KR101957370B1 - An episomal vector harboring suicide gene and method for reprogramming using the same - Google Patents

Abstract

The present invention relates to an episome vector containing a suicide gene and a reprogramming method using the episome vector. More particularly, the present invention relates to an episome vector containing a reprogramming factor and a suicide gene, And selecting cells that do not contain foreign DNA.
Since the reprogramming method of the present invention can produce reprogrammed cells containing no foreign DNA by using an episome vector containing reprogramming factors and suicide genes, Can be usefully used.

Description

An episomal vector containing a suicide gene and an episomal vector harboring suicide gene and method for reprogramming using the same,

The present invention relates to an episome vector containing a suicide gene and a reprogramming method using the episome vector. More particularly, the present invention relates to an episome vector containing a reprogramming factor and a suicide gene, Lt; / RTI >

Human induced pluripotent stem cells (iPSCs) have been regarded as a tool for direct research into human diseases and as a source of regenerative medicine since its development (Takahashi et al., Cell, 2007, 131: 861-872). One method for producing iPSCs is to use an episomal vector. This method shows relatively low reprogramming efficiency compared to other reprogramming methods, but it is a method that can obtain a safe iPSC at a low cost. Since episomal vectors can naturally escape from the cells during cell culture, they enable the production of exogene-free iPSCs. However, it has been reported that the degenerated cells prepared using the episome vector are inserted into the chromosome more frequently than the vector was expected, and the residual episome vector may remain even when cultured over 10 passages (Schlaeger et al. Nat Biotechnol, 2015, 33: 58-63). The presence of a residual vector in a cell for an extended period of time increases the probability of insertion into the genome of the host, thereby causing cancer, destroying the characteristics of normal cells, and further causing cell death. For this reason, researchers need to isolate iPSCs that do not have a residual vector for application in regenerative medicine applications, but the separation process can take a lot of labor, time, and cost. In order to easily screen for iPSCs that do not contain foreign DNA, mRNA transfection or Sendai viruses have been developed as reprogramming tools, but these methods are expensive and development of new reprogramming systems is still required.

Meanwhile, scURA3 The gene is called < RTI ID = 0.0 > Saccharomyces < / RTI > is a gene encoding orotidine 5'-phosphate decarboxylase (ODCase) necessary for cell growth in S. cerevisiae. When 5-fluoroorotic acid (5-FOA) is added to the medium, ODCase converts 5-FOA to 5-fluorouracil (5-FU), which is toxic to cells, leading to apoptosis. S. cerevisiae with scURA3 is known to abrogate this gene for survival under conditions in which 5-FOA is applied.

Similarly, scFCY1 , a suicide gene that encodes cytosine deaminase (CD), is able to convert the non-toxic pro-drug 5-fluorocytosine (5-FC) to the toxic drug 5-FU . The 5-FU cytotoxic mechanism is characterized by the misincorporation of 5-fluoro-uridine-triphosphate (5-FUTP) and 5-fluoro-deoxyuridine-triphosphate (5-FdUTP), and the nucleotide synthesis enzyme thymidylate synthetase (thymidylate synthase). The suicide gene has been reported to have been applied to cancer treatment to have a tumor killing effect (Altanerova et al., Int J Cancer, 2012, 130: 2455-2463; Yi et al., Stem Cell Res, 2014, 12: 36-48) , It has not been reported so far to produce reprogrammed cells that do not contain foreign DNA using such suicide genes.

As a result of intensive efforts to develop a method for producing reprogrammed cells containing no foreign DNA using an episome vector, the present inventors have found that reprogramming and reprogramming of cells using an episome vector containing a suicide gene It has been confirmed that reprogrammed cells containing no foreign DNA can be easily obtained by negative selection of cells having a residual episome vector through pro-drug treatment, thereby completing the present invention .

One object of the present invention is to provide an episome vector comprising (i) a reprogramming factor and (ii) a suicide gene.

It is another object of the present invention to provide a method for the treatment and prophylaxis of cancer, comprising introducing the episome vector into isolated cells to reprogram the cells and treating the pro-drug to select cells that do not contain foreign DNA , A method of reprogramming a cell.

Each description and embodiment disclosed in the present invention can be applied to each other description and embodiment. That is, all combinations of various elements disclosed in the present invention fall within the scope of the present invention. Further, the scope of the present invention is not limited by the detailed description described below.

The method of reprogramming a cell by introducing a foreign gene into a cell is a very useful method for securing a patient-customized cell particularly as a regenerative medicine resource. Since the first development of the iPSC manufacturing method by the Yamanaka Group in 2006, research and clinical trials related to reprogramming have been very active worldwide. However, by introducing a foreign gene into cells, not only the possibility of cancer induced by the introduced gene itself, but also foreign DNA can be inserted into the genome of cells, resulting in problems such as cancer formation caused therefrom.

As a method for solving such a problem, there has been developed a method of introducing a reprogramming factor into a cell using an episome vector, but it takes much time to remove the episome vector from the introduced cell, and further, Lt; / RTI > vector may be inserted into the genome of the cell.

Accordingly, the present inventors have developed episome vectors containing reprogramming factors and suicide genes to solve the problems of episome vectors in reprogramming cells. Since the vector expresses a suicide gene as well as a reprogramming factor, the vector may be introduced to treat the reprogrammed cell with a pro-drug against the suicide gene to rapidly remove the cells containing the episome vector, Rapid removal of episomal vectors remaining in reprogrammed cells has the advantage of ensuring reprogrammed cells that do not contain foreign DNA within a short period of time.

Hereinafter, the present invention will be described in detail.

One aspect of the present invention for achieving the above object is a method for producing a cell, comprising: (a) introducing an episome vector comprising (i) a reprogramming factor and (ii) a suicide gene into isolated cells; (b) culturing the cells into which the episome vector has been introduced in a medium to reprogram the cells; And (c) culturing the reprogrammed cells in a medium containing a pro-drug to select cells that do not contain foreign DNA.

The term " reprogramming " in the present invention means a method of converting a global gene expression pattern of a specific cell into a desired cell. In other words, in the present invention, reprogramming refers to a method of artificially manipulating cells to convert them into cells having completely different characteristics. For the purpose of the present invention, the reprogramming is performed by introducing a foreign gene or a vector containing DNA into cells ≪ / RTI > By way of example, reprogramming may include, but is not limited to, dedifferentiation or transdifferentiation of cells. The cells that can be produced by the reprogramming method include not only stem cells such as induced pluripotent stem cells, neural stem cells, mesenchymal stem cells, etc. but also fully differentiated cells such as neurons, pancreatic cells, liver cells, All of which can be included in the scope of the present invention and can be produced by varying the reprogramming factor depending on the kind of the desired cell.

