KR20090055953A - Expression vectors for monitering differentiation of mesenchymal stem cell into the cardiomyogenic lineage, and monitering methods of differentiating into the cardiomyogenic lineage by using the same - Google Patents

Abstract

A recombinant expression vector for monitoring the differentiation of stem cell to a cardiomyogenic cell is provided to detect the differentiation and use for cell therapy of cardiovascular disease and stability and efficiency of stem cell therapy. A recombinant expression vector pDsRed-Express-1/mMLC, pDsRed-Express-1/rMLC, pEGFP-1/cTn-1, or pDsRed-Express-1/cTn-1 for monitoring the differentiation to a cardiomyogenic cell comprises a promoter inducing the cardiomyogenic cell-specific expression and reporter gene which is operably linked. The promoter inducing the cardiomyogenic cell-specific expression is the promoter myosin light chain 2v promoter of the sequence number 1(SEQ ID NO:1), rat myosin light chain 2v promoter of the sequence number 2, or myocardial troponin I promoter of the sequence number 3. A method for monitoring the differentiation to cardiomyogenic cell comprises: a step of preparing a mesenchymal stem cell; a step of transducing the recombinant expression vector into a prepared stem cell; and a step of confirming the expression of reporter gene in the transduced stem cell.

Description

Expression vectors for monitering differentiation of mesenchymal stem cell into the cardiomyogenic lineage, and monitering methods of differentiating into the cardiomyogenic lineage by using the same}

The present invention relates to a recombinant expression vector for monitoring the differentiation of stem cells to cardiomyocytes, and more specifically to monitoring the differentiation into cardiomyocytes comprising a promoter inducing cardiomyocyte specific expression and a reporter gene operably linked thereto. The present invention relates to a recombinant expression vector for and a method for monitoring the differentiation of cardiomyocytes using the same.

The differentiation monitoring system based on the cardiomyocyte specific promoter developed by the present invention can be used not only for the study of differentiation into cardiomyocytes on board but also useful for tracking the differentiation of cells transplanted in vivo by applying molecular imaging technology. Can be.

The present invention constructs an expression vector having a reporter gene attached to a cardiomyocyte specific promoter to construct a system capable of monitoring only cells differentiated into cardiomyocytes, and isolates the specificity of action of the constructed expression vector from mouse bone marrow. It relates to the development of technology to verify after introduction into -1 + mesenchymal stem cells.

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 treatment and cardiac transplantation, which are the ultimate treatment methods, are limited by cost and donor limitations. Recent studies have shown that adult stem cells, such as adult bone marrow, are capable of differentiating into various cell lines, and thus the interest in the treatment of cardiovascular diseases using stem cells is increasing. Attempts have been made to improve the regeneration and damage of damaged heart tissue.

Cell therapy for cardiovascular disease was performed by Tomita S et al. (1999, Circulation 00: 11247-11256) after a first animal experiment in which cardiac function was improved after transplanting mesenchymal stem cells isolated from rat bone marrow into a myocardial infarction model animal. Hematopoietic stem cells that are-/ c-kit + (Orlic D et al., Nature 2001, 410: 701-705), and side population cells (Jackson KA et al. 2001, J. Clin Invest, which represent hematopoietic stem cell characteristics) 107: 1395-1402) and vascular endothelial progenitor cells (Kocher AA et al. 2001, Nature Med 7: 430-636), which are CD34 +, and then transplanted into cardiovascular model animals. Promising research results that differentiate into cells and vascular smooth muscle cells, improve blood vessel formation and proliferation, and left ventricular function have been demonstrated in many animal experiments.

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 who underwent autologous bone marrow mesenchymal cells 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) were reported in patients with reduced infarct area compared with the control group, increased blood flow rate and improved left ventricular function in the infarct area. ), And the implantation of autologous skeletal myoblasts (Seletal myoblast, Menasche et al. 2001, Lancet 357: 279-280) improved the function of the left ventricle and reported that metabolic activity in the infarcted heart was enhanced. Recently, the recovery of mononuclear cells that have been activated into peripheral blood from bone marrow of cardiovascular patients by injection of cytokine (G-CSF) has been reported to improve the contractility and motor capacity of the left ventricle (Kang HJ et al. 2004, Lancet). 363: 751-756).

