagaacc tgagctgctc 120 tgacgcggcc gtctggtgcg tttcactgat cctggtgctg cagcttcctt acacttccca 180 agaggagaag cagtttggaa aaacaaaatc agaataagtt ggtcctgagt tctaactttg 240 gctcttcacc tttctagtcc ccaatttata ttgttcctcc gtgcgtcagt tttacctgtg 300 agataaggcc agtagccagc cccgtcctgg cagggctgtg gtgaggaggg gggtgtccgt 360 gtggaaaact ccctttgtga gaatggtgcg tcctaggtgt tcaccaggtc gtggccgcct 420 ctactccctt tctctttctc catccttctt tccttaaaga gtccccagtg ctatctggga 480 catattcctc cgcccagagc agggtcccgc ttccctaagg ccctgctctg ggcttctggg 540 tttgagtcct tggcaagccc aggagaggcg ctcaggcttc cctgtccccc ttcctcgtcc 600 accatctcat gcccctggct ctcctgcccc ttccctacag gggttcctgg ctctgctctt 660 cagactgagc cccgttcccc tgcatccccg ttcccctgca tcccccttcc cctgcatccc 720 ccagaggccc caggccacct acttggcctg gaccccacga gaggccaccc cagccctgtc 780 taccaggctg ccttttgggt 800 <210> 9 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> left recognition site <400> 9 atctggtgac acacccccat ttcc 24 <210> 10 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> right recognition site <400> 10 gccagagagg atcctgggag gg 22 <210> 11 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> talen cleavage site <400> 11 tggagccatc tctctccttg ccagaacctc taaggtttgc ttacgatgga 50 <210> 12 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> Fragment 1 FW Primer <400> 12 ttgggcccga cgtcgcatgc cgcggccgct taattaac 38 <210> 13 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Fragment 1 RV Primer <400> 13 aattaagcgg ccgcggatgg ccgccaccgc gga 33 <210> 14 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Fragment 2 FW Primer <400> 14 cgcggccgct taattaac 18 <210> 15 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Fragment 2 RV Primer <400> 15 cgatcgcaga tccttgatgg ccgccaccgc gga 33 <210> 16 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Fragment 3 FW Primer <400> 16 aaggatctgc gatcgctc 18 <210> 17 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Fragment 3 RV Primer <400> 17 gggagctctc ccatatggcg gccgcaaaaa acctc 35

Claims

VEGF 유전자의 상동성 재조합을 유도하고 상기 상동성 재조합을 통해 VEGF 유전자를 삽입하는 서열번호 2의 HA-L (homology arm-L) 염기서열, rtTA 가 결합되어 있는 상태에서 CMV 프로모터의 발현을 조절하는 Tet-오퍼레이터(Tet-O), 혈관내피 성장인자 유전자의 발현 프로모터인 CMV 프로모터, 상기 CMV 프로모터와 작동 가능하게 연결되는 혈관내피성장인자 (Vascular endothelial growth factor, VEGF) 유전자 염기서열, Tet-O/CMV 프로모터를 활성화 시킬수 있는 rtTA 를 지속적으로 발현시키는 hEF1a 프로모터, 상기 hEF1a 프로모터와 작동 가능하게 연결되며, 독시사이클린 존재하에서 Tet-오퍼레이터에 결합하는 rtTA 유전자 염기서열, VEGF 유전자의 상동성 재조합을 유도하고 상기 상동성 재조합을 통해 VEGF 유전자를 삽입하는 서열번호 8의 HA-R (homology arm-R) 염기서열을 포함하는 것을 특징으로 하는 독시사이클린의 처리 유무에 따라 혈관내피성장인자의 발현조절이 가능한 TALEN (Transcription activator-like effector nuclease)에 의해 숙주 염색체 (host chromosome)에 삽입(integration) 가능한 재조합 발현벡터.(Homolog arm-L) sequence of SEQ. ID. NO: 2 which induces homologous recombination of the VEGF gene and inserts the VEGF gene through the homologous recombination, and regulates the expression of the CMV promoter in the state in which rtTA is bound (Tet-O), a CMV promoter that is an expression promoter of a vascular endothelial growth factor gene, a vascular endothelial growth factor (VEGF) gene sequence operably linked to the CMV promoter, a Tet-O / An hEF1a promoter that continuously expresses rtTA capable of activating the CMV promoter, an rtTA gene sequence operably linked to the hEF1a promoter, which binds to the Tet-operator in the presence of a doxycycline, a homologous recombination of the VEGF gene, (Homolog arm-R) sequence of SEQ ID NO: 8 that inserts the VEGF gene through homologous recombination. Treatment or without expression control of vascular endothelial growth factor according to the doxycycline is possible TALEN (Transcription activator-like effector nuclease) in the host chromosome (host chromosome) insertion (integration) available by the recombinant expression vector according to.

청구항 1에 있어서, 상기 Tet 오퍼레이터는 서열번호 3이고, CMV 프로모터는 서열번호 4이고, 혈관내피성장인자(Vascular endothelial growth factor, VEGF)유전자는 서열번호 5이고, hEF1a 프로모터는 서열번호 6이고, rtTA 유전자는 서열번호 7인 것인 재조합 발현벡터.The vascular endothelial growth factor (VEGF) gene is SEQ ID NO: 5, the hEF1a promoter is SEQ ID NO: 6, the rtTA promoter is SEQ ID NO: 3, the CMV promoter is SEQ ID NO: Wherein the gene is SEQ ID NO: 7.

청구항 1에 있어서, 상기 재조합 발현벡터는 서열번호 1인 것인 재조합 발현벡터. 2. The recombinant expression vector according to claim 1, wherein the recombinant expression vector is SEQ ID NO:

청구항 1 내지 청구항 3 중 어느 한 항의 재조합 발현벡터로 TALEN에 의해 형질전환되어 독시사이클린의 처리 유무에 따라 혈관내피성장인자의 발현조절이 가능한 세포주.A cell line capable of regulating the expression of a vascular endothelial growth factor according to the presence or absence of treatment with a toxin, which is transformed by TALEN with the recombinant expression vector of any one of claims 1 to 3.

청구항 4에 있어서, 상기 세포주는 제대혈 유래 줄기세포(hUCB-MSC cell)인 것인 세포주.
The cell line according to claim 4, wherein the cell line is a cord blood-derived stem cell (hUCB-MSC cell).