In the present invention, the term " differentiation " refers to a phenomenon in which the structure or function of a cell is specialized. In other words, it refers to a process in which cells, tissues, etc. of a living organism are changed into appropriate forms and functions to perform their respective roles. For example, the process of transforming pluripotent stem cells such as embryonic stem cells into ectoderm, mesoderm, and endoderm cells is also differentiation. Narrowly, the processes in which hematopoietic stem cells change into red blood cells, white blood cells, platelets, etc. are also differentiated.

The term " dedifferentiation " in the present invention means a process by which a differentiated cell can be restored to a state of having a potential of a new type of differentiation, and " transdifferentiation " Is converted into a cell. These cell degenerative and cross-differentiating mechanisms are characterized by a different set of epigenetic markers after deletion of the markers in the nucleus (epigenetics, DNA conditions associated with genetic changes in function without changes in the nucleotide sequence) And the like.

The term " pluripotent stem cell " in the present invention refers to a pluripotent stem cell capable of pluripotent or totipotent self-renewal capable of differentiating into cells of all tissues of an individual Stem cells, including, but not limited to, embryonic stem cells and induced pluripotent stem cells.

The term " embryonic stem cell " in the present invention refers to an embryo obtained by extracting an inner cell mass from a blastocyst embryo immediately before embryo transfer into the uterus of a mother and culturing the same in vitro, Refers to a cell that has a self-renewal ability that is pluripotent or totipotent.

The term " induced pluripotent stem cell " (iPSC) in the present invention refers to cells induced to have universal differentiation ability through artificial reprogramming from differentiated cells. An artificial reprogramming process may be performed by introduction of a non-viral-mediated reprogramming factor using virus-mediated or non-viral vector utilization, retroviruses and lentiviruses, proteins and cell extracts, or by stem cell extracts, And includes a de-differentiation process. Induced pluripotent stem cells have almost the same characteristics as embryonic stem cells. Specifically, they show the same cell shape. They are similar in gene and protein expression pattern, and are universal in in vitro and in vivo , Which forms a teratoma and is inserted into a blastocyst of a mouse to form a chimera mouse and enable germline transmission of the gene. The inducible pluripotent stem cells of the present invention may include all the induced pluripotent stem cells derived from human, monkey, pig, horse, cow, sheep, dog, cat, mouse, rabbit and the like, May be, but are not limited to, pluripotent stem cells.

Specifically, each step of the reprogramming method includes: (a) introducing an episome vector containing (i) a reprogramming factor and (ii) a suicide gene into isolated cells; to be.

As used herein, the term " reprogramming factor " refers to a gene (or a polynucleotide encoding it) capable of being introduced into a cell to induce reprogramming. The reprogramming factor may vary depending on the target cell from which reprogramming is to be induced, and on the type of isolated cell from which the reprogramming is induced. For example, in the case of producing induced pluripotent stem cells, reprogramming factors include one or more genes selected from the group consisting of Oct4, Sox2, Klf4, Lin28, C-myc and L-myc And may include all factors known in the art as being capable of producing induced pluripotent stem cells. In addition, Ascl1, Pitx3, Nurr1, and Lmx1a can be introduced into cells as reprogramming factors to induce cross-differentiation into dopaminergic neurons. In the cross-differentiation methodology, there is a method using an allotropic agent such as Oct4, Sox2, Klf4, Lin28, and C-myc, and the vector of the present invention can be utilized for such an application. Therefore, a person skilled in the art can select an appropriate factor depending on the target cell and the type of the cell before being reprogrammed, and it is within the scope of the present invention within the range known to those skilled in the art, Do not. As described above, reprogramming regulates the entire gene expression pattern of a cell to induce conversion to a target cell. Therefore, when the reprogramming factor is introduced into a cell and the cell is cultured for a predetermined period of time, The cells can be reprogrammed with the target cells having the expression pattern.

The term " suicide gene " in the present invention refers to a gene encoding a protein (e.g., an enzyme) having an activity of converting a non-toxic pro-drug into a cytotoxic drug. In addition, the suicide gene can be used regardless of its origin if it has only a function of coding a protein (for example, an enzyme) having an activity of converting a non-toxic pro-drug into a cytotoxic drug. In addition, the suicide genes and proteins can be used without limitation as long as they are known in the art, and the sequence can be obtained from GenBank of NCBI, a known database.

The suicide gene may be, but is not limited to, cytosine deaminase (CD), thymidine kinase, or cytochrome P450. The prodrug may specifically be, but is not limited to, 5-fluorocytosine (5-FC), ganciclovir or cyclophosphamide. More specifically, cytosine deaminase (CD) in the suicide gene reacts with 5-fluorocytosine (5-FC) as a prodrug and thymidine kinase is a prodrug of Ganciclovir as a prodrug. And cytochrome P450 is a prodrug and may react with cyclophosphamide, but is not limited thereto.

The cytosine diaminase is an enzyme that catalyzes the reaction of producing uracil by hydrolyzing an amino group using cytosine as a substrate. The cytosine diaminase produces 5-fluorouracil by 5-fluorocytosine (5-FC), a non-toxic prodrug, by deamination with 5-FU, Lt; RTI ID = 0.0 > cytotoxicity. ≪ / RTI > In the present invention, the cytosine deaminase is specifically Saccharomyces Roman Isis three Levy jiae (Saccharomyces cerevisiae , but is not limited thereto.

In the present invention, an episomal vector is a nonviral non-insertion vector. It is known that the episomal vector is capable of expressing a gene contained in a vector without being inserted into a chromosome. However, when the episome vector is introduced into the cell as described above, it may remain in the cell for a long time or may be inserted into the genome and become a problem. In the present invention, the reprogramming factor capable of inducing reprogramming in the episome vector And suicide genes were included to selectively remove cells containing the episome vector. In the present invention, the cells comprising the episome vector include all cases in which the episome vector is inserted into the genome or existing in the cell without being inserted into the genome. In one example, the episome vector may comprise one or two reprogramming factors with suicide genes.

The episomal vector may be specifically an Epstein-Barr Virus-based vector, and more specifically, it may be a pCXLE vector or a pCEP4 vector, but a person skilled in the art can select an appropriate episome vector according to his purpose Can be applied.

By introducing the episome vector into the isolated cells, cells containing epiposomal vectors including reprogramming factors and suicide genes can be prepared, and the cells can be reprogrammed by the introduced episome vector Suicide genes can be expressed. The isolated cells may be somatic cells or stem cells, and may be suitably selected by those skilled in the art depending on the purpose and are not particularly limited to the types. Intracellular introduction of the episome vector in the present invention can be carried out by appropriately selecting a person skilled in the art by a known transformation method.

In one specific embodiment of the present invention, an episome vector comprising cytosine diaminase as a suicide gene and OCT4 as a reprogramming factor, or SOX2 and KLF4, or L-MYC and LIN28, respectively, was prepared in three episomal vectors And introduced into human-derived cancer cell lines to confirm the expression of each gene (FIG. 2).