However, reports of arrhythmia due to the absence of electrophysiological connections between transplanted cells and peripheral cells after transplantation of autologous skeletal myoblasts, and reports of stent-coronary restenosis were found in patients transplanted after G-CSF administration. Therefore, the necessity of research on the safety and the effects of treating stem diseases using stem cells has been raised.

Recently, when bone marrow-derived hematopoietic stem cells were transplanted into a cardiovascular disease model animal, differentiation into the blood cell line instead of the cardiomyocytes, unlike the initial report, improved cardiac function by angiogenesis, Claims have been made that transdifferentiation does not occur (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.

Recognizing the importance of stem cell research in spite of the insignificant effect of cardiovascular disease on cell therapy, advanced countries in the field of biotechnology and medicine have led the government to provide economic, political and legal support for stem cell research. have. The stem cell treatment market for cardiovascular disease is expected to reach $ 5.9 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.

Therefore, in order to apply the cardiovascular disease treatment to the clinic, it is necessary to develop technologies to improve the cardiac function after cell transplantation and to secure the efficacy and safety such as the viability, fate, differentiation, mobility of the transplanted cells and the possibility of differentiation into tumor cells. Required by the industry.

Accordingly, the present invention addresses technical issues related to cross-differentiation and cell fusion, which are controversial by developing a technology to monitor in-flight and in vivo whether stem cells transplanted for cell therapy of cardiovascular disease differentiate into functional cardiomyocytes. To contribute to secure the efficacy and safety of stem cell treatment.

Therefore, the main object of the present invention is to provide a recombinant expression vector for monitoring the differentiation into cardiomyocytes that can determine whether the stem cells transplanted for cell therapy of cardiovascular disease has differentiated into functional cardiomyocytes.

Another object of the present invention to provide a method for monitoring cardiomyocyte differentiation using the recombinant expression vector.

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

In the present invention, the expression vector refers to a vector into which a promoter inducing cardiomyocyte specific expression and a reporter gene operably linked thereto may be inserted or introduced. In addition, the reporter gene may be operably linked to a cardiomyocyte specific promoter, wherein the operably linked reporter gene sequence and promoter sequence are in one expression vector containing a selection marker and a replication origin together. May be included.

By “operably linked” is meant 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 may include 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 / detoxification. Sequences that control termination.

In the present invention, the promoter for inducing cardiomyocyte-specific expression refers to a promoter of marker proteins specifically expressed in cardiomyocytes, that is, a promoter capable of expressing cardiomyocyte-specific proteins by binding a transcription factor to these promoters. do.

In the present invention, in order to secure the mouse myosin light chain-2v promoter region corresponding to the rat myosin light chain-2v, rats were identified based on the complementarity of the nucleotide sequences based on the rat myosin light chain-2v promoter sequence and mouse gene bank information (NT_078458.5). A mouse myosin light chain-2v promoter region with a size of 327 bp showing approximately 90% identity to the sequence was selected. Considering cloning into the reporter gene, a primer pair unique to the mouse myosin light chain-2v promoter containing a specific restriction enzyme sequence was prepared and amplified by PCR using mouse genomic DNA as a template and identified as a TA cloning vector (FIG. 1). And 2). The sequencing assay confirmed that the promoter region identical to the sequence reported to the GenBank was cloned correctly. In the next step, the 327 bp mouse myosin light chain-2v promoter identified in the TA cloning vector was cloned into pDsRed-Express-1 having a reporter gene, and the cloning direction and size were confirmed by restriction enzyme treatment (FIG. 3). .

In the present invention, the rat myosin light chain-2v promoter region capable of reflecting cardiomyocyte differentiation is based on the existing research report and the information of the rat gene bank (GenBank # RNU26708) in consideration of cloning into the reporter gene, a specific restriction enzyme sequence. A unique primer pair was prepared and amplified by PCR using genomic DNA as a template, and then identified as a TA cloning vector (FIGS. 1 and 2). In addition, the sequencing assay confirmed that the promoter region identical to the sequence reported to the GenBank was correctly cloned. The next step is to clone the 309 bp rat myosin light chain-2v promoter identified into the TA cloning vector into pDsRed-Express-1, which contains the reporter gene DsRed but does not have its own promoter, and then cloned by restriction enzyme treatment. Direction and size were confirmed (FIG. 4).

The myocardial troponin-I promoter region that may reflect cardiomyocyte differentiation is based on previous research reports and mouse gene bank information (# NT_039413.7), considering the cloning into the reporter gene, and thus including a specific restriction enzyme sequence. Primer pairs were prepared, amplified by PCR using genomic DNA as a template, and identified as TA cloning vectors (FIGS. 1 and 2). In addition, the sequencing assay confirmed that the promoter region identical to the sequence reported to the GenBank was cloned correctly. The next step was to identify the 388 bp myocardial troponin-I promoter identified in the TA cloning vector into a vector (pEGFP-1 or pDsRed-Express-1) that contains the reporter genes EGFP and DsRed but does not have its own promoter. After cloning, the cloning direction and size were confirmed by restriction enzyme treatment (FIGS. 5 and 6).

In the recombinant expression vector of the present invention, the promoter inducing the cardiomyocyte-specific expression may be a promoter of any gene specific only when the stem cell is differentiated from the cardiomyocyte, but preferably the mouse myocardium having SEQ ID NO: 1 Myocin light chain 2v promoter, rat myocardium having SEQ ID NO: 2 Myosin light chain 2v promoter or mouse myocardium troponin I promoter having SEQ ID NO: 3.

In 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 the presence or absence of the protein expressing the reporter gene encoding the fluorescent protein Good to check 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 a preferred embodiment 2 of the present invention, a fluorescent protein, green fluorescent protein EGFP (enhanced GFP), and in Example 3, a red fluorescent protein DsRed was used, respectively, the base sequence of the EGFP gene (GenBank Accession No U55761) and the base sequence of the DsRed gene (GenBank 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 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 the green fluorescence protein (GFP), blue fluorescence It is preferably selected from the group consisting of genes of blue fluorescence protein (BFP), yellow fluorescence protein (YFP) and red fluorescence protein (RFP). The base sequence of the EGFP gene (GenBank Accession No. U55761) and the base sequence of the DsRed gene (GenBank Accession No. AF506025) used in the present invention are 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 shows 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 a method using immunostaining or confocal microscopy, the differentiation of stem cells to cardiomyocytes can be easily monitored without killing (or non-invasively).

In the recombinant expression vector of the present invention, the expression vector is pDsRed-Express-1 / mMLC having a cleavage map of Figure 3, pDsRed-Express-1 / rMLC having a cleavage map of Figure 4, having a cleavage map of Figure 5 It is preferred that it is selected from the group consisting of pEGFP-1 / cTn-1 and pDsRed-Express-1 / cTn-1 having a cleavage map of FIG.

According to another aspect of the present invention, the present invention provides a bone marrow-derived mesenchymal stem cells in which the reporter gene is expressed when the recombinant expression vector is transduced and differentiated into cardiomyocytes.

As a method of introducing the recombinant expression vector into a host cell, calcium chloride (CaCl ₂ ) and heat shock method (particle gun bombardment), silicon carbide whiskers, ultrasonic treatment (sonication), electroporation (electroporation), precipitation method by polyethylenglycol (PEG) and the like can 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 cardiomyocytes 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 a cardiac region of a cardiovascular disease patient, and may be transplanted using blood vessels of heart tissue. In animals, mobilized reactions secreted by the damaged heart after injecting stem cells into the tail vein (for example, when the SDF-1 molecule, the most well known cell mobilizing factor, is expressed at high levels in the damaged heart, It can also be used to confirm the hypothesis that stem cells from CXCR4-expressing bone marrow, peripheral blood, and the like move into the damaged heart and are involved in regeneration of damaged tissue. In addition, in order to enhance the engraftment rate of stem cells, various embedding materials such as sodium alginate, hyaluronic acid, collagen and the like can be used after forming a stem cell complex. After transplanting the stem cells into which the recombinant expression vector is introduced into a patient, blood and a very small amount of tissue samples may be taken to determine whether the transplanted cells are differentiated and to what extent.

According to another aspect of the invention, the invention provides a method of monitoring differentiation into cardiomyocytes comprising the following steps:

a) preparing mesenchymal stem cells;

b) transducing the recombinant expression vector of the present invention into the prepared stem cells; And

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

In the method for monitoring differentiation into cardiomyocytes of the present invention, a method for preparing mesenchymal stem cells in step a), in the present invention, to solve the limitation of stem cell research due to the absence of characteristic markers of mesenchymal stem cells For this purpose, 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. 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 of step c) is characterized by specific differentiation of stem cells to cardiomyocytes by confirming whether the expression of the reporter gene, such as fluorescent protein, by immunostaining or confocal microscopy. Can be monitored.

According to another aspect of the invention, the invention provides a primer set for amplifying a mouse myosin light chain 2v promoter having SEQ ID NOs: 4 and 5.

According to another aspect of the present invention, the present invention provides a primer set for amplifying the rat myosin light chain 2v promoter having SEQ ID NOs: 6 and 7.

According to another aspect of the present invention, the present invention provides a primer set for amplifying the myocardial troponin I promoter having SEQ ID NOs: 8 and 9.

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 incubated for 3 days in 0.1% gelatin coated cell vessel (10 cm) 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 culture for 6-7 days until the cell density reached 80% or more of the culture vessel.

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, the bone marrow cells were reacted with PE-bound Sca-1 antibody (D7 monoclonal antibody, BD Pharmingen, San Diego, CA, 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, followed by magnetization 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 steps of passing the magnetoactivity column were repeated four times to enhance the cell isolation purity.

Example 2 Construction of Expression Vector with DsRed Reporter Gene Attached to Mouse Myocardial Myosin Light Chain-2v Promoter

2-1. Amplification of the Mouse Myosin Light Chain-2v Promoter for Cloning into a DsRed Reporter Gene Expression Vector

Amplifying the mouse myosin light chain-2v promoter moiety that can induce specific expression upon differentiation into cardiomyocytes in the myosin light chain-2v promoter sequence (GenBank # NT_078458.5) via PCR from mouse C57BL / 6J genomic DNA In order to prepare a new primer pair as follows, PCR was performed. PCR reactions were performed on mouse genomic DNA with a primer set forth in SEQ ID NO: 4 comprising an Xho I restriction enzyme sequence, a primer set forth in SEQ ID NO: 5 comprising an EcoR I restriction enzyme sequence, dNTP, 10 × PCR buffer and Taq polymerase. The reaction was performed at 94 DEG C for 5 minutes, 35 seconds at 94 DEG C, 40 seconds at 56 DEG C, and 40 seconds at 72 DEG C, followed by reaction at 72 DEG 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 had a size of 327 bp corresponding to the gene bank sequence information (FIG. 1).

Mouse MLC-2v 1F- Xho I: 5'-ctcgagTCCTCCTCCTCCTGGGTTAC-3 '(SEQ ID NO: 4)

Mouse MLC-2v 1R- EcoR I: 5'-gaattcCCTCTGGAGAGTTCGAGGAG-3 '(SEQ ID NO: 5)

The 327 bp mouse myosin light chain-2v promoter gene fragment amplified by PCR was cloned into the pGEM-T Easy vector (Promega). FIG. 2 shows that the mouse myosin light chain-2v gene fragment having a size of 327 bp was correctly cloned after cleavage with a restriction enzyme to confirm cloning. As a result of sequencing using the T7 / SP6 primer position, the myosin light chain-2v was identified. It was confirmed to have a nucleotide sequence described as a promoter sequence.

2-2. Cloning the Mouse Myosin Light Chain-2v Promoter into a DsRed Reporter Gene Expression Vector

The mouse myosin light chain-2v promoter gene amplified in Example 2-1 was cloned into a pDsRed-Express-1 vector (Clontech, # 632412) without a promoter. Specifically, the mouse myosin light chain-2v fragment amplified in Example 2-1 was digested with Xho I and EcoR I and purified using a gel elution kit (Quieagen), and then digested with the same restriction enzyme. One pDsRed-Express-1 vector was ligated (T4 DNA ligase, Promega) to produce an expression vector comprising a mouse myosin light chain-2v promoter and a reporter gene, DsRed (FIG. 3). In addition, it was confirmed by performing electrophoresis whether the promoter was introduced into the expression vector, the results are shown in FIG.

Example 3 Construction of Expression Vector with DsRed Reporter Gene Attached to Rat Myocardial Myosin Light Chain-2v Promoter

3-1. Amplification of the Rat Myosin Light Chain-2v Promoter for Cloning into DsRed Reporter Gene Expression Vector

A portion of the rat myosin light chain-2v promoter that can induce specific expression upon differentiation into cardiomyocytes of the myosin light chain-2v promoter sequence (GenBank # RNU26708) via PCR from Sprague-Dawley rat genomic DNA. To amplify, a new primer pair was prepared as follows and PCR was performed. The PCR reaction was carried out using a primer described in SEQ ID NO: 6 comprising an Xho I restriction enzyme sequence, a primer described in SEQ ID NO: 7 comprising an EcoR I restriction enzyme sequence, dNTP, 10 × PCR buffer and Taq polymerase in rat genomic DNA. The reaction was performed 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, it was confirmed that the DNA fragment had a size of 309 bp corresponding to the gene bank sequence information (FIG. 1).

Rat MLC-2v 1F- Xho I: 5'-ctcgagTCCCTTCCTGGGTTACTTT-3 '(SEQ ID NO: 6)

Rat MLC-2v 1R- EcoR I: 5'-gaattcAGAATTCAAGGAGCCTGCTG-3 '(SEQ ID NO: 7)

The 309 bp rat myosin light chain-2v promoter gene fragment amplified by PCR was cloned into the pGEM-T Easy vector. Figure 2 shows that the 309 bp rat myosin light chain-2v gene fragment was correctly cloned after cleavage with restriction enzymes to confirm cloning, and sequence analysis using T7 / SP6 primer position revealed that myosin light chain-2v. It was confirmed to have a nucleotide sequence described as a promoter sequence.

3-2. Cloning the Rat Myosin Light Chain-2v Promoter into a DsRed Reporter Gene Expression Vector

The rat myosin light chain-2v promoter gene amplified in Example 3-1 was cloned into a pDsRed-Express-1 vector without a promoter. Specifically, the pDsRed-Express-1 vector digested with the same restriction enzyme after cleavage of the rat myosin light chain-2v fragment amplified in Example 3-1 with Xho I and EcoR I and then purified using a gel recovery kit. The ligation was performed to prepare an expression vector comprising the rat myosin light chain-2v promoter and the reporter gene DsRed (FIG. 4). In addition, it was confirmed by performing electrophoresis whether the promoter was introduced into the expression vector, the results are shown in FIG.

Example 4 Preparation of Expression Vector with EGFP Reporter Gene Attached to Mouse Myocardial Troponin-I Promoter

4-1. Amplification of Myocardial Troponin-I Promoter for Cloning into EGFP Reporter Gene Expression Vector

Mouse myocardial troponin-I promoter portion capable of inducing specific expression upon differentiation into cardiomyocytes of the mouse cardiomyocytes troponin-I promoter sequence (GenBank # NT_039413.7) via PCR from mouse C57BL / 6J genomic DNA. In order to amplify the new primer pairs were prepared as follows and PCR was performed. The PCR reaction was carried out using a primer described in SEQ ID NO: 8 containing a Sac I restriction enzyme sequence, a primer described in SEQ ID NO: 9 comprising a Kpn I restriction enzyme sequence, dNTP, 10 × PCR buffer and Taq polymerase in mouse genomic DNA. The reaction was performed 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, it was confirmed that the DNA fragment had a size of 388 bp corresponding to the gene bank sequencing information (FIG. 1).

cTn-I 1F- Sac I: 5'-agtagagctcGGCATGGATTTCTGGGTATG-3 '(SEQ ID NO: 8)

cTn-I 1R- Kpn I: 5' -atagggtaccCTCCAGAGGCAGAGAACAGG-3 '( SEQ ID NO: 9)

The 388 bp myocardial troponin-I promoter gene fragment amplified by PCR was digested with EcoR V restriction enzyme and cloned into CIAP treated pBlueScript II SK (+) vector (Stratagene). Figure 2 shows that 388 bp myocardial troponin-I gene fragment was correctly cloned after cleavage by restriction enzyme to confirm cloning, and sequence analysis using T7 / SP6 primer position, myocardial troponin- It was confirmed to have the nucleotide sequence described as the I promoter sequence.

4-2. Cloning Myocardial Troponin-I Promoter into EGFP Reporter Gene Expression Vector

The myocardial troponin-I promoter gene amplified in Example 4-1 was cloned into a pEGFP-1 vector consisting of a reporter gene sequence without a promoter. Specifically, the myocardial troponin-I fragment amplified in Example 4-1 was digested with Xho I and BamH I, purified using a gel recovery kit, and then purified with the same restriction enzyme pEGFP-1 vector. The ligation was performed to prepare an expression vector containing the myocardial troponin-I promoter and the reporter gene EGFP (FIG. 5). In addition, it was confirmed by performing electrophoresis whether the promoter was introduced into the expression vector, the results are shown in FIG.

Example 5 Construction of Expression Vector with DsRed Reporter Gene Attached to Mouse Myocardial Troponin-I Promoter

Myocardial troponin-I promoter portion ( Sac I-cTn-I- EcoR) by Sac I / EcoR I restriction enzyme treatment in an expression vector having EGFP attached to the myocardial troponin-I promoter constructed in Example 4-2. I) was cut and purified using a gel recovery kit. The expression vector in which 388 bp myocardial troponin-I promoter was inserted into Sac I / EcoR I of the reporter gene pDsRed-Express-1 was digested with the pDsRed-Express-1 vector digested with the same restriction enzyme. (FIG. 6). In addition, it was confirmed by performing electrophoresis whether the promoter was introduced into the expression vector, the results are shown in FIG.

Example 6 Verification of specificity of cardiomyocyte differentiation after introduction of cardiomyocyte specific expression vector into Sca-1 + bone marrow mesenchymal cells

Sca-1 + bone marrow mesenchymal stem cells (2 × 10 ⁴ cells / well) isolated by Example 1 using DMEM medium containing 10% fetal bovine 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 incubation, the cardiomyocyte promoter expression vectors (mMLC-DsRed, rMLC-DsRed, and cTn-I-EGFP) of 1 ug, respectively, constructed in FIGS. After mixing to react 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 mixing the expression vector and lipofectamine solution, the reaction was performed at room temperature for 20 minutes, and then cultured for 48 hours after addition into the culture solution inoculated with Sca-1 + bone marrow mesenchymal stem cells. After 48 hours, the medium containing DMEM + 10% fetal bovine serum was washed twice, treated with 5-azacytidine at a concentration of 1 μM, and then cultured for 14 days to induce differentiation into cardiomyocytes. Cultures containing 5-azacytidine were exchanged every 3 days.

To determine whether the reporter gene expressed in the cardiomyocyte promoter showed specific expression only in cells differentiated into cardiomyocytes, Sca-1 + bone marrow mesenchymal stem cells attached to 0.1% gelatin-coated coverslip were subjected to cardiomyocyte-specific antibody. It confirmed by the immunostaining method used. 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, incubated for 1 hour with 5% goat serum, incubated for 1 hour using myosin light chain-2v antibody (Simga) and troponin-IC antibody (Santa Cruz Biotechnology), primary antibody, washed with PBST solution, 1 hour incubation with secondary antibody Alexa 488 antibody (Molecular Probes, Eugene, OR, USA) or Cy3 antibody (Zymed Laboratories Inc., San Francisco, USA), washed with PBST solution, 1 hour with DAPI solution for nuclear staining After culturing and washing with PBST solution, a sample was prepared using a mounting solution for maintaining the intensity of the fluorescence signal under fluorescence microscopy and observed under fluorescence microscopy (FIG. 8).

As described above, according to the present invention, expression vectors having an EGFP or DsRed reporter gene attached to a cardiomyocyte specific promoter sequence capable of detecting whether stem cells have been differentiated into a cardiomyocyte system can be constructed. After introduction into stem cells, differentiation into cardiomyocytes was induced by 5-azacytidine treatment, and then stem cells were identified by immunostaining to express reporter gene expression only in cells differentiated into cardiomyocytes. It has been demonstrated that it can detect whether it has differentiated into a lineage. Therefore, the cardiomyocyte-specific expression vector constructed in the present invention shows that it can specifically reflect the differentiation of cardiomyocytes, and is expected to contribute greatly to the study of cell therapy of cardiovascular diseases. In addition, by integrating the cardiomyocyte differentiation detection technology and molecular imaging technology developed by the present invention, non-invasive in vivo stem cell differentiation, fate research, etc. will greatly contribute to securing the efficacy and safety of stem cell treatment. .

Figure 1 is amplified by PCR using genomic DNA promoter (mMLC, cTn-I, rMLC) inducing cardiomyocyte specific expression according to the present invention, electrophoresis that the DNA fragment of the expected size was produced Photograph confirmed by: Lane M is 100 bp DNA ladder size marker.

Figure 2 shows that promoters (mMLC, cTn-I, rMLC) inducing cardiomyocyte specific expression amplified by PCR according to the present invention was cloned into pGEM-T Easy, which is a TA cloning vector. Photograph confirmed by: Lane M is 100 bp DNA ladder size marker.

3 is a cleavage map of the recombinant expression vector pDsRed-Express-1 / mMLC according to the present invention.

4 is a cleavage map of the recombinant expression vector pDsRed-Express-1 / rMLC according to the present invention.

5 is a cleavage map of the recombinant expression vector pEGFP / cTn-1 according to the present invention.

6 is a cleavage map of the recombinant expression vector pDsRed-Express-1 / cTn-1 according to the present invention.

Figure 7 is a photogram confirmed by electrophoresis after cleavage of a cardiomyocyte promoter (mMLC, rMLC, cTn-I) cloned into a pEGFP-1 or pDsRed-Express-1 vector using restriction enzymes: pDsRed-Express-1 Lane M cloned into the vector is a λ / Hind III size marker. Lane M cloned into pEGFP-1 vector is a 100 bp DNA ladder size marker.

Figure 8 is after the introduction of the expression vector DsRed and EGFP marker vector attached to the Sca-1 + mesenchymal stem cells promoters (mMLC, rMLC, cTn-I) inducing cardiomyocyte specific expression according to the present invention After inducing differentiation into cardiomyocytes by 5-azacytidine treatment, immunostaining was performed to verify that the cells expressing reporter genes were simultaneously stained with cardiomyocyte specific antibodies.

<110> Korea University Industry and Academy Cooperation Foundation <120> Expression vectors for monitering differentiation of mesenchymal          stem cell into the cardiomyogenic lineage, and monitering methods          of differentiating into the cardiomyogenic lineage by using the          same <160> 9 <170> KopatentIn 1.71 <210> 1 <211> 327 <212> DNA <213> Mus musculus <400> 1 tcctcctcct cctgggttac tcttccccat tagacaatgg cagggaagag agcacacccc 60 atcatcccca ggccaggccc cagccactga ctctttaacc ttgaaggcat ttttgggtct 120 cacgtgtcca cccaggcggg tggccgcctt tgagcagctc ttacttcaga agaacggcat 180 ggagtggggg gtggggggct taggtggcct ccgcctcacc tacaactgcc aaaagtggtc 240 atggggttat ttttaacccc aggggagagg tatttattgt tccacagcag gggcagaggc 300 cagcaggctc ctcgaactct ccagagg 327 <210> 2 <211> 309 <212> DNA <213> Rattus norvegicus <400> 2 tccccttcct gggttacttt tccccattag acaatggcag gacccagagc acagagcatc 60 gttcccaggc caggccccag ccactgtctc tttaaccttg aaggcatttt tgggtctcac 120 gtgtccaccc aggcgggtgt cggactttga acggctctta cttcagaaga acggcatggg 180 gtgggggggc ttaggtggcc tctgcctcac ctacaactgc caaaagtggt catggggtta 240 tttttaaccc cagggaagag gtatttattg ttccacagca ggggccggcc agcaggctcc 300 ttgaattct 309 <210> 3 <211> 388 <212> DNA <213> Mus musculus <400> 3 ggcatggatt tctgggtatg aacatgggaa gctgaagtct agactcttgg atttgagaga 60 agagggacct tgctccgggt tttcctaagt ttgagggagg agggagctgg ggcgctagag 120 tcaaaggagg aggggtgtag atcctgggca ccttggttga cccaactgga gctttgcaca 180 cggctcccct cacaccctgt tatcgcttat cctgggcagg ggaggagaca gcagtatatt 240 tagtctttgt cctcgcccct tatctcagtg tcctcagtga ggcttgagca gcccagagga 300 aacccaacct ctagagacct ccaaggtcac cagggacacc cttccaggac cctccaggaa 360 tctccgatcc tgttctctgc ctctggag 388 <210> 4 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> forward primer for mM LC <400> 4 ctcgagtcct cctcctcctg ggttac 26 <210> 5 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for mM LC <400> 5 gaattccctc tggagagttc gaggag 26 <210> 6 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> forward primer for rMLC <400> 6 ctcgagtccc ttcctgggtt acttt 25 <210> 7 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for rMLC <400> 7 gaattcagaa ttcaaggagc ctgctg 26 <210> 8 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> forward primer for cTn-I <400> 8 agtagagctc ggcatggatt tctgggtatg 30 <210> 9 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for cTn-I <400> 9 atagggtacc ctccagaggc agagaacagg 30  

Claims (9)

A recombinant expression vector for monitoring differentiation into cardiomyocytes comprising a promoter inducing cardiomyocyte specific expression and a reporter gene operably linked thereto. According to claim 1, wherein the promoter for inducing cardiomyocyte specific expression is a mouse myosin light chain 2v promoter having SEQ ID NO: 1, rat myosin light chain 2v promoter having SEQ ID NO: 2 or myocardial troponin I promoter of SEQ ID NO: 3 Characterized by a recombinant expression vector. According to claim 1, wherein the reporter gene is a group consisting of genes of green fluorescence protein (green fluorescence protein), blue fluorescence protein (blue fluorescence protein), yellow fluorescence protein (red fluorescence protein) and red fluorescence protein (red fluorescence protein) Recombinant expression vector, characterized in that selected from. According to claim 1, wherein the expression vector pDsRed-Express-1 / mMLC having a cleavage map of Figure 3, pDsRed-Express-1 / rMLC having a cleavage map of Figure 4, pEGFP-1 having a cleavage map of Figure 5 Recombinant expression vector, characterized in that selected from the group consisting of / cTn-1 and pDsRed-Express-1 / cTn-1 having a cleavage map of FIG. Bone marrow-derived mesenchymal stem cells wherein the reporter gene is expressed when the recombinant expression vector according to claim 1 is transduced and differentiated into cardiomyocytes. A method for monitoring differentiation into cardiomyocytes comprising the following steps: a) preparing mesenchymal stem cells; b) transducing the recombinant expression vector according to claim 1 to the prepared stem cells; And c) confirming the expression of the reporter gene in the transduced stem cells. A primer set for amplifying the mouse myosin light chain 2v promoter having SEQ ID NOs: 4 and 5. A primer set for amplifying the rat myosin light chain 2v promoter having SEQ ID NOs: 6 and 7. A primer set for amplifying the myocardial troponin I promoter with SEQ ID NOs: 8 and 9.

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