KR101456998B1 - Method for producing induced pluripotent stem cell using intercellular protein delivery - Google Patents

Abstract

The present invention relates to a method for producing a stem cell with a high efficiency of non-EE using a protein-secreted amino acid sequence of a homeode domain in the intracellular introduction of a degenerating stem cell inducer, A transfected cell expressing a fusion protein of a secretion-permeation-related site and a dedifferentiation inducer capable of migrating into a cell is prepared and co-cultured with the somatic cell of the differentiated cell, whereby the cell is transformed by a dedifferentiation factor Since the dedifferentiated stem cells have been prepared from the somatic cells, the gene construct encoding the fusion protein of the present invention and the transformant cells expressing the same are useful for preparing undifferentiated stem cells from differentiated somatic cells have.

Description

TECHNICAL FIELD The present invention relates to a method for producing a stem cell,

The present invention relates to a method for producing degenerative stem cells by high-efficiency non-nuclear transformation using proteinaceous amino acid sequences of the homeomain or secretory alkaline dephosphorylase for intracellular introduction of a degenerated stem cell inducer .

Most of the cells that constitute the body of animals, including humans, are not regenerated, and if human cells are lost by aging or other external factors, they can not be restored and normal life phenomena become impossible.

In contrast, some tissues, including the liver and skin, have lifelong cell death and new cell growth, and these tissues contain stem cells that are capable of producing new cells. These are called tissue-specific stem cells.

The tissue-specific stem cells are characterized by being differentiated into defined cells. For example, liver stem cells are made up only of hepatocytes, and skin stem cells make up only skin cells. In contrast, embryonic stem cells in the early stages of development are allergic non-differentiated cells capable of differentiating into all cells in vivo with various functions.

Therefore, in order to regenerate tissues that can not be regenerated, it is necessary to induce differentiation into desired cells using embryonic stem cells and transplant them into damaged tissues. However, since embryonic stem cells are derived from embryos, There is a limit to introducing embryonic stem cells into regenerative medicine in a state where controversy has not yet been solved. In addition, since embryonic stem cells are basically non-autologous cells of other people, it is also difficult to overcome the immunity fitness of individual individuals.

These moral and technical limitations have been somewhat resolved by the induction pluripotent stem cell (iPSC) developed by Sinya Yamanaka of Japan in 2006. Degenerative stem cells are prepared by exogenously introducing specific genes (e.g., Oct4, Sox2, Klf4, and c-Myc) that allow embryonic stem cell status to remain in completely differentiated cells, It is possible to induce the cells to cells having the characteristics of undifferentiated state again. Therefore, it is possible to apply the present invention to a regenerative medicine treatment method in which undifferentiated stem cells are obtained by introducing these genes into somatic cells of an adult, and then differentiated into desired cells and introduced into the body of the subject again.

Degeneration stem cell induction is largely divided into direct gene transfer using viruses and mRNA or proteins produced by these genes. Although the direct gene introduction method has the highest induction efficiency, it has a very limited clinical application because of the side effects that can induce intracellular gene degeneration. Furthermore, the induction method using mRNA is extremely unstable in the mRNA structure, and there is a technical difficulty to mass-produce it and stably introduce it into the cell. Protein-mediated transfer can avoid the side effects of genetic modification, and at the same time it is possible to mass-produce the protein. However, since protein can not permeate through the cell membrane, the intracellular introduction efficiency is extremely low, which is a fatal weak point .

To solve this problem, a method of delivering a deoxyribed stem cell-derived protein to a cell through a membrane permeable peptide is being developed. However, the proteins labeled with transmembrane peptides are extremely low in water solubility, and most proteins are inactivated. This is a low-efficiency method in which such inactivated dedifferentiation-inducing proteins must be administered in large amounts into cell culture fluids. Therefore, genetically safe, high-efficiency protein delivery is an essential technique for inducing degenerative stem cells in the future.

Accordingly, the inventors of the present invention have conducted studies to efficiently induce degenerated stem cells through a genetically safe protein transduction method. As a result, they have found that Secretion-Penetration (SecPen) or secretory alkaline dephosphorylation of homeodomain By co-culturing the transfected cells expressing the fusion gene and the differentiated somatic cells to which the gene encoding the signal peptide-penetration (SigPen) region of the enzyme and the gene encoding the dedifferential induction factor are ligated, The present inventors have completed the present invention by confirming that the dedifferentiation factor secreted from the cells migrates into differentiated somatic cells and effectively induces the differentiation.