In the reprogramming method of the present invention, step (b) is a step of reprogramming cells by culturing the cells into which the episome vector has been introduced in a medium. the cells into which the episome vector of step (a) has been introduced can be reprogrammed by reprogramming factors expressed by the episome vector during the culture process.

In the present invention, the term " cultivation " means that the cells are grown under moderately controlled environmental conditions. The culturing process of the present invention can be carried out according to a suitable culture medium and culture conditions known in the art. Such a culturing process can be easily adjusted by those skilled in the art depending on the cell to be selected.

Particularly, since step (b) is a process of culturing the cells into which the reprogramming factor has been introduced, the culture medium for culturing the cells is a composition suitable for reprogramming cells, that is, . Therefore, for example, in the case of producing induced pluripotent stem cells using the reprogramming method of the present invention, the medium of step (b) may have a medium composition suitable for culturing induced pluripotent stem cells or embryonic stem cells. In addition, if the neural stem cells are to be produced, the medium of step (b) may have a medium composition suitable for culturing the neural stem cells. Such a medium composition can be suitably selected and applied by those skilled in the art within the range already known in the art.

In particular, when an inducible pluripotent stem cell is to be produced using the reprogramming method of the present invention, the medium may further include a substance capable of enhancing reprogramming efficiency. Such a substance may be, for example, an ALK5 inhibitor, a GSK-3 inhibitor, a histone deacetylase (HDAC) inhibitor or nicotinamide, and specifically, A83-01, CHIR99021, sodium butyrate or nicotine Amide, but is not limited thereto.

In one specific embodiment of the present invention, an episome vector containing the reprogramming factor and a cytosine deaminase gene is introduced into a human-derived fibroblast and cultured to induce induction pluripotent stem cells (CD-iPSC) (Fig. 3). In the present invention, an inducible pluripotent stem cell prepared by introducing an episome vector containing the cytosine deaminase gene was named CD-iPSC.

In the reprogramming method of the present invention, step (c) includes culturing the reprogrammed cells through the step (b) in a medium containing a pre-drug to select cells that do not contain foreign DNA to be. In step (c), the suicide gene contained in the episome vector introduced into the cell is expressed, whereby the prodrug is converted into a cytotoxic substance by the protein encoded by the suicide gene. Accordingly, the cells in which the dielectric insertion of the episome vector is removed, and the episome vector remaining without the insertion of the dielectric in the reprogrammed cell are rapidly removed, and only the cells not containing the episome vector can be selected.

When the suicide gene is cytosine diamine, the prodrug may be 5-fluorocytosine (5-FC). In the case of thymidine kinase, the prodrug may be Ganciclovir. In the case of cytochrome P450, May be cyclophosphamide. That is, the prodrug used in step (c) is determined in correspondence with the suicide gene, and is converted into a cytotoxic substance by the suicide gene to selectively remove the episome vector, that is, the cell into which the foreign DNA has been inserted And the foreign DNA remaining in the cell without the insertion of a dielectric substance is rapidly removed, and cells without foreign DNA can be selected.

When 5-FC is used as a prodrug, the 5-FC may be contained in the medium in a concentration of 20 μM to 100 μM, specifically, 50 μM to 100 μM, but is not limited thereto. 5-FC does not show toxicity when treated with cells, but can be toxic to cells when converted to 5-fluorouracil by a protein encoded by a suicide gene.

In the step (c), the process of selectively removing the cells containing the episome vector by treating the prodrug may include culturing the reprogrammed cells in one or more, two or more, three or more, or four or more Or culturing for 7 days to 28 days, but the present invention is not limited thereto. Also, the time required for screening may vary depending on the cells used, suicide genes, prodrugs, and the like.

In a specific example of the present invention, it was confirmed that the amount of the episome vector remaining in the CD-iPSC by the treatment of 5-FC rapidly decreased (Fig. 4), and the cytosine diaminase- Note the human-derived embryos were treated for 5-FC to stem cells confirmed the selective removal is possible (FIG. 7 and 8), induced pluripotent stem cells that do not contain the foreign gene were created using the above-mentioned procedure, in vitro (in in vitro and in vivo , and found no abnormality of the genome of the genome (FIG. 9).

In a specific embodiment of the present invention, an episome vector containing the reprogramming factor and a cytosine deaminase gene is introduced into human-derived fibroblasts and cultured to produce inducible neural stem cells 10). In the present invention, an inducible neural stem cell prepared by introducing an episome vector containing the cytosine deaminase gene was named CD-iNSC.

It was confirmed that the amount of the remaining episome vector was rapidly decreased by 5-FC treatment in CD-iNSC, and the inducible neural stem cells without the external gene generated through the above process were found to be similar to the representative neural stem cell marker proteins PAX6 and N- The expression of CADHERIN was confirmed and the expression of KI67, a typical marker protein for proliferation, was confirmed (FIG. 10).

In general, it is known that a complete removal of an episome vector is carried out after culturing at least 10 passages or more. However, when cultured at a single cell level using the method of the present invention, the episome vector can be removed by only one passage As a result, it can be seen that reprogrammed cells containing no foreign DNA can be obtained within a short period of time.

Therefore, the reprogramming method using the episome vector of the present invention can rapidly remove the foreign DNA from the reprogrammed cell, and thus, compared with the cell reprogramming method using the existing episome vector, . Furthermore, the inducible pluripotent stem cells and inducible neural stem cells prepared by the reprogramming method of the present invention can be usefully used for studying the mechanism of a specific disease or for developing a cell therapy agent.

Another aspect of the present invention is an episome vector comprising a gene of (i) reprogramming factor and (ii) cytosine deaminase (CD), a suicide gene.

Reprogramming factor, suicide gene and episome vector are as described above.

Since the reprogramming method of the present invention can produce reprogrammed cells containing no foreign DNA by using an episome vector containing reprogramming factors and suicide genes, Can be usefully used.

FIG. 1 is a graph showing the results of a process for inducing a fibroblast isolated from a human to an inducible pluripotent stem cell using an episome vector comprising the cytosine deaminase of the present invention, Induced pluripotent stem cells. ≪ Desc / Clms Page number 2 >
2A and 2B are diagrams showing the production of an episome vector containing cytosine deaminase, FIG. 2A is a schematic diagram of an episome vector containing cytosine diaminase, FIG. 2B is a schematic diagram of an episome vector containing cytosine deaminase, In order to confirm the function of the episome vector containing minase, the expression of the gene contained in each episome vector was confirmed.
FIG. 3 is a diagram illustrating a process of inducing fibroblasts isolated from humans to inducible pluripotent stem cells using an episome vector comprising cytosine deaminase of the present invention.
FIGS. 4A and 4B are graphs showing the effect of 5-FC on the cytosine deaminase activity of the episomal vector and 5-FC treated exogenously in the inducible pluripotent stem cell The amount is confirmed. Specifically, FIG. 4A shows the amount of the remaining episome vector when 5-FC is treated on the induced pluripotent stem cells produced using an episome vector not containing cytosine diaminase. FIG. 4 B shows the amount of remaining episome vector when 5-FC is treated on induced pluripotent stem cells produced using an episome vector containing cytosine diamidinase.
FIG. 5 shows the time point at which the episomal vector was completely removed due to the presence or absence of cytosine diaminase and exogenously treated 5-FC in each case of induction of pluripotent stem cells into a large-scale culture or single cell level It is also confirmed.
6A to 6C are diagrams showing the toxicity of 5-FC and 5-FU in embryonic stem cells. Specifically, FIG. 6A shows the concentration of 5-FC or 5-FU treated with HuES9 cells at concentrations of 0 to 1, 10, 50, and 100 μM to show the toxicity of each substance. FIG. 6C is a graph showing the concentrations of 5-FC or 5-FU treated with H9 cells at concentrations of 0, 2.5, 5.0, 7.5 and 10 μM, B shows the state of the cells under a microscope.
FIGS. 7A to 7C show that 293T cells inserted with a CD gene as a genome selectively induce apoptosis when 5-FC was treated by inserting EGFP or CD-EGFP into the genome of 293T cells using lentivirus . Specifically, FIG. 7A shows the state of cells and the expression of EGFP after microscopic examination of 293T cells in which EGFP or CD-EGFP was inserted into the genome for 0 days, 10, 50 μM 5-FC for 6 days FIG. 7B shows that 293T cells in which EGFP or CD-EGFP was inserted into the genome were treated with 0, 10, and 50 μM 5-FC for 6 days, respectively. After collecting the cells, cells expressing green fluorescence were analyzed by flow cytometry FIG. 7C is a graph showing the relative number of cells after the treatment of 293T cells in which EGFP or CD-EGFP was inserted into the genome for 0 days, 10, and 50 μM 5-FC for 6 days, respectively Fig.
FIGS. 8A and 8B are graphs showing that H9 cells in which CD is inserted into a genome selectively induce apoptosis by inserting EGFP or CD-EGFP into the genome of H9 cells using lentivirus and treating 5-FC to be. Specifically, FIG. 8A shows the number of cells directly counted after 48 hours of treatment with 50 μM 5-FC to H9 cells in which EGFP or CD-EGFP was inserted into the genome. FIG. 8B shows EGFP Or H9 cells in which CD-EGFP was inserted into the genome were observed microscopically at intervals of 12 hours from before treatment with 50 μM 5-FC.
FIGS. 9A to 9F are graphs showing the characteristics of the foreign gene-free inducible pluripotent stem cells selected in the above experiment. Specifically, FIG. 9A shows the expression of a pluripotency maker protein in CD-iPSC, and FIG. 9B shows CD-iPSC with alkaline phosphatase staining (AP staining) Fig. 9C shows the expression of the ectoderm, mesoderm and endodermic marker proteins of the embryoid body (EB) differentiated from CD-iPSC. FIG. 9D shows the differentiation of ectoderm, mesoderm and endoderm in vivo of mice. E of FIG. 9 shows the karyotype analysis result of CD-iPSC And F in FIG. 9 is a diagram showing a result of short tandem repeat (STR) analysis of fibroblasts and CD-iPSC before reprogramming.
FIG. 10 is a view for confirming the amount of an episome vector remaining in the induction neural stem cells when 5-FC is treated in CD-iNSC and confirming the characteristics of selected foreign gene-free induction neural stem cells. Specifically, FIG. 10A shows the amount of the remaining episome vector when 5-FC is treated or not treated on the induction neural stem cells prepared using the episome vector containing cytosine deaminase. FIG. 10B shows the expression of the neural stem cell marker protein in the CD-iNSC selected in FIG. 10A.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are intended to illustrate the present invention, and the scope of the present invention is not limited to these examples.

Example  1: cytosine Diaminazine  Included Episome  Vector production and production Episome  Check the function of the vector

<1-1> Cytosine Diaminazine  Included Episome  Production of vector

The following method was used to prepare the pCXLE-hOCT4-CD vector (FIG. 2A). The OCT4 gene DNA was obtained from the pHAGE2-EF1a-hSTEMCCA-W-loxP vector by polymerase chain reaction. The DNA of the suicide gene Cytosine deaminase (CD) gene was obtained from the genome of Saccharomyces cerevisiae through polymerase chain reaction. The resulting DNA fragments were ligated into a single piece using the Gibson assembly reaction kit (Gibson Assembly reaction kit; New England Biolabs, USA) and amplified in a large amount by polymerase chain reaction. Eoteonaen Purification of the amplified DNA fragments was performed following the TOPO cloning reaction ^(TM pENTR Directional TOPO ^® Cloning Kits; Invitrogen, USA). The reaction was then diluted with the Escherichia coli ) cells and cultured in a 37 ° C incubator. Plasmid DNA was obtained from some of the transgenic strains that appeared the following day, and sequencing was requested by Genotech Corp. (Korea) to analyze the nucleotide sequence. The analyzed plasmid DNA was cloned into pCXLE-GW vector through LR cloning (Gateway ™ LR Clonase ™ II Enzyme Mix; Invitrogen, USA). The reaction was then transformed into DH5alpha E. coli cells and cultured in a 37 &lt; 0 &gt; C incubator. Plasmid DNA was obtained from some of the transgenic strains that appeared the following day, and sequencing was requested by Genotech Corp. (Korea) to analyze the nucleotide sequence.

The following method was used to prepare the pCXLE-hSK-CD vector (Fig. 2A). The DNA of the SOX2 and KLF4 genes was obtained from the pHAGE2-EF1a-hSTEMCCA-W-loxP vector by polymerase chain reaction. The subsequent steps are the same as those for producing the pCXLE-hOCT4-CD vector.

The pCXLE-hUL-CD vector (A in Fig. 2) was produced by Enzynomics Corp. (Korea).

The list of primers used in the vector production process is shown in Table 1 below.

Name of the primer purpose order TOPO-hOCT4-CD-F OCT4 PCR 5'-CACCATGGCGGGACAC-3 '(SEQ ID NO: 1) GA_Oct_CD_R2 OCT4 PCR 5'-CGCCCTTACCCCTACTCACCAATATCTTCAAACC-3 '
(SEQ ID NO: 2) TOPO-GA-SOX2-P2A-F SOX2-2A PCR 5'-CACCATGTACAACATGAT-3 '(SEQ ID NO: 3) TOPO-GA-SOX2-P2A-R SOX2-2A PCR 5'-GTCGCTGACAGCCATTGGCCCGGGATTCTCTTCGACAT-3 '(SEQ ID NO: 4) TOPO-GA-P2A-KLF4-IRES-F KLF4-IRES PCR 5'-GAGAATCCCGGGCCAATGGCTGTCAGCGACGCGCTGCT-3 '(SEQ ID NO: 5) TOPO-GA-P2A-KLF4-IRES-R KLF4-IRES PCR 5'-TCCCCCTGTCACCATCATATGTGTGGCCATATTATCAT-3 '(SEQ ID NO: 6) TOPO-GA-IRES-CD-F IRES-CD PCR 5'-ATGGTGACAGGGGGAATG-3 '(SEQ ID NO: 7) TOPO-GA-IRES-CD-R IRES-CD PCR 5'-CTACTCACCAATATCTTCAAACCA-3 '(SEQ ID NO: 8)

&Lt; 1-2 > Episome  Check the function of the vector

To confirm the function of the produced episome vector in human cells, 293T cells were transfected with each episome vector and the expression of the gene was confirmed at the level of mRNA.

Specifically, 293T cells were cultured in DMEM medium (Dulbecco's modified Eagle`s medium, DMEM; Invitrogen, USA) containing 10% FBS (fetal bovine serum; Invitrogen, USA). 500 ng of each episome vector was transformed into 293T cells using the TransIT 2020 reagent (Mirus, USA) according to the manufacturer's protocol. After 3 days of culture, RNA was extracted from the cells cultured according to the manufacturer's protocol using the RNeasy Mini Plus Kit (Qiagen, USA). Then, cDNA was synthesized according to the manufacturer's protocol using iScript cDNA synthesis kit (Bio-Rad, USA). RT-PCR was performed using the primers shown in Table 2 below. The PCR product was confirmed on 2% agarose gel. As a control, an experiment not containing reverse transcriptase was carried out and PCR was performed using the same method as described above and confirmed on 2% agarose gel.

 Target gene Forward sequence Reverse sequence OCT4 5'-GAGGAGTCCCAGGACATCAA-3 '
(SEQ ID NO: 9) 5'-AATAGAACCCCCAGGGTGAG-3 '
(SEQ ID NO: 10) SOX2 5'-GCCGAGTGGAAACTTTTGTCG-3 '(SEQ ID NO: 11) 5'-GGCAGCGTGTACTTATCCTTCT-3 '
(SEQ ID NO: 12) KLF4 5'-CCCACATGAAGCGACTTCCC-3 '
(SEQ ID NO: 13) 5'-CAGGTCCAGGAGATCGTTGAA-3 '
(SEQ ID NO: 14) yCD 5'-TGACAGGGGGAATGGCAAGC-3 '
(SEQ ID NO: 15) 5'-ACATCCGCCAATAGGAACACCAC-3 '
(SEQ ID NO: 16) GAPDH 5'-GGAGCGAGATCCCTCCAAAAT-3 '(SEQ ID NO: 17) 5'-GGCTGTTGTCATACTTCTCATGG-3 '
(SEQ ID NO: 18) LIN28 5'-TGCGGGCATCTGTAAGTGG-3 '
(SEQ ID NO: 19) 5'-GGAACCCTTCCATGTGCAG-3 '
(SEQ ID NO: 20) L- MYC 5'-CTGCGGGGAGGATTTCTACC-3 '
(SEQ ID NO: 21) 5'-CATGCAGTCACGGCGTATGAT-3 '
(SEQ ID NO: 22)

As a result, as shown in Fig. 2, it was confirmed that the gene contained in each of the episome vectors was well expressed in human-derived cells (Fig. 2B).

Example  2: cytosine Diaminazine  (Cytosine deaminase ) Episome  Induced pluripotent stem cells

<2-1> Production of induced pluripotent stem cells

Human fibroblast cell line CRL2097 (ATCC, USA) was cultured in DMEM containing 10% FBS (fetal bovine serum, Invitrogen, USA) and 1 mM L-glutamine (Invitrogen, USA). (PCXLE-hOCT4-CD, pCXLE-hSK-CD and pCXLE-hUL) were injected into 100,000 CRL2097 cells using a Neon-electroporator (Invitrogen, USA) for the production of inducible pluripotent stem cells Transformed by perforation method. Electroporation was performed at 1,650 V, 10 ms, and 3 pulses. Transfected CRL2097 cells were inoculated into 6-well plates coated with Geltrex (Invitrogen, USA) for 1 hour. For 5 days and then replaced with reprogramming medium in CRL2097 culture medium. The reprogramming medium was supplemented with 1 mM nicotinamide (Sigma-Aldrich, USA), 0.5 μM A83-01 (Tocris Bioscience, UK), 3.0 μM CHIR99021 (Tocris Bioscience, UK), and 0.2 mM sodium butyrate Sigma-Aldrich, USA) was used. The colonies of the induced pluripotent stem cells were separated and transferred to a 4-well plate coated with geltrex and then further cytosine (Sigma-Aldrich, USA) was added based on TeSR-E8 (Stemcell Technologies, USA) Lt; / RTI &gt; culture medium. The inventors named inducible pluripotent stem cells prepared by the above process as CD-iPSC.

A brief schematic diagram of the above process is shown in Fig. 3 (Fig. 3).

Example 3: Elimination of episome vector remaining in induced pluripotent stem cells

<3-1> Removal of episome vector by 5-FC treatment and identification of remaining episome vector

Induced pluripotent stem cells produced using episomal vectors with or without cytosine deaminase were isolated and cultured using TeSR-E8 medium containing 0, 10, 50 μM 5-FC (Abcam, USA) . Each cell was then separated from the cell culture dish using Accutase (Millipore, Germany) and subcultured again into a gel culture coated cell culture dish. The passage was performed at intervals of 5 to 7 days, and the remaining cells in the process of passage were collected for the purpose of obtaining DNA.

The remaining cells in the passages were centrifuged and the supernatant was removed to obtain the total DNA that the cells had. The remaining cells were resuspended using DirectPCR Lysis Reagent (Viagen, USA) and proteinase K (Invitrogen, USA) was added and reacted at 56 ° C for 3 hours. The reactants were stored at -20 ° C and used for quantitative PCR analysis (qPCR).

A standard curve for FBXO15 and EBNA-1 was generated using the pCXLE-hFbx15-cont2 vector to determine the number of episomal vectors. The Ct (Threshold cycle) values observed in each sample were used to estimate the number of FBXO15 and EBNA-1. Since FBXO15 has two alleles per cell to calculate the number of cells used in one qPCR reaction, the measured value was finally divided by 2. The number of episomal vectors per cell was calculated by dividing the total number of EBNA-1 measured by the number of cells used.

As a result, as shown in Fig. 4, it was confirmed that all the episomal vectors were removed before the passage of the subcultures (about 20 to 28 days) under the condition of treating 50 μM 5-FC (FIG. As a control, it was confirmed that the episomal vector remaining continuously in the subculture of more than 6 times (about 30 to 42 days) was observed under the condition of 0 μM 5-FC treatment under the same conditions, and the cytosine diaminase- Induced pluripotent stem cells did not increase the rate at which the episomal vector escaped even after treatment with 50 [mu] M 5-FC (Fig. 4A).

<3-2> Through single-cell culture Episome  The removal of the vector and the remaining Episome  Vector Check

Induced pluripotent stem cells prepared using an episome vector containing cytosine diamidinase were isolated and cultured at a single cell level using TeSR-E8 medium containing 0, 50 [mu] M 5-FC (Abcam, USA). Transfection was performed at 7-day intervals, and the remaining cells in the process of passage were collected for the purpose of obtaining DNA. The subsequent process is the same as the process of the embodiment <3-1>.

As a result, as shown in FIG. 5, it was confirmed that all of the episomal vectors were removed before 1 to 2 passages (7 to 14 days) under the condition of 50 μM 5-FC treatment (FIG. As a control, it was confirmed that the remaining episome vector appeared at the same time under the condition that 0 μM 5-FC was treated under the same conditions (FIG. 5). In general, it has been reported that for the complete removal of the episome vector, verification is started after incubation of at least 10 passages (about 70 days).

<3-3> Toxicity of embryonic stem cells according to 5-FC and 5-FU concentrations

5-FC was treated with human embryonic stem cells HuES9 and H9 cells at various concentrations in order to confirm whether 5-FC treatment caused toxicity to embryonic stem cells. 5-FU (Sigma-Aldrich, USA) was used as a control because 5-FC was converted to 5-FU by cytosine deaminase and is known to exhibit toxicity.

Embryonic stem cells were inoculated into 96-well plates and various concentrations of 5-FC or 5-FU were added to the culture medium for the next 48 hours. WST-1 cell proliferation assay system (Takara, Japan) was used to measure cytotoxicity of 5-FC and 5-FU.

As a result, it was confirmed that 5-FU showed cytotoxicity in HuES9 cells and H9 cells, while 5-FC did not show toxicity (Fig. 6). This proves that the concentrations of 5-FC used in Examples <3-1> and <3-2> are in a range that does not exhibit toxicity.

Example  4: cytosine Diaminase  Induce selective cell death of cells inserted into the genome

<4-1> Production of lentivirus

In order to construct lentivirus, we include or include the packaging vector psPAX2 (Addgene, USA), envelope vector pMD2.G (Addgene, USA) and pLX301 (Addgene, USA) containing CD-EGFP or EGFP as an expression vector and CD PWPXL (Addgene, USA) was used. Production of lentivirus was carried out by co-transfection of 293T cells with 8.75 μg of expression vector, 3.75 μg of envelope vector and 2.5 μg of packaging vector using TransIT-2020 Reagent (Mirus, USA) . Simultaneous nucleic acid transfer was performed. After 24 hours, the cells were replaced with fresh medium. At 48 hours and 72 hours, whole cell culture was collected to obtain lentiviral particles. The obtained lentiviral particles were concentrated by ultracentrifugation at 4 ° C for 2 hours at a rate of 25,000 rpm and stored at -80 ° C until just before use.

<4-2> 5- Of FC  Cytosine by treatment Diaminase  Identification of selective cell death of 293T cells inserted into the genome

To determine whether apoptosis was induced by treatment with cytosine diaminase and 5-FC in human cells, EGFP and CD-EGFP were transfected into 293T cells using the lentivirus prepared in the above Example <4-1> Respectively. Cells transfected with each gene were inoculated on a 12-well plate and replaced with culture medium containing 5-FC the following day. Fluorescence activated cell sorter (FACS) was used to count cells expressing EGFP in each transduced cell. Specifically, 293T cells were separated into single cells by treatment with Accutase (Milipore, Germany) at 37 占 폚 for 10 minutes or longer, washed with DPBS (Welgene, Korea) and centrifuged. The number of resuspended 293T cells was measured using an automated cell counter (Invitrogen, USA) and 200,000 cells per sample were prepared for FACS analysis. Analysis was performed using a FACSCalibur flow cytometer (BD, USA) and the results were summarized using FlowJo 10.1 software.

As a result, it was confirmed that the number of cells expressing EGFP was significantly decreased only in the condition that 50 μM 5-FC was treated with CD-EGFP-expressing cells (FIGS. 7A and 7B). In the cells expressing EGFP used as the control group, it was confirmed that the number of cells expressing EGFP did not change even after treatment with 50 μM 5-FC. In addition, in the counting of the number of cells, it was confirmed that the number of living cells in CD-EGFP-expressing cells was significantly reduced under the condition of 50 μM 5-FC treatment (FIG. 7C).

<4-3> 5- Of FC  Cytosine by treatment Diaminase  Identification of selective cell death of embryonic stem cells inserted into the genome

In order to confirm that apoptosis was induced by treatment with cytosine diaminase and 5-FC in the same manner as in 293T cells in embryonic stem cells, lentivirus produced by the method of Example <4-1> was used in H9 cells EGFP and CD-EGFP. Cells transfected with each gene were inoculated into a 12-well plate, and after two days, the medium was replaced with a culture medium containing 5-FC.

As a result, it was confirmed that the number of living cells decreased significantly after 48 hours in the condition of treating 50 μM 5-FC with CD-EGFP expressing cells (FIGS. 8A and 8B). In the cells expressing EGFP used as the control group, it was confirmed that the cells were grown under conditions of 50 μM 5-FC treatment (FIGS. 8A and 8B).

Example  5: Not containing foreign DNA ( exogene -free) Induced pluripotent stem cells  Identify characteristics

<5-1> Identification of pluripotency of selected pluripotent stem cells

To confirm whether the undifferentiated CD-iPSC derived from fibroblasts exhibits pluripotency, expression of a pluripotency maker protein in CD-iPSC was confirmed.

Specifically, a solution prepared by diluting 16% paraformaldehyde (Electron microscopy science, USA) in DPBS to 4% was applied to the CD-iPSC selected in Example 3, and the reaction was allowed to proceed at room temperature for 10 minutes to fix , PBS solution containing 3% BSA (Bovine serum albumin; Invitrogen, USA) and 0.3% Triton X-100 (Sigma-Aldrich, USA) for 1 hour to impart permeability to the cell membrane Respectively. After treatment, anti-OCT4 antibody (1: 500 dilution, Santa Cruz Biotechnology, USA), anti-NANOG antibody (1:70 dilution, R & D systems, USA), anti-TRA- (1: 500 dilution, Milipore, Germany), anti-SSEA3 antibody (1: 500 dilution, Milipore, Germany), anti- SSEA4 antibody Antibodies (1: 500 dilution, Millipore, Germany) were each treated and left overnight at 4 占 폚. The cells were washed three times with 0.1% BSA and treated with a secondary antibody conjugated with Alexa Fluor 488 or Alexa Fluor 594 (Invitrogen, USA) and left at room temperature for 1 hour to obtain CD-iPSC Were immunofluorescently stained and observed with a fluorescence microscope to confirm the expression of OCT4, NANOG, SSEA4, TRA-1-80 and TRA-1-61 proteins. Cells were stained with Hoechst 33342 (Invitrogen, USA) and stained for comparison.

As a result, it was confirmed that the universal markers OCT4, NANOG, TRA-1-81, SSEA3, SSEA4 and TRA-1-60 proteins were expressed in CD-iPSC (FIG.

<5-2> Alkaline phosphatase (AP) of CD-iPSC dyeing

In order to confirm the characteristics of CD-iPSC derived from fibroblasts, alkaline phosphatase staining (AP staining), which is one of the methods for verifying the characteristics of pluripotent stem cells, was performed.

Specifically, an AP staining kit (Alkaline phosphatase kit; Sigma Aldrich, USA) was used. 10% formalin (Sigma Aldrich, USA) was added to the CD-iPSC selected in Example 3 as a fixative solution and reacted at room temperature for 30 seconds. Immobilized cells were washed once with TBST. Then, 10 μl of a sodium nitrate solution and 10 μl of an FRV-alkaline solution were mixed and allowed to stand for 2 minutes. Then, 0.15% sodium chloride (Sigma-Aldrich, USA) And 10 [mu] l of Naphthol AS-BI alkaline solution were added to prepare an AP staining mixture. The fixed CD-iPSC was prepared by adding the above-prepared AP staining mixture, wrapped with foil, blocked with light, and allowed to react at room temperature for 20 minutes. After the reaction, the cells were washed twice with PBS and AP stained cells were observed with a phase contrast microscope. As a result, it was confirmed that CD-iPSC stained positive for AP (Fig. 9B).

<5-3> In vitro In vitro ) To confirm the triploidity of CD-iPSC

In order to confirm the ability of the CD-iPSC-derived soma (CD-soma) to differentiate, the ectoderm marker proteins TUJ1, OTX2, endodermic marker proteins FOXA2, AFP, mesoderm ) Marker proteins, ASMA and BRACHYURY, and the expression of three germ layer marker proteins was confirmed.

Specifically, the colonies of CD-iPSC were cultured in an embryoid body differentiation medium (DMED / F12 medium containing 10% serum replacement (SR) for 7 days to induce differentiation into CD-somata . The cells were inoculated onto the plate coated with geltrex and CD for culturing for an additional 7 days to induce differentiation. The differentiated cells were subjected to immunofluorescence staining in the same manner as in Example <5-1> to confirm the expression of TUJ1, OTX2, ASMA, BRACHYURY, AFP or FOXA2 protein. (1: 5000 dilution; PRB-435P, Covance, USA), anti-OTX2 antibody (1: 100 dilution; ab21990, Abcam, USA) as the primary antibody for the immunofluorescent staining, anti-ASMA antibody (1: 200 dilution; A0008, Dako, USA), anti-BRACHYURY antibody (1: 300 dilution; sc-17745, Santa Cruz Biotechnology, (1: 100 dilution; AF2400, R & D systems, USA) were used, respectively. To compare the degree of expression, Hoechst 33342 was treated to stain the nuclei of the cells.

As a result, the cells differentiated from CD-iPSC express all of the triple-lobe marker of TUJ1, OTX2, ASMA, BRACHYURY, AFP or FOXA2 protein, thus confirming that CD-iPSC is universal (FIG.

<5-4> In vivo In vivo ) To confirm the triploidity of CD-iPSC

In order to confirm the differentiation potential of CD-iPSC, it was confirmed whether or not teratomas were generated in the mice after cell transplantation.

Specifically, CD-iPSCs were implanted near the shoulders of nonobese diabetic / severe combined immunodeficient mice. After 12 weeks, the resulting teratoma was isolated and fixed with 10% formalin (Sigma-Aldrich, USA), and paraffin blocks were prepared. Tissue sections were obtained from the prepared blocks and the characteristics of cells and specific tissues were confirmed by hematoxylin / eosin staining.

As a result, it was observed that endoderm, mesoderm, and ectodermal cells were formed in one teratoma, and it was confirmed that CD-iPSC had a triploidal differentiation ability (FIG. 9D).

<5-5> Dielectric karyotype analysis

The following analysis was performed to confirm that the selected CD-iPSC treated with 5-FC did not cause dielectric anomalies during the procedure of the above example. A genomic analysis was performed by Chromosomal G-banding analysis with reference to GenDix Inc., Korea.

As a result, it was confirmed that the selected CD-iPSC exhibits a normal human karyotype (FIG. 9E).

<5-6> Analysis of STR (short tandom repeat) of CD-iPSC

STR analysis was performed to confirm that the selected CD-iPSC was a cell prepared from the fibroblast as the starting cell.

STR genotyping of fibroblast cell line and CD-iPSC was performed by HumanPass Inc. (Korea) after isolating genomic DNA from fibroblast cell line (CRL2097) and CD-iPSC, respectively .

As a result, the results of STR analysis of fibroblast cells before reprogramming with CD-iPSC showed the same result, and it was confirmed that CD-iPSC selected from this was originated from starter cells (F in FIG. 9).

Example  6: cytosine Diaminazine  (Cytosine deaminase ) Episome  Production of inducible neural stem cells using vector

<6-1> Production of inducible neural stem cells

Human fibroblast cell line CRL2097 (ATCC, USA) was cultured in DMEM containing 10% FBS (fetal bovine serum, Invitrogen, USA) and 1 mM L-glutamine (Invitrogen, USA). 10 μg of an episome vector mixture (pCXLE-hOCT4-CD, pCXLE-hSK-CD and pCXLE-hUL-CD) was added to 2,000,000 CRL2097 cells using an NEPA21 Super Electroporator (Nepagene, Japan) And transformed by electroporation. Transfected CRL2097 cells were inoculated into 6-well plates coated with Geltrex (Invitrogen, USA) for 1 hour. 3 days and then replaced with reprogramming medium in CRL2097 culture medium. The reprogramming medium was supplemented with 0.05% AlbuMAX-I (Invitrogen, USA), 1x N2 (Invitrogen, USA) in a 1: 1 mixture of Advanced DMEM / F-12 (Invitrogen, USA) and Neurobasal medium (Invitrogen, USA) 1 mM B27 without vitamin A (Invitrogen, USA), 1 mM Glutamax (Invitrogen, USA), 0.11 mM β-mercaptoethanol (Sigma-Aldrich, USA), 0.5 μM A83-01 (Tocris Bioscience, , 3.0 μM CHIR99021 (Tocris Bioscience, UK), 0.2 mM sodium butyrate (Sigma-Aldrich, USA) and 10 ng / ml human LIF (Peprotech, USA). Colonies of the induced induction neural stem cells were separated and transferred to a 4-well plate coated with geltrex and then proliferated using a medium except for sodium butyrate alone in reprogramming medium. The present inventors named the induction neural stem cells prepared by the above process as CD-iNSC.

Example 7: Elimination of episomal vectors remaining in inducible neural stem cells

<7-1> 5- fluorocytosine  (5- FC ) Processing Episome  Removal of vector and identification of remaining episome vector

Inducible neural stem cells prepared using episomal vectors containing cytosine deaminase were isolated and cultured using media containing 0, 50 [mu] M 5-FC (Abcam, USA). Each cell was then separated from the cell culture dish using Accutase (Millipore, Germany) and subcultured again into a gel culture coated cell culture dish. Transfection was performed at 7-day intervals, and the remaining cells in the process of passage were collected for the purpose of obtaining DNA.

The subsequent process is the same as in the above-mentioned embodiment <3-1>.

As a result, as shown in Fig. 10, it was confirmed that all of the episomal vectors were removed before two passages (14 days) under the condition of 50 占 5 5-FC treatment. As a control, it was confirmed that an episome vector still remaining in 3 or more subculture (21 days) under the condition of 0 μM 5-FC treated under the same conditions (FIG. 10A).

<7-2> Characterization of selected induction neural stem cells

In order to confirm whether CD-iNSC derived from fibroblasts exhibits the characteristics of neural stem cells, expression of neural stem cell marker protein was confirmed in CD-iNSC.

Specifically, a solution prepared by diluting 16% paraformaldehyde (Electron microscopy science, USA) in DPBS to 4% was applied to the CD-iNSC selected in the above <Example 7-1>, followed by reaction at room temperature for 10 minutes And permeabilized to a cell membrane by treating with PBS solution containing 3% BSA (Bovine serum albumin; Invitrogen, USA) and 0.3% Triton X-100 (Sigma-Aldrich, USA) . (1: 100 dilution, Covance, USA), anti-KI67 antibody (1: 200 dilution, BD, USA) and anti-N-CADHERIN antibody (1: 200 dilution, Santa Cruz Biotechnology, USA) and left overnight at 4 ° C. Washed three times with 0.1% BSA and treated with secondary antibody conjugated with Alexa Fluor 488 (Alexa Fluor 488) or Alexa Fluor 594 (Invitrogen, USA) and left at room temperature for 1 hour to obtain CD-iNSC Were immunofluorescently stained and observed with a fluorescence microscope to confirm the expression of PAX6, KI67 and N-CADHERIN protein. Cells were stained with Hoechst 33342 (Invitrogen, USA) and stained for comparison.

As a result, it was confirmed that neural stem cell markers PAX6, KI67 and N-CADHERIN protein were expressed in CD-iNSC (FIG. 10B).

From the above description, it will be understood by those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. In this regard, it should be understood that the embodiments described above are illustrative in all aspects and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention without departing from the scope of the present invention as defined by the appended claims.

<110> KOREA RESEARCH INSTITUTE OF BIOSCIENCE AND BIOTECHNOLOGY An episomal vector harboring suicide gene and method for          reprogramming using the same <130> KPA160583-KR-P1 <150> KR 10-2016-0074749 <151> 2016-06-15 <160> 22 <170> Kopatentin 2.0 <210> 1 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> TOPO-hOCT4-CD-F <400> 1 caccatggcg ggacac 16 <210> 2 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> GA_Oct_CD_R2 <400> 2 cgcccttacc cctactcacc aatatcttca aacc 34 <210> 3 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> TOPO-GA-SOX2-P2A-F <400> 3 caccatgtac aacatgat 18 <210> 4 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> TOPO-GA-SOX2-P2A-R <400> 4 gtcgctgaca gccattggcc cgggattctc ttcgacat 38 <210> 5 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> TOPO-GA-P2A-KLF4-IRES-F <400> 5 gagaatcccg ggccaatggc tgtcagcgac gcgctgct 38 <210> 6 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> TOPO-GA-P2A-KLF4-IRES-R <400> 6 tccccctgtc accatcatat gtgtggccat attatcat 38 <210> 7 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> TOPO-GA-IRES-CD-F <400> 7 atggtgacag ggggaatg 18 <210> 8 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> TOPO-GA-IRES-CD-R <400> 8 ctactcacca atatcttcaa acca 24 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> OCT4 primer F <400> 9 gaggagtccc aggacatcaa 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> OCT4 primer R <400> 10 aatagaaccc ccagggtgag 20 <210> 11 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> SOX2 primer F <400> 11 gccgagtgga aacttttgtc g 21 <210> 12 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> SOX2 primer R <400> 12 ggcagcgtgt acttatcctt ct 22 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> KLF4 primer F <400> 13 cccacatgaa gcgacttccc 20 <210> 14 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> KLF4 primer R <400> 14 caggtccagg agatcgttga a 21 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> yCD primer F <400> 15 tgacaggggg aatggcaagc 20 <210> 16 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> yCD primer R <400> 16 acatccgcca ataggaacac cac 23 <210> 17 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> GAPDH primer F <400> 17 ggagcgagat ccctccaaaa t 21 <210> 18 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> GAPDH primer R <400> 18 ggctgttgtc atacttctca tgg 23 <210> 19 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> LIN28 primer F <400> 19 tgcgggcatc tgtaagtgg 19 <210> 20 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> LIN28 primer R <400> 20 ggaacccttc catgtgcag 19 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> L-MYC primer F <400> 21 ctgcggggag gatttctacc 20 <210> 22 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> L-MYC primer R <400> 22 catgcagtca cggcgtatga t 21

Claims (13)

(a) introducing into an isolated cell an episome vector comprising (i) a reprogramming factor and (ii) a cytosine deaminase (CD) as a suicide gene;
(b) culturing the cells into which the episome vector has been introduced in a medium to reprogram the cells; And
(c) culturing the reprogrammed cells in a culture medium containing 5-fluorocytosine (5-FC) as a pro-drug for an introduced suicide gene to select cells that do not contain foreign DNA Lt; RTI ID = 0.0 &gt; reprogramming &lt; / RTI &gt;
2. The method of claim 1, wherein the reprogramming factor comprises at least one gene selected from the group consisting of Oct4, Sox2, Klf4, Lin28 and L-myc.
delete The method of claim 1, wherein the suicide gene is derived from Saccharomyces cerevisiae .
delete 2. The method of claim 1, wherein said isolated cells are somatic or stem cells.
2. The method of claim 1, wherein said reprogramming is dedifferentiation or transdifferentiation of cells.
2. The method of claim 1, wherein the episome vector is a pCXLE vector or a pCEP4 vector.
The method of claim 1, wherein the medium of step (b) comprises at least one selected from the group consisting of an ALK5 inhibitor, a GSK-3 inhibitor, a histone deacetylase (HDAC) inhibitor and nicotinamide.
The method according to claim 1, wherein the 5-FC is contained in a medium at a concentration of 20 μM to 100 μM.
The method of claim 1, wherein step (c) is performed for 7 to 28 days.
delete delete

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