KR20090055868A - Construction of the vectors expressing reporter genes driven by the endothelial cell-specific promoters and their application in monitoring of mesenchymal stem cells differentiating into the endothelial lineage - Google Patents

Abstract

A monitoring method of the differentiation to an endothelial cell using a recombinant expression vector containing a promoter and reporter gene inducing the endothelial cell is provided to treat as a gene therapy of cardiovascular disease and stem cell therapy. A recombinant expression vector pEGFP/Flk-1 or pEGFP/Tie-2 for monitoring the differentiation of endothelial cell comprises a promoter which induces the endothelial cell-specific expression and reporter gene which is connected to be operatable. The endothelial cell-specific promoter is the Flk-1 promoter of sequence number 1 (SEQ ID NO:1) and Tie-2 promoter of sequence number 2. The reporter gene is green fluorescence protein(GFP), CFP, YFP, or DsRed. A method for monitoring the differentiation of the endothelial cell comprises: a step of preparing a mesenchymal stem cell; a step of inducing a recombinant expression vector for monitoring the differentiation of endothelial cell into the mesenchymal stem cell; and a step of confirming the expression of reporter gene in the transformed mesenchymal stem cell.

Description

Expression vectors with reporter genes attached to vascular endothelial cell-specific promoters and methods for monitoring differentiation into vascular endothelial cells using the same differentiating into the endothelial lineage}

The present invention relates to an expression vector having a reporter gene attached to a vascular endothelial cell-specific promoter and a method for monitoring differentiation into vascular endothelial cells using the same. It relates to a recombinant expression vector for monitoring vascular endothelial cell differentiation comprising a linked reporter gene.

Cardiac diseases including myocardial infarction are rapidly increasing in Korea, not only in the US and Europe, but also in Korea, due to changes in eating habits, lack of exercise, and increasing number of older people due to aging society. have. However, drug therapy and cardiac transplantation, which are the ultimate treatment, are limited by cost and donor limitations. Recently, the results of adult stem cells that can differentiate into various cell lines in adult tissues including adult bone marrow have been reported, and there is increasing interest in the possibility of treating cardiovascular diseases using stem cells. Investigators have reported positive studies showing improved cardiac function and regeneration of damaged heart tissue. Cell therapy for cardiovascular disease was performed by Tomita S et al. (1999, Circulation 100: 11247-11256) after the first animal experiments showed that cardiac function improved after transplanting mesenchymal stem cells isolated from rat bone marrow into a myocardial infarction model animal. Many animals have hoped that after transplanting stem cells into cardiovascular disease models, the transplanted cells differentiated into cardiomyocytes, vascular endothelial cells, and vascular smooth muscle cells, and improved blood vessel formation, proliferation, and left ventricular function. It is proven through experiments. In addition, the first clinical trial using bone marrow mesenchymal stem cells was performed by Strauer BE et al. (2002, Circulation 106: 1913-1918) in 10 patients with autologous bone marrow mesenchymal cells transplanted through the artery of myocardial infarction. Autologous bone marrow mesenchymal stem cells and autologous blood progenitors (Assmus B et al. 2002, Circulation 106: 3009- 3017), autologous skeletal myoblasts (Merasche et al. 2001, Lancet 357: 279-280) have been reported to improve left ventricular function and enhance metabolic activity in infarcted heart. Recently, the injection of cytokines (G-CSF) from the bone marrow of cardiovascular patients recovered peripheral cells that have been activated into peripheral blood and transplanted them, suggesting the improvement of left ventricular contractility and motor performance (Kang HJ et al. 2004, Lancet). 363: 751-756).

However, there were reports of arrhythmia due to the absence of electrophysiological linkage between transplanted cells and peripheral cells after transplantation of autologous skeletal myoblasts and stent-coronary stenosis was found in patients transplanted after G-CSF administration. Therefore, the need for a study on safety and the therapeutic effect of heart disease using stem cells has been raised. Recently, when bone marrow-derived hematopoietic stem cells were transplanted into a cardiovascular disease model animal, cardiomyocytes are improved by angiogenesis by differentiating into a blood cell line rather than by differentiating cardiomyocytes. It has been argued that transdifferentiation of furnaces is not achieved (Balsam LB et al. 2004, Nature 428: 668-673). In addition, some researchers claim that the rate at which stem cells differentiate into cardiomyocytes is minimal and appears to have been differentiated into cardiomyocytes by cell fusion between cardiomyocytes in the heart and transplanted stem cells ( Nygren JM et al. 2004, Nat Med 10: 494-501). On the other hand, the rate of differentiation of transplanted cells into blood vessels and cardiomyocytes after stem cell transplantation in animal models is minimal, and the survival, proliferation and differentiation of cells in the heart damaged by factors secreted from the transplanted cells are promoted. There are many reports on the paracrine effect of improved cardiac function.

As mentioned above, although the cell therapy effect of cardiovascular disease is less than expected and the side to enhance the efficacy and safety of stem cell treatment, it is recognized the importance of stem cell research and biotechnology and medicine field. In developed countries, the government has led the government in providing economic, political and legal support for stem cell research. The stem cell treatment market for cardiovascular disease is expected to reach $ 3.9 billion in 2015 from $ 3.8 billion in 2010 (A Jain PharmaBiotech Report 2005), and the potential beneficiary and size of stem cell research in the US for heart disease is 58 million. (D. Perry 2000, "Patients' Voices: The powerful Sound in the Stem Cell Debate", Science 287: 1423). In the field of stem cell treatment, commercialization clinical trials are actively progressing around bioventures, and recently, multinational pharmaceutical companies have entered the field of cell therapy through mergers and acquisitions between ventures.

Analysis of promoters and enhancer regions capable of inducing vascular endothelial cell-specific expression, and the construction of expression vectors using them to verify expression specificity in vascular endothelial cells mainly block the neovascularization of tumor tissues. Has been studied. To detect molecules that regulate angiogenesis by attaching highly sensitive enzymes to specific promoters that induce the expression of vascular endothelial cells or to attach target molecules to vascular endothelial specific promoters to kill only cancer cells. There have been studies of expression in blood vessels. For example, the Tie-2 vascular endothelial promoter and enhancer regions have been useful in the development of signaling systems related to vascular endothelial cell-specific expression in adult tissues (Fadel BM et al. 1998, Biochem J 330: 335). -343; Kisanuki YY et al. 2001, Dev Biol 230: 230-242). De Palma M et al. (2003, Hum Gene Ther 14: 1193-1206) construct a lentiviral vector with a GFP marker gene attached to the Tie1, Tie2, Flk-1, VE-Cad and ICAM-2 promoter regions and then vascular endothelial cells. In comparison with expression specificity after introduction into islet cell, nerve, lymphocyte, hematopoietic stem and cancer cell lines, Tie-2 promoter and enhancer have been shown to induce the highest specific vascular endothelial cell expression. . In addition, specific target genes are attached to Tie-2 promoters and enhancers to specifically express them only at the blood-brain barrier, and have been used as a tool for verifying the effects of target genes (Patent paper, JP2006141228). .

Heidenreich R et al. (2000, Cancer Res 60: 6142-6147) introduced expression vectors with beta-galactosidase marker genes at 939 bp Flk-1 promoter and 2.3 kbp enhancer regions into various carcinoma tissues. Investigations showed specific expression in blood vessels present in tumors, whereas most blood vessels in normal tissues did not express marker genes (Heidenreich R et al. 2000, Cancer Res 60: 6142-6147). In addition, Licht AH et al. (2004, Dev Dyn 229: 312-318) express a specific expression during vascular development during development through a transgenic mouse model in which a beta-galactosidase marker gene is attached to the Flk-1 promoter region. (Licht AH et al. 2004, Dev Dyn 229: 312-318).

Inducing specific expression of vascular endothelial cells may be said to be most important in the presence of transcriptional activity and inhibitory sites present in the promoter and enhancer sites. However, even when using the same promoter and enhancer region, expression specificity of vascular endothelial cell specific gene regulatory sites may be influenced by the intracellular environment which can regulate the activity of transcription factors present in the introduced cells. In this context, expression vector construction and specific expression can be specifically monitored for the differentiation of mesenchymal stem cells into vascular endothelial cells, which are widely used in the treatment of cardiovascular diseases due to their differentiation ability to myocardial and vascular endothelial cells. The study to validate the results could greatly contribute to the development of gene therapy and stem cell therapy research for cardiovascular disease.

Thus, the present inventors have made intensive research to overcome the problems of the prior art, when transforming mesenchymal stem cells with a vector containing a reporter gene operably linked with a promoter for inducing vascular endothelial cell specific expression, It was confirmed that the differentiation into vascular endothelial cells can be specifically monitored, and the present invention was completed.

Therefore, the main object of the present invention is to express an expression vector capable of specifically monitoring the differentiation of mesenchymal stem cells into vascular endothelial cells, which are widely used in the treatment of cardiovascular diseases due to their differentiation ability into myocardial and vascular endothelial cells. To provide.

Another object of the present invention is to provide a method for monitoring vascular endothelial cell differentiation using the recombinant expression vector.

According to one aspect of the invention, the invention provides a recombinant expression vector for monitoring vascular endothelial cell differentiation comprising a promoter inducing vascular endothelial cell specific expression and a reporter gene operably linked thereto.

In the present invention, the expression vector refers to a plasmid, virus or other medium into which a promoter for inducing vascular endothelial cell specific expression and a reporter gene operably linked thereto may be inserted or introduced. The reporter gene sequence according to the present invention may be operably linked to the vascular endothelial cell specific promoter, wherein the operably linked gene sequence and the promoter sequence include a selection marker and a replication origin together. It can be included in the expression vector of. "Operably linked" means that the appropriate gene sequence is linked in such a way as to enable gene expression when bound to the promoter sequence. Thus, the expression vector is a promoter for transcription, any operator sequence for controlling transcription, a sequence for fitting in frame of the coding sequence, a sequence encoding a suitable mRNA ribosomal binding site, and transcription and translation It may comprise a sequence that controls the termination of. In the present invention, the promoter for inducing vascular endothelial cell-specific expression refers to promoters of marker proteins that are characteristically expressed in vascular endothelial cells, and promoters that bind to these promoters to express vascular endothelial cell-specific marker proteins. Means. Examples of the plasmids include E. coli-derived plasmids (pBR322, pBR325, pUC118 and pUC119, pET-22b (+)), Bacillus subtilis-derived plasmids (pUB110 and pTP5) and yeast-derived plasmids (YEp13, YEp24 and YCp50). The virus may be an animal virus such as a retrovirus, adenovirus or vaccinia virus, or an insect virus such as a baculovirus. A vector suitable for introducing a polynucleotide according to the present invention into a host cell can be used, and preferably a vector designed to facilitate protein expression induction and isolation of the expressed protein.

In the recombinant expression vector for monitoring vascular endothelial cell differentiation of the present invention, the vascular endothelial cell specific promoter may be any promoter that regulates transcription of marker proteins specifically expressed in vascular endothelial cells, preferably SEQ ID NO: It is preferred that it is the Flk-1 promoter of 1 or the Tie-2 promoter of SEQ ID NO: 2.

In the recombinant expression vector for monitoring vascular endothelial cell differentiation of the present invention, the reporter gene may be any reporter gene capable of confirming the transcribed and translated protein in vivo or in vitro, but preferably, encodes a fluorescent protein. The presence or absence of a reporter gene expressed protein may be confirmed without going through a separate process. Examples of the fluorescent protein include green fluorescence protein (GFP), blue fluorescent protein (CFP), yellow fluorescent protein (YFP) or red fluorescent protein (DsRed). The green fluorescent protein comes from a luminescent jellyfish called Aequorea Victoria, which is genetically engineered to mutate blue (CFP), yellow (YFP), and red (DsRed) fluorescent proteins engineered to have different excitation and emission wavelengths. And the like can be used. In the preferred embodiment 2 of the present invention, the fluorescent protein, green fluorescent protein EGFP (enhanced GFP), and in Example 3, the red fluorescent protein DsRed was used, respectively, the base sequence of the EGFP gene (GeneBank Accession No. U55761) and the base of the DsRed gene. The sequence (GeneBank Accession No. AF506025) is well known in the art. The red fluorescent protein DsRed shows the highest excitation wavelength at 558 nm and the highest emission wavelength at 583 nm, and the green fluorescent protein GFP has the highest excitation wavelength at 488 nm and the highest emission wavelength at 507 nm. When using the fluorescent protein gene as a reporter gene, by using immunostaining or confocal microscopy, the differentiation of stem cells into vascular endothelial cells can be easily monitored without killing (or non-invasively). .

In the recombinant expression vector for monitoring vascular endothelial cell differentiation of the present invention, the expression vector is a pEGFP / Flk-1 vector having a cleavage map of FIG. 3, a pDsRed / Flk-1 vector having a cleavage map of FIG. 5, and FIG. 6. It is preferably selected from the group consisting of pEGFP / Tie-2 vectors with cleavage maps.

According to another aspect of the present invention, the present invention provides bone marrow mesenchymal stem cells wherein the recombinant expression vector is transduced to express the reporter gene upon differentiation into vascular endothelial cells. The method of introducing the recombinant vector according to the present invention into a host cell may include, but is not limited to, calcium chloride (CaCl ₂ ) and heat shock, particle gun bombardment, and silicon carbide. Silicon carbide whiskers, sonication, electroporation, and precipitation with polyethylenglycol (PEG) may be used. The stem cells into which the recombinant expression vector of the present invention is introduced may be used to determine how much differentiation of the transplanted stem cells into vascular endothelial cells is performed after transplantation into a patient in need of cell therapy. To this end, the stem cells into which the recombinant expression vector is introduced may be directly administered or transplanted to the heart region of a patient with vascular disease, and may be transplanted using blood vessels of heart tissue. In animals, stem cells are injected intravenously into the tail vein, followed by mobilization reactions from the damaged heart (e.g., the highest known cell mobilizing factor, SDF-1, is expressed at high levels in the damaged heart. Stem cells from the bone marrow, the peripheral blood, and the like, expressing the reaction to the damaged heart (reaction in the regeneration of the damaged tissue) can also be used for research. In addition, in order to enhance the engraftment rate of stem cells, various embedding materials (e.g., sodium alginate, hyaluronic acid, or collagen, etc.) may be used by forming complexes with stem cells. Thereafter, blood or very small amounts of tissue samples can be taken to determine the progress and degree of differentiation of the transplanted cells.

According to another aspect of the present invention, the present invention provides a method for monitoring vascular endothelial cell differentiation comprising the following steps.

(a) separating the mesenchymal stem cells;

(b) introducing a recombinant expression vector for monitoring vascular endothelial cell differentiation according to claim 1 into the prepared mesenchymal stem cells; And

(c) confirming the expression of the reporter gene in the transduced mesenchymal stem cells.

In the method for monitoring differentiation into vascular endothelial cells of the present invention, as a method for preparing mesenchymal stem cells in step a), the present invention solves the limitation of stem cell research due to the absence of characteristic markers of mesenchymal stem cells. To this end, Sca-1, a specific marker capable of separating mesenchymal stem cells with high purity, was used. Previously reported mouse, animal and human-derived bone marrow mesenchymal stem cells are CD29 +, CD44 +, and CD90 +, and CD34- and CD45-, which are the hematopoietic stem cell markers, have not been expressed, but there are differences in the expression of cell surface proteins. It is not fully established as reported. Although cell surface proteins such as CD29, CD44, and CD90 are positive for mesenchymal stem cells, these markers, unlike the characteristic markers such as CD34, CD117, and CD133, are widely used to separate hematopoietic stem cells. It is also reported to be expressed in relation to cell contact, cognition, and the like. Therefore, in the present invention, after identifying the cell surface characteristics of the cells attached to the cell container after separating the bone marrow cells, Sca-1 was used as a specific cell surface marker capable of separating the bone marrow mesenchymal stem cells with high purity.

In the present invention, the process of confirming the expression of the reporter gene in step c) is specific for stem cell to vascular endothelial cells by confirming whether the expression of the reporter gene, for example, fluorescent protein, by immunostaining or confocal microscopy. Differentiation can be monitored.

According to another aspect of the present invention, the present invention provides a primer set of SEQ ID NO: 3 and SEQ ID NO: 4 and primers of SEQ ID NO: 4 and SEQ ID NO: 5 for polymerase chain reaction (PCR) of the Flk-1 promoter gene of SEQ ID NO: A set and primer sets of SEQ ID NO: 5 and SEQ ID NO: 6 for polymerase chain reaction (PCR) of the Tie-2 promoter gene of SEQ ID NO: 2 are provided.

Hereinafter, the production of a recombinant expression vector for monitoring vascular endothelial cell differentiation and monitoring vascular endothelial cell differentiation using the same will be described in more detail step by step.

In the present invention, instead of amplifying a promoter region of a known sequence using a previously reported primer set, amplifying specific vascular endothelial cells by amplifying 844 bp Flk-1 and 1,061 bp Tie gene regulatory regions, respectively, using an original primer set. Expression vectors were constructed. The prior art reports the expression specificity of vascular endothelial cell gene regulatory sites during angiogenesis of the generator or tumor, but shows differences in expression specificity depending on the promoter enhancer region used or the type of cell introduced. In the present invention, after introducing the vascular endothelial cell-specific expression vectors constructed in the present invention into highly available mesenchymal stem cells for the treatment of cardiovascular diseases, the differentiation of mesenchymal stem cells into vascular endothelial cells is specifically Prove that it can be monitored. Differentiation of high-purity bone marrow Sca-1 + mesenchymal stem cells into vascular endothelial cells can be specifically monitored, which may be useful for gene therapy and cell therapy research of cardiovascular diseases.

In order to achieve the above object, the present invention includes the following steps.

(a) high purity isolation of Sca-1 + bone marrow mesenchymal stem cells using magnet activation;

(b) constructing an expression vector having a reporter gene attached to a vascular endothelial cell specific promoter;

(c) verifying whether the expression vector monitors specific differentiation into vascular endothelial cells after introducing the vascular endothelial cell specific expression vector into the Sca-1 + bone marrow mesenchymal stem cells.

Through literature searches on the promoters of genes that can specifically monitor differentiation into vascular endothelial cells, mouse Flk-1 gene regulatory sites are found in vivo (Heidenreich R et al. 2000, Cancer Res 60: 6142-6147) as well as in vivo. Licht AH et al., 2004, Dev Dyn 229: 312-318) induces specific differentiation in vascular endothelial cells. In the present invention, the mouse Flk-1 promoter region, which may reflect vascular endothelial cell differentiation, is based on the existing research report and mouse gene bank information (# NT_039306.6), and based on the cloning of the reporter gene into a specific restriction enzyme sequence. A unique primer set including was prepared and amplified by PCR using genomic DNA as a template and identified as a TA cloning vector (FIG. 1, FIG. 2). Since there is a BglII restriction site in the mouse Flk-1 promoter sequence, a strategy of using restriction enzymes other than BglII restriction enzyme was selected for cloning into the reporter gene. The sequencing assay confirmed that the promoter region identical to the sequence reported to the GenBank was cloned correctly. In the next step, the 844 bp mouse Flk-1 promoter identified in the TA cloning vector was cloned into pEGFP-1 and pDsRed-Express-1 vectors, and the cloning direction and size were confirmed by restriction enzyme treatment (FIG. 3 and FIG. 5). ).

In addition, the literature search on the promoters of genes that can specifically monitor differentiation into vascular endothelial cells resulted in not only mouse Tie-2 gene regulatory regions inflight (Fadel BM et al. 1998, Biochem J 330: 335-343). In vivo (De Palma M et al. 2003 Hum Gene Ther 14: 1193-1206; Kisanuki YY et al. 2001, Dev Biol 230: 230-242) it was confirmed that the specific induction in vascular endothelial cells. In the present invention, the mouse Tie-2 promoter region, which may reflect vascular endothelial cell differentiation, was selected based on the existing research report and mouse gene bank information (# NT_039280.5) in consideration of cloning into a reporter gene to select a specific restriction enzyme sequence. A unique primer set was prepared, amplified by PCR using genomic DNA as a template, and identified as a TA cloning vector (FIGS. 1 and 2). Since the SacI and BamHI restriction enzyme sites are present in the mouse Tie-2 promoter sequence, a strategy of using restriction enzymes other than the restriction enzymes was selected for cloning into the reporter gene. The sequencing assay confirmed that the promoter region identical to the sequence reported to the GenBank was cloned correctly. The next step was to clone the 1,061 bp mouse Tie-2 promoter identified in the TA cloning vector into the pEGFP-1 vector and to confirm the cloning direction and size by restriction enzyme treatment (FIG. 6).

The expression vectors (Flk-1-DsRed, Flk-1-EGFP, Tie-2-EGFP) to which the reporter gene is attached to the vascular endothelial cell promoter constructed by the present invention specifically monitor the differentiation into vascular endothelial cells. Introduction of expression vectors into Sca-1 + bone marrow mesenchymal cells, induction of differentiation into vascular endothelial cells by 20ng / ml VEGF treatment, Flk-1 antibody expressed by promoter cloned into expression vector And immunostaining using Tie-2 protein antibody were performed (Example 5). As a result, the expression of the protein antibody of the promoter regulating the expression of the reporter gene was proved to be consistent (Fig. 7, Fig. 8).

It is important that the vascular endothelial promoter regions amplified using the unique primer set prepared in the present invention were specifically expressed only in Sca-1 + bone marrow mesenchymal cells differentiated into the vascular endothelial cell line. Even if the same promoter region is used to induce tissue specific expression, tissue specific expression may vary depending on the introduced cells. In other words, embryonic stem cells and adult stem cells have different stem cell titers and differentiation capacity, and the presence and distribution of molecules that may affect tissue-specific expression, including transcription factors that bind expression promoters and regulate expression. This is because the concentration, the control mode, and the like may be different.

Hereinafter, the present invention will be described in more detail with reference to Examples. Since these examples are only for illustrating the present invention, the scope of the present invention is not to be construed as being limited by these examples.

Example 1 High Purity Isolation and Cell Surface Characterization of Sca-1 + Bone Marrow Mesenchymal Stem Cells

1.1 Isolation of Bone Marrow Cells

After the internal bone marrow cells were isolated from the mouse femur by perfusion using a syringe, tissue fragments and the like were removed using a 70 μm cell mesh and only individual cells were recovered. Bone marrow cells were cultured for 3 days in 0.1% gelatin coated 10-cm cell vessels using Dulbecco's Modified Eagle's Media (DMEM) + 10% fetal bovine serum + 100 U / ml penicillin, 100 μg / ml streptomycin medium. After 3 days of culture, the culture medium was replaced to remove cells that did not adhere to the cell container, followed by further culturing until the cell density reached 80% or more of the culture container (6 to 7 days).

1.2 Isolation of Sca-1 + Bone Marrow Mesenchymal Stem Cells by Magnetic Activation

Sca-1 + bone marrow mesenchymal stem cells from the bone marrow cells attached to the cell culture vessels were isolated using a magnetic activity method. Specifically, after three passages, bone marrow cells were reacted with PE-bound Sca-1 antibody (D7 monoclonal antibody, BD Pharmingen, San Diego, Calif., USA) at 4 ° C. for 15 minutes, followed by washing (PBS + 0.5% BSA). + 2 mM EDTA), reaction with anti-PE microbeads (anti-PE microbeads, Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) for 15 minutes at 4 ° C, washing with cleaning solution and then magnetoactivation column (Miltenyi Biotec GmbH, Bergisch) Only Sca-1 + cells were isolated by passing through Gladbach, Germany, MACS Separation Column, LS Columns Cat # 130-042-401. The step of passage through the magnetoactivity column was repeated four times to enhance cell separation purity.

Example 2 Preparation of Expression Vector with EGFP Reporter Gene Attached to the Mouse Vascular Endothelial Flk-1 Promoter

2.1 EGFP  To reporter gene expression vector Cloning  Mouse Flk -1 promoter amplification

The inventors have identified a mouse Flk-1 promoter moiety that can induce specific expression upon differentiation into vascular endothelial cells of the Flk-1 promoter sequence (GenBank # NT_039306.6) via PCR from mouse C57BL / 6J genomic DNA. PCR was performed after constructing a new primer set for amplification. The PCR reaction was performed at 94 DEG C by adding to the mouse genomic DNA the primer set forth in SEQ ID NO: 3 containing the HindIII restriction enzyme sequence, the primer set forth in SEQ ID NO: 4 comprising the EcoRI restriction enzyme sequence, dNTP, 10x PCR buffer and Taq polymerase. The reaction was carried out for 5 minutes at, 30 seconds at 94 ° C., 40 seconds at 56 ° C. and 40 seconds at 72 ° C., followed by reaction at 72 ° C. for 7 minutes. As a result of electrophoresis of the reaction product obtained after the PCR reaction, an 844 bp DNA fragment corresponding to the Flk-1 promoter of SEQ ID NO: 1 was confirmed (FIG. 1).

Mouse Flk-1 1F-HindIII: 5'-aagcttAGGGCAAATGCACAGAGAAG-3 '(SEQ ID NO: 3)

Mouse Flk-1 1R-EcoRI: 5'-gaattcCAGGGGAACACCGATAAAAA-3 '(SEQ ID NO: 4)

The 844 bp Flk-1 promoter gene fragment amplified by PCR was cloned into pGEM-T Easy vector (Promega). FIG. 2 shows that 844 bp Flk-1 gene fragment was correctly cloned after cleavage with restriction enzyme to confirm cloning, and sequenced using T7 / SP6 primer position to describe the Flk-1 promoter sequence. It was confirmed that it has a nucleotide sequence.

2.2 mouse Flk -1 promoter EGFP  To reporter gene expression vector Cloning

The present inventors cloned the mouse Flk-1 promoter gene amplified in Example 2.1 into a pEGFP-1 vector (Clontech). Specifically, the Flk-1 fragment amplified in Example 2.1 was digested with HindIII and EcoRI, purified using a gel recovery kit, and then ligated with the pEGFP-1 vector digested with the same restriction enzyme. An expression vector including the -1 promoter and the reporter gene EGFP was constructed (FIG. 3). Electrophoresis after restriction enzyme treatment to confirm that the Flk-1 promoter was inserted into the vector confirmed the DNA size of the Flk-1 promoter and confirmed that the vector including the Flk-1 promoter was constructed (FIG. 4 left 1). lane).

3. Mouse Vascular Endothelial Specificity Flk -1 to promoter DsRed  Construction of Expression Vectors Attached with Reporter Genes

3.1 Amplification of the Mouse Flk-1 Promoter for Cloning into DsRed Reporter Gene Expression Vectors

We amplify the mouse Flk-1 promoter, which can induce specific expression upon differentiation into vascular endothelial cells in the Flk-1 promoter sequence (GenBank # NT_039306.6) via PCR from mouse C57BL / 6J genomic DNA. To prepare a new primer set to perform a PCR. The PCR reaction was performed by adding primers described in SEQ ID NO: 5 containing XhoI restriction enzyme sequences, primers described in SEQ ID NO: 4 comprising EcoRI restriction enzyme sequences, dNTP, 10 × PCR buffer and Taq polymerase to mouse genomic DNA. The reaction was carried out at 94 ° C. for 5 minutes, 35 seconds at 94 ° C., 40 seconds at 56 ° C., and 40 seconds at 72 ° C., followed by reaction at 72 ° C. for 7 minutes. As a result of electrophoresis of the reaction product obtained after the PCR reaction, an 844 bp DNA fragment corresponding to the Flk-1 promoter of SEQ ID NO: 1 was confirmed (FIG. 1).

Mouse Flk-1 1F-XhoI: 5'-ctcgagAGGGCAAATGCACAGAGAAG-3 '(SEQ ID NO: 5)

Mouse Flk-1 1R-EcoRI: 5'-gaattcCAGGGGAACACCGATAAAAA-3 '(SEQ ID NO: 4)

The 844 bp Flk-1 promoter gene fragment amplified by PCR was cloned into the pGEM-T Easy vector. FIG. 2 shows that 844 bp Flk-1 gene fragment was correctly cloned after cleavage with restriction enzyme to confirm cloning. As a result of sequencing using the T7 / SP6 primer position, the Flk-1 promoter sequence was described. It was confirmed that it has a nucleotide sequence.

3.2 mouse Flk -1 promoter DsRed  To reporter gene expression vector Cloning

The present inventors cloned the mouse Flk-1 promoter gene amplified in Example 3.1 into pDsRed-1 vector (Clontech # 632412). Specifically, the Flk-1 fragment amplified in Example 3.1 was digested with XhoI and EcoRI, purified using a gel recovery kit, and then ligated with the pDsRed-Express-1 vector digested with the same restriction enzyme. The expression vector containing the Flk-1 promoter and the reporter gene DsRed was prepared (FIG. 5). Electrophoresis after restriction enzyme treatment to confirm that the Flk-1 promoter was inserted into the vector confirmed the DNA size of the Flk-1 promoter and confirmed that the vector containing the Flk-1 promoter was constructed (Fig. 4 right). .

Example  4 Mouse Endothelial Cell Specificity Tie -2 to promoter EGFP  Construction of Expression Vector with Reporter Gene Attached

4.1 EGFP  To reporter gene expression vector Cloning  Mouse Tie -2 promoter amplification

We amplified the mouse Tie-2 promoter capable of inducing specific expression upon differentiation into vascular endothelial cells in the Tie-2 promoter sequence (gene bank # NT_039280.5) via PCR from mouse C57BL / 6J genomic DNA. To prepare a new primer set to perform a PCR. The PCR reaction was performed by adding primers described in SEQ ID NO: 6 including HindIII restriction enzyme sequences, primers described in SEQ ID NO: 7 including EcoRI restriction enzyme sequences, dNTP, 10 × PCR buffer and Taq polymerase to mouse genomic DNA. The reaction was carried out at 94 ° C. for 5 minutes, at 35 ° C. for 30 seconds at 94 ° C., 40 seconds at 56 ° C., and for 40 seconds at 72 ° C., followed by reaction at 72 ° C. for 7 minutes. As a result of electrophoresis of the reaction product obtained after the PCR reaction, it was confirmed that the DNA fragment was 1,061bp corresponding to the Tie-2 promoter sequence information of SEQ ID NO: 2 (FIG. 1).

Mouse Tie-2 1F-HindIII: 5'-aagcttGTCTTGGTTTTACCGGCTGA-3 '(SEQ ID NO: 6)

Mouse Tie-2 1R-EcoRI: 5'-gaattcACTCACAACTTTGCGACTTCC-3 '(SEQ ID NO: 7)

The 1,061 bp Tie-2 promoter gene fragment amplified by PCR was cloned into the pGEM-T Easy vector. Figure 2 confirms that the Tie-2 gene fragment of 1,061bp size was correctly cloned after cleavage with restriction enzymes to confirm cloning, and sequenced using T7 / SP6 primer position as a Tie-2 promoter sequence. It was confirmed that it has a nucleotide sequence.

4.2 Mouse Tie -2 promoters EGFP  To reporter gene expression vector Cloning

The present inventors cloned the mouse Tie-2 promoter gene amplified in Example 4.1 into pEGFP-1 vector. Specifically, the Tie-2 fragment amplified in Example 4.1 was digested with HindIII and EcoRI, purified using a gel recovery kit, and then ligated with a pEGFP-1 vector digested with the same restriction enzyme. An expression vector comprising the -2 promoter and the reporter gene EGFP was constructed (FIG. 6). Electrophoresis after restriction enzyme treatment to confirm that the Tie-2 promoter was inserted into the vector confirmed the DNA size of the Tie-2 promoter and confirmed that the vector containing the Tie-2 promoter was constructed (FIG. 4 left 2). lane).

Example  5. Sca -1+ bone marrow Into mesenchymal cells  Verification of specificity of differentiation into vascular endothelial cells after introduction of vascular endothelial cell specific expression vector

Sca-1 + bone marrow mesenchymal stem cells (2 × 10 ⁴ cells / well) isolated by Example 1 using DMEM medium containing 10% fetal calf serum without antibiotics to enhance DNA transduction efficiency Were inoculated into 24-well cell containers placed with 0.1% gelatin coated coverslips. After 24 hours of culture, 1 ug of vascular endothelial promoter expression vectors (Flk-1-EGFP and Tie-2-EGFP), respectively, constructed in FIGS. After reacting for 5 minutes. 2 ul of lipofectamine (Lipofectamine 2000, Invitrogen) solution and 50 ul of Opti-Mem culture were mixed and allowed to react for 5 minutes. After the expression vector and the lipofectamine solution were mixed and reacted at room temperature for 20 minutes, Sca-1 + bone marrow mesenchymal stem cells were added into the culture solution inoculated and incubated for 48 hours. Differentiation was induced for 14 days in DMEM + 10% fetal bovine serum medium in the presence of 20 ng / ml VEGF for induction of differentiation into vascular endothelial cells. Cultures containing 20ng / ml VEGF were changed every 3 days.

To determine whether the reporter gene expressed in the vascular endothelial cell promoter showed specific expression only in cells differentiated into vascular endothelial cells, Sca-1 + bone marrow mesenchymal stem cells attached to 0.1% gelatin-coated coverslips It confirmed by the immunostaining method using a specific antibody. Specifically, differentiated Sca-1 + bone marrow mesenchymal stem cells were treated with 2% paraformaldehyde fixative for 15 minutes, washed with PBS + 0.1% Tween20 (PBST) solution, and treated with 0.25% Triton X-100 solution for 30 minutes. , Washed with PBST solution, 1 hour incubation with 5% goat serum, 1 hour incubation with primary antibodies Flk-1 antibody (Santa Cruz Biotechnology) and Tie-2 antibody (Upstate), washed with PBST solution, 2 1 hour incubation with Cy3 antibody (Zymed Laboratories Inc., San Francisco, USA), a secondary antibody, washed with PBST solution, 1 hour incubation with DAPI solution, PBST solution for nuclear staining, and the intensity of fluorescence signal under fluorescence microscope Samples were prepared using a mounting solution for maintenance and observed under a fluorescence microscope. As a result, it was demonstrated that the expression of the protein antibody of the promoter regulating the expression of the reporter is consistent with the expression (Figs. 7 and 8).

As described above, according to the present invention, after introducing into the bone marrow mesenchymal stem cells using an expression vector attached to the vascular endothelial cell specific promoter sequence capable of detecting differentiation into a vascular endothelial cell line, the EGFP or DsRed reporter gene is attached. Differentiation can be monitored by vascular endothelial cells.

 Therefore, the vascular endothelial cell-specific expression vector constructed in the present invention is able to specifically reflect the differentiation of vascular endothelial cells. In addition, by integrating vascular endothelial cell differentiation detection technology and molecular imaging technology developed by the present invention, non-invasive in vivo stem cell differentiation, fate research, etc., can greatly contribute to securing the efficacy and safety of stem cell treatment. will be.

Figure 1 is a photograph confirming by electrophoresis that the vascular endothelial promoters (Flk-1, Tie-2) was amplified by PCR using genomic DNA to generate a DNA fragment of the expected size.

Figure 2 is a photograph confirmed by electrophoresis after cleavage with restriction enzymes that the vascular endothelial promoter (Flk-1, Tie-2) amplified by PCR was cloned into pGEM-T Easy, a TA cloning vector.

Figure 3 is a schematic diagram of the recombinant vector generated by inserting the vascular endothelial promoter (Flk-1) into the parent vector pEGFP-1 vector.

4 is a photograph confirmed by electrophoresis after cleavage of vascular endothelial promoters (Flk-1, Tie-2) cloned into pEGFP-1 or pDsRed-Express-1 vector using restriction enzymes.

5 is a schematic diagram of a recombinant vector generated by inserting the vascular endothelial cell promoter (Flk-1) into the parent vector pDsRed-Express-1 vector.

6 is a schematic diagram of a recombinant vector generated by inserting a vascular endothelial cell promoter (Tie-2) into the parent vector pEGFP-1 vector.

7 is a reporter after introducing an expression vector having the reporter gene EGFP attached to Sca-1 + mesenchymal stem cells to the vascular endothelial cell promoter (Flk-1) and inducing differentiation into vascular endothelial cells by VEGF treatment. In order to verify that the gene expressing cells are stained with vascular endothelial specific antibodies at the same time, it is a photograph confirmed under confocal microscopy after immunostaining.

FIG. 8 is a reporter after introducing an expression vector having a reporter gene EGFP attached to Sca-1 + mesenchymal stem cells to a vascular endothelial cell promoter (Tie-2) and inducing differentiation into vascular endothelial cells by VEGF treatment. In order to verify that the gene expressing cells are stained simultaneously with vascular endothelial cell-specific antibodies, the photographs were confirmed under confocal microscopy after immunostaining.

<110> Korea University Industry Academy Cooperation Foundation <120> Construction of the vectors expressing reporter genes driven by          the endothelial cell-specific promoters and their application in          monitoring of mesenchymal stem cells differentiating into the          endothelial lineage <160> 7 <170> KopatentIn 1.71 <210> 1 <211> 844 <212> DNA <213> Mus Musculus <400> 1 agggcaaatg cacagagaag agcccccggt ccttctccag tggctgctag agtgcttttg 60 tctacccagt tccatgacca cggtccccta cagtaacttt ggccaggccc cttttcagag 120 ctgacctttc agccaagata tgcttggggg gaagagaagg gagagtttct tgccagaggc 180 gccagtgtag gaacaaagag gaaaccggtc ccatgtgaac agtccccccc ccccccccag 240 ctatgtagac ttcaagtagc gactagagag gaacagcagc cttcacccct cgttttcttt 300 ccattccttt ccttccacgc ctgggaaatg acaacaaata tcttgtgaca tgttttactc 360 ctggcaaaaa ccaaatgcat ttagaatgat agtttaaagg acagtctttc agccagacaa 420 agagataaga ttattgtctc tacatcggat ctaaactaca ctgatatgca tgattaatga 480 tgtaacatgg tgcttttctt taaaaaaaat aaggggggaa acaggcatgc tcctttatca 540 cgtgcctaag cctcaagtat ggtgccagaa gcaggtggca aaccaggagg gtttgtaaaa 600 tccaggttca ctataaaaac atgtcaacac atagcgaatc tatataataa aattatactt 660 gaagatcttg ggcatgtttg gtgtgggcat cctcatttag cagaggcatg cctggtctgc 720 tcacatttag ctttgttgta tattcagctc ctcacgaatg aaaactccgt atgaaggctg 780 cttggtgtac ctatcgacag cgttctgaag gttctggctt gttcttttta tcggtgttcc 840 cctg 844 <210> 2 <211> 1061 <212> DNA <213> Mus musculus <400> 2 gtcttggttt taccggctga gccatctcac ctgcctgatt atttaaaaat ctccggagta 60 atccaagagt gtggtttatg attgtagtat caacactcgg gaggctgagg gagcatcgtt 120 atcatgagct ccaggctagt tccaggcttg cctaagctgt agagcaagtc actctcttaa 180 aaagtgcctc tcccatattt ttgtatataa tttgcatctg aaattctgtt tgccaataac 240 tatgaaatta ttcacattac taaaatcttc ctgtgccaag ttctccaacg aattagatca 300 cactcagatg aaatgctaat aaaaattaaa gctgtagcca gtagcatgcg tatatttggg 360 ctcagggcca acaggcaggc gatctgggtg taagaaaata ggctaatggc tgtggaatct 420 ggtctctagt ggctccgctg agagctgacc tcaaccacgc tccctcaaat tgattgcctt 480 ccaggttatg atttctcatc acaggaaact ttgttgccca attcaaaccc tgtgagtgaa 540 aacaaaaaca ggagagcaag tgctgctccc cgtgccccaa agccccttct gtcagggatc 600 ccaaatgcac cccagagaac agcttagcct gcaagggctg gtcctcatcg cataccatac 660 ataggtggag ggcttgttat tcaattcctg gcctatgaga ggatacccct attgttcctg 720 aaaatgctga ccaggacctt acttgtaaca aagatccctc tgccccacaa tccagttaag 780 gcaggagcag gagccggagc aggagcagaa gataagcctt ggatgaaggg caagatggat 840 agggctcgct ctgccccaag ccctgctgat accaagtgcc tttaagatac agcctttccc 900 atcctaatct gcaaaggaaa caggaaaaag gaacttaacc ctccctgtgc tcagacagaa 960 atgagactgt taccgcctgc ttctgtggtg tttctccttg ccgccaactt gtaaacaaga 1020 gcgagtggac catgcgagcg ggaagtcgca aagttgtgag t 1061 <210> 3 <211> 26 <212> DNA <213> Artificial Sequence <220> 5 prime PCR primer for Flk1 <400> 3 aagcttaggg caaatgcaca gagaag 26 <210> 4 <211> 26 <212> DNA <213> Artificial Sequence <220> 3 prime PCR primer for Flk1 <400> 4 gaattccagg ggaacaccga taaaaa 26 <210> 5 <211> 26 <212> DNA <213> Artificial Sequence <220> 5 prime PCR primer for Flk1 <400> 5 ctcgagaggg caaatgcaca gagaag 26 <210> 6 <211> 26 <212> DNA <213> Artificial Sequence <220> 5 prime PCR primer for Tie2 <400> 6 aagcttgtct tggttttacc ggctga 26 <210> 7 <211> 27 <212> DNA <213> Artificial Sequence <220> 3 prime PCR primer for Tie2 <400> 7 gaattcactc acaactttgc gacttcc 27  

Claims (8)

A recombinant expression vector for monitoring vascular endothelial cell differentiation comprising a promoter inducing vascular endothelial cell specific expression and a reporter gene operably linked thereto. The recombinant expression vector of claim 1, wherein the vascular endothelial cell-specific promoter is a Flk-1 promoter of SEQ ID NO: 1 or a Tie-2 promoter of SEQ ID NO: 2. 6. The method of claim 1, wherein the reporter gene is selected from the group consisting of green fluorescence protein (GFP), blue fluorescence protein (CFP), yellow fluorescence protein (YFP) and red fluorescence protein (DsRed) Recombinant expression vector. The pEGFP / Flk-1 vector having a cleavage map of FIG. 3, the pDsRed / Flk-1 vector having a cleavage map of FIG. 5, or the pEGFP / Tie- having a cleavage map of FIG. 6. Recombinant expression vector for monitoring vascular endothelial cell differentiation, characterized in that 2 vectors. Bone marrow mesenchymal stem cells wherein the reporter gene is expressed when the recombinant expression vector of claim 1 is transduced and differentiated into vascular endothelial cells. A method for monitoring vascular endothelial cell differentiation comprising the following steps: (a) preparing a mesenchymal stem cell; (b) introducing a recombinant expression vector for monitoring vascular endothelial cell differentiation according to claim 1 into the prepared mesenchymal stem cells; And (c) confirming the expression of the reporter gene in the transduced mesenchymal stem cells. A primer set of SEQ ID NO: 3 and SEQ ID NO: 4 or a primer set of SEQ ID NO: 4 and SEQ ID NO: 5 for polymerase chain reaction (PCR) of the Flk-1 promoter gene of SEQ ID NO: 1. Primer sets of SEQ ID NO: 5 and SEQ ID NO: 6 for polymerase chain reaction (PCR) of the Tie-2 promoter gene of SEQ ID NO: 2.

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