It is an object of the present invention to provide a method for producing degenerative stem cells by high-efficiency non-electron modification.

In order to achieve the above object, the present invention provides a gene construct comprising a fusion gene comprising a gene consisting of a nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2 and a gene encoding a dedifferentiation inducer linked thereto.

The present invention also provides a recombinant expression vector in which the gene construct is operably linked.

The present invention also provides a transformant obtained by introducing the recombinant expression vector into a host cell.

In addition, the present invention provides a method for inducing undifferentiated cells from differentiated cells, comprising the step of co-culturing the transformant or its culture with the target cells differentiated.

In addition,

1) mixing and co-culturing the transformant or a culture thereof with target cells differentiated; And

2) isolating embryonic stem cell-like colonies derived from the target cell differentiated from the co-culture of step 1) from the differentiated cells.

In addition, the present invention provides degenerated stem cells prepared by the above method.

The fusion protein expressing transfected cells, which are secreted to the outside of the cells of the present invention and capable of migrating into the surrounding cells, are linked to the dedifferentiation-inducing factor and co-cultured with the differentiated somatic cells, Since the dedifferentiated stem cells are derived from the somatic cells by the dedifferentiation inducer protein, they are genetically more safe than the gene transfer method restricted to clinical application by genetic modification and the protein delivery method with low efficiency of intracellular introduction Since it is a method of inducing stem cell differentiation by high-efficiency protein transfer, it can be effectively used for producing undifferentiated stem cells of differentiation from differentiated cells.

FIG. 1 shows the SecPen-OSKM gene construct. FIG.
Fig. 2 is a diagram showing the SigPen-OSKM gene construct.
Fig. 3 shows the results of introduction of SecPen or SigPen into Oct4, Sox2, Klf4 and c-myc, respectively.
FIG. 4 is a diagram showing a SecPen-OSKM recombination vector. FIG.
FIG. 5 is a diagram showing a SigPen-OSKM recombination vector. FIG.
6 is a diagram showing the SecPen-OSKM-EGFP-TK gene cassette.
Fig. 7 is a diagram showing the SigPen-OSKM-EGFP-TK gene cassette.
Figure 8 is a diagram showing the SecPen-OSKM retrovirus vector.
Fig. 9 shows the SecPen-OSKM-EGFP-TK retrovirus vector.
Fig. 10 shows secretion of SecPen-OSKM protein into cell culture medium.
11 is a diagram for confirming the ability of the gene cassette of the present invention to regenerate stem cells.
FIG. 12 shows the expression of SecPen-OSKM gene cassette through immunocytochemistry.

Hereinafter, the present invention will be described in detail.

As used herein, the term " degenerated stem cell "refers to a pluripotent cell obtained by differentiation from differentiated cells (for example, somatic cells), and capable of differentiating into various organ cells.

The present invention relates to a method for detecting signal peptide-penetration (SigPen) consisting of Secretion-Penetration (SecPen) of a homeo domain consisting of the nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence of SEQ ID NO: A gene coding for a site and a gene encoding a dedifferential inducer are connected to each other, and a gene construct comprising the fusion gene.

The genetic construct may include two or more kinds of fusion genes in which a gene encoding SecPen or SigPen and a gene encoding a dedifferential induction factor are linked, wherein the fusion gene encodes a gene fragment encoding a protease cleavage site Can be mediated. The cleavage site of the protease may be a 2A protease recognition site derived from foot-and-mouth disease virus (FMDV), but is not limited thereto.

The Secretion-Penetration (SecPen) site may be comprised of the nucleotide sequence of SEQ ID NO: 1 as part of the homeostatic domain of the evolutionarily well-preserved homeostatic domain from invertebrates to humans. The SecPen can be excreted outside the cell and migrate into surrounding cells. Thus, if the amino acid sequence of SecPen is labeled with a demultiplexing inducer, the proteins are released into the cell You can move.

The signal peptide-penetration (SigPen) site is the secretion signal sequence (Sig) of the secreted alkaline phosphatase (SEAP) and the penetration sequence (Pen) of the home auto main, 2 < / RTI >

The protein of the SecPen gene described in the nucleotide sequence of SEQ ID NO: 1 is capable of repeated cell-to-cell movement, and the protein of the SigPen gene described in the nucleotide sequence of SEQ ID NO: 2 is secreted outside the cell, It is possible. Thus, SecPen is secreted through the cell membrane directly from the cytoplasm and can migrate through the cell membrane of the surrounding cells and then migrate to other cells, whereas SigPen is secreted outside the cell using the endoplasmic reticulum endoplasmic reticulum and Golgi apparatus, The Sig site may be cleaved and secreted outside the cell. Sig is an abbreviation of a signal peptide. It is an amino acid sequence contained in a protein secreted outside the cell. It serves to transfer secretory proteins to the endoplasmic reticulum during protein translation.

The dedifferentiation inducing factor includes all factors having a function of inducing cell degeneration, and proteins commonly used in the art having such characteristics can be used without limitation. Preferably a combination of reprogramming induction factors known to be involved in de-differentiation, for example, one or more selected from the group consisting of Oct4, Sox2, Klf4 and c-myc known to induce de- differentiation .

The gene construct includes a gene encoding SecPen or SigPen and a gene encoding Oct4, a gene encoding SecPen or SigPen, a gene encoding Sox2, a gene encoding SecPen or SigPen, and a gene encoding Klf4 And a fusion gene in which a gene encoding SecPen or SigPen and a gene encoding c-myc are linked, wherein the fusion genes are linked through a gene fragment encoding a protease cleavage site Structure, more preferably a structure disclosed in Fig. 1 or Fig. 2, but it is not limited thereto.

In a specific embodiment, the present inventors have attempted to exclude the mass production process of the dedifferentiation factor using microorganisms or animal cells which lose their structural stability during the separation and purification process by utilizing intercellular protein transfer phenomena. In order to develop a highly efficient induction of differentiation induction, SecPen or SigPen was fused to the N-terminus of decellularized stem cell-inducible transcription factors Oct4, Sox2, Klf4 and c-myc (OSKM) Or SigPen-OSKM), each of the four fusion genes was ligated to form a single gene structure. This was done so that each protein could be synthesized in cells with the same efficiency. However, since one protein translated by the gene construct to which the above four genes (OSKM) are linked can not perform normal protein functions, it is possible to isolate four reprogramming inducing factors fused with SecPen as independent transcription factor proteins The gene construct was prepared by inserting a protease-recognition sequence derived from foot-and-mouth disease virus (FMDV) into each transcription factor cDNA (see FIGS. 1 and 2). As a result, it was confirmed that the nucleotide sequence encoding SecPen or SigPen was successfully bound to Oct4, Sox2, Klf4 and c-myc (see FIG. 3).

Therefore, the gene construct produced as described above is linked to the amino acid sequence of the dedifferentiation inducer having specificity for secretion and infiltration and is capable of inducing the differentiation inducing factor having intercellular mobility Can be usefully used.

The present invention also provides a recombinant expression vector in which the gene construct is operably linked.

The genetic construct can be produced using recombinant expression vectors containing the construct using conventional methods known in the art. The recombinant expression vector of the present invention can use a commercially available vector, and a pcDNA3 vector can be used, but the present invention is not limited thereto, and a recombinant vector suitable for a person skilled in the art may be prepared and used. As the expression vector, a virus-derived vector such as retrovirus, adenovirus, Herns virus or adeno-associated virus may also be used. Preferably, the virus-derived vector is a retrovirus vector, and in particular, it may be a pRevTRE vector. I never do that.

The recombinant expression vector may additionally comprise a reporter gene operably linked as a selection marker or an antibiotic resistance gene. The reporter gene may be expressed in the form of an enhanced green fluorescent protein (EGFP), a green fluorescent protein (GFP), an enhanced red fluorescent protein (ERFP), a red fluorescent protein (RFP) (EBFP), blue fluorescent protein (BFP), enhanced yellow fluorescent protein (EYFP), yellow fluorescent protein (EYFP), enhanced blue fluorescent protein (ECFP), indigo fluorescent protein (CFP), luciferase, But are not limited to, those selected from the group consisting of gaussia luciferase,? -Galactosidase, and alkaline phosphatase. The antibiotic resistance gene may be a gene showing resistance to hygromycin, geneticin, neomycin, kanamycin or gentamicin, but is not limited thereto.

The present inventors prepared a recombinant expression vector expressing a dedifferentiation factor by inserting the above gene construct into an expression vector. Specifically, the gene construct was inserted into a pcDNA3 vector expressing in animal cells and selectively cultured by G418, and the SecPen-OSKM or SigPen-OSKM gene cassette specifically removed only the cells introduced into the chromosome of the cell (SEQ ID NO: 2). The expression vector for SecPen-OSKM or SigPen-OSKM was prepared by further inserting tyrosine kinase (TK) gene (see FIGS. 4 and 5). In addition, the SecPen-OSKM or SigPen-OSKM gene is secreted from a mixed culture of degenerated stem cells inserted into the chromosome of the cell and the degenerated stem cells formed by receiving the SecPen-OSKM or SigPen-OSKM protein secreted therefrom In order to isolate stem cells derived from SecPen-OSKM or SigPen-OSKM proteins from genetically engineered stem cells expressing EGFP and TK, the green fluorescent protein gene and TK (Fig. 6 and Fig. 7). The expression vector of SecPen-OSKM-EGFP-TK or SigPen-OSKM-EGFP-TK was constructed.

The present inventors have found that the gene construct can be expressed only in the presence of doxycyclin, an analogue of tetracyclin, in order to express a defective inducing factor and produce a viral-secreting cell line that can be used for various target cells A tetracycline-reponsible element (TRE) was inserted into the pRevTRE vector located in front of the CMV promoter to produce a retroviral vector (see FIGS. 8 and 9).

Since the recombinant expression vector prepared above can express the gene construct containing a specific amino acid sequence related to secretion and infiltration and a fusion gene of a dedifferentiation inducer having the ability to induce dedifferentiation, And may be useful for the expression of a demultiplexing factor having intercellular mobility.

The present invention also provides a transformant obtained by introducing the recombinant expression vector into a host cell.

The host cell may be an animal cell and preferably a mouse embryonic fibroblast (MEF), but is not limited thereto.

The transformant can secrete a reprogramming factor having intercellular mobility by introducing the recombinant expression vector capable of expressing a differentiation inducing factor.

The present inventors transfected the SecPen-OSKM recombinant expression vector prepared above into mouse embryonic fibroblast (MEF) cells to prepare transformed cells. The secretion of SecPen-OSKM protein from the transfected cells into the culture medium was analyzed by Western blot. As a result, the gene cassette was inserted into animal cells and SecPen-OSKM was synthesized as a long protein in the cells. Then, 2A protease recognition Oct4, SecPen-Sox2, SecPen-Klf4, and SecPen-cMyc, respectively, by the intracellular protease recognizing the site of the protein (Fig. 10).

Therefore, the transformant prepared as described above can be used as a transformant expressing the differentiation inducing factors having intercellular mobility.

In addition, the present invention provides a method for inducing undifferentiated cells from differentiated cells, comprising the step of co-culturing the transformant or its culture with the target cells differentiated.

In addition, the present invention provides a method for producing degenerative stem cells from differentiated cells and a degenerated stem cell prepared thereby.

1) mixing and co-culturing the transformant or a culture thereof with target cells differentiated; And

2) isolating embryonic stem cell-like colonies from the target cell differentiated from the co-culture of step 1).

In the above method, the transformant may secrete a dediffering inducer having intercellular mobility.

The differentiated target cells are selected from the group consisting of adipocytes, bone marrow cells, osteoblasts, chondrocytes, fibroblasts, muscle fiber bands, neurons, skeletal muscle cells, cardiomyocytes, vascular endothelial cells, vascular smooth muscle cells, hepatocytes and kidney cells It is more preferable that it is a fibroblast, but it is not limited thereto, and it may be a cell in differentiated form such as a somatic cell or a differentiated mature cell.

The present inventors constructed a method for inducing degeneration stem cells through the mixed culturing of SecPen-OSKM or SigPen-OSKM-expressing MEF transformants and EGFP-expressing MEF cells prepared as described above. Only colonies expressing green fluorescence among the colonies appearing after co-culturing were collected, and only the degenerated stem cells formed by infiltrating Oct4, Sox2, Klf4 and c-myc proteins secreted from the surrounding cells expressing SecPen-OSKM or SecPen-OSKM , And treated with Gancyoclovir, which is innocuous to normal cells but kills TK gene-expressing cells, can inhibit the expression of some SecPen-OSKMs, which may be mixed with degenerated stem cells expressing the separated green fluorescent protein Cells containing the gene cassette can be completely removed. Then, the stem cells expressing green fluorescence and resistant to Genciclovir were separated and cultured in culture medium containing normal medium and G418 antibiotic, and neomycin (SigPen-OSKM) contained in the SigPen-OSKM gene cassette neomycin resistance gene-deficient cells are selected as decidualized stem cells, and the dedifferentiated stem cells induced by the dedifferentiation factors secreted by the transformant can be finally obtained.

In addition, when a transformant into which the SecPen-OSKM-EGFP-TK or SigPen-OSKM-EGFP-TK gene cassette is introduced is used, the reverse differentiation induced by the dedifferentiation factor secreted by the transformant As a stem cell, a colony that does not express green fluorescence can be selected. These derived stem cells were analyzed for cell characteristics using cell-specific markers and confirmed to exhibit the characteristics of undifferentiated stem cells. Thus, they were derived from the differentiated MEFs into embryonic stem cell-like degenerated stem cells Able to know.

Therefore, it is useful for the production of degenerated stem cells by co-culturing the transformant or its culture with the differentiated cells to induce undifferentiated cells and isolating the differentiated embryonic stem cell-like colony from the target cells Can be used.

Hereinafter, the present invention will be described in detail with reference to examples.

However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

< Example  1> Home  Domain iPSC  Gene fusion of inducible proteins Construct  Produce

<1-1> SecPen - OSKM Construct  making

Secretion & Penetration (SecPen) amino acid sequence of the homeome domain, which is part of the homeo domain transcription factor and can be exported to the outside of the cell, Sox2, Klf4, and c-myc (OSKM), which are derivatives of stem cells capable of inducing degeneration stem cells, using the primers described in SEQ ID NOS: 3 to 18 and Cold fusion cloning (System Bioscienc Inc.) ) Was labeled with the gene coding for the SecPen site represented by SEQ. ID. NO. 1, and then each of the four transcription factor genes was ligated to produce one gene. At this time, a foot-and-mouth disease virus (FMDV) sequence, which is a target of 2A protease, is transcribed into four transcription factors labeled with SecPen so that it can be cleaved in the translation process as an independent transcription factor, To introduce a SecPen-OSKM gene cassette (Fig. 1). Then, tyrosine kinase (TK) gene was added to the pcDNA3 vector (Invitrogen) to specifically remove only the cells into which the SecPen-OSKM gene cassette was introduced, and introduction of SecPen into each of the OSKM cDNAs was confirmed (Lane 2 in FIG. 3).

As a result, a pcDNA3-SecPen-OSKM vector into which the SecPen-OSKM gene cassette was successfully introduced was prepared as shown in Fig. 4 (Fig. 4).

Hind III-SecPen-H1 forward primer: ggg aga ccc aag cttATG CAG AGC CTG CGG CAG GAG (SEQ ID NO: 3);

Hind III-SecPen-H1 reverse primer: agt gcc taa aac cat acg CTT CTT CCA CTT CAT GCG (SEQ ID NO: 4);

H1-Oct4 / 2A-H2 forward primer: cgt atg gtt tta ggc act gct gga cac ct ggc ttca (SEQ ID NO: 5);

H1-Oct4 / 2A-H2 Reverse primer: ctg act tga gcc gta tat agg ccc ggg gtt ttc ttc (SEQ ID NO: 6);

H2-secpen-H3 forward primer: ata tac ggc tca agt cag CAG AGC CTG CGG CAG GAG (SEQ ID NO: 7);

H2-secpen-H3 reverse primer: cac cgc tct gtc aaa att CTT CTT CCA CTT CAT GCG (SEQ ID NO: 8);

H3-Sox2 / 2A-H4 forward primer: aat ttt gac aga gcg gtg Gca tgc atg tat aac atg (SEQ ID NO: 9);

 H3-Sox2 / 2A-H4 reverse primer: gct ata att gga acc gct tgg gcc ggg att ttc ctc (SEQ ID NO: 10);

 H4-Secpen-H5 forward primer: agc ggt tcc aat tat agc CAG AGC CTG CGG CAG GAG (SEQ ID NO: 11);

  H4-Secpen-H5 reverse primer: ctg ctc taa agc gag ata CTT CTT CCA CTT CAT GCG (SEQ ID NO: 12);

H5-Klf4 / 2A-H6 forward primer: tat ctc gct tta gag cag ctc gag atg agg cag cca (SEQ ID NO: 13);

H5-Klf4 / 2A-H6 reverse primer: ttg acg ctg tat agg tct ggg acc tgg gtt gct ctc (SEQ ID NO: 14);

H6-Secpen-H7 forward primer: aga cct ata cag cgt caa CAG AGC CTG CGG CAG GAG (SEQ ID NO: 15);

H6-Secpen-H7 Reverse primer: gag tga agg gta atc aga CTT CTT CCA CTT CAT GCG (SEQ ID NO: 16);

H7-Cmyc / 2A-XbaI forward primer: tct gat tac cct tca ctc Ggc gcg cca ctg gat ttc (SEQ ID NO: 17); And

H7-Cmyc / 2A-XbaI reverse primer: aga ata ggg ccc tct aga tta tgc acc aga gtt tcg (SEQ ID NO: 18).

<1-2> Production of drug-induced SecPen-OSKM construct

In order to selectively express the SecPen-OSKM gene cassette only when necessary, a tetracycline-responsible element (TRE) is introduced into CMVs to express genes only in the presence of doxycycline, a tetracycline analogue The SecPen-OSKM gene cassette prepared in Example <1-1> was introduced into a pREV-TRE vector (Clonetech), which is a retroviral vector inserted in front of the promoter.

As a result, a pREV-TRE-SecPen-OSKM vector into which the SecPen-OSKM gene cassette was introduced was prepared as shown in FIG. 8 (FIG. 8).

<1-3> SigPen - OSKM Construct  Produce

Cold fusion cloning (System Bioscienc Inc.) was used to fuse the SigPen amino acid sequence, which is part of the homeo-domain transcription factor, Method was used to label genes coding for the SigPen sites represented by SEQ ID NO: 2 at the N-terminus of Oct4, Sox2, Klf4 and c-myc (OSKM), transcription factors having dedifferentiated stem cell induction ability, Each transcription factor gene was ligated to produce one gene. At this time, the squamous cell virus target of 2A protease was introduced between four SigPen-labeled transcription factor genes (Fig. 2) so that it could be cleaved in the translation process as an independent transcription factor of the respective transcription factors. Then, tyrosine kinase (TK) gene was added to the pcDNA3 vector (Invitrogen) to specifically remove only the cells into which the SigPen-OSKM gene cassette was introduced, and introduction of SigPen into each of the OSKM cDNAs was confirmed (Lane 3 in Fig. 3).

As a result, a pcDNA3-SigPen-OSKM vector into which the SigPen-OSKM gene cassette was introduced was prepared as shown in Fig. 5 (Fig. 5).

Hind III-SigPen-H1 forward primer: ggg aga ccc aag cttATG ATG CTG CTG CTG CTG CTG (SEQ ID NO: 19);

Hind III-SigPen-H1 reverse primer: agt gcc taa aac cat acg CTT CTT CCA CTT CAT GCG (SEQ ID NO: 20);

H1-Oct4 / 2A-H2 forward primer: cgt atg gtt tta ggc act gct gga cac ct ggc ttca (SEQ ID NO: 21);

H1-Oct4 / 2A-H2 Reverse primer: ctg act tga gcc gta tat agg ccc ggg gtt ttc ttc (SEQ ID NO: 22);

H2-SigPen-H3 forward primer: ata tac ggc tca agt cag ATG CTG CTG CTG CTG CTG (SEQ ID NO: 23);

H2-SigPen-H3 Reverse primer: cac cgc tct gtc aaa att CTT CTT CCA CTT CAT GCG (SEQ ID NO: 24);

H3-Sox2 / 2A-H4 forward primer: aat ttt gac aga gcg gtg Gca tgc atg tat aac atg (SEQ ID NO: 25);

 H3-Sox2 / 2A-H4 reverse primer: gct ata att gga acc gct tgg gcc ggg att ttc ctc (SEQ ID NO: 26);

 H4-SigPen-H5 forward primer: agc ggt tcc aat tat agc ATG CTG CTG CTG CTG CTG (SEQ ID NO: 27);

  H4-SigPen-H5 reverse primer: ctg ctc taa agc gag ata CTT CTT CCA CTT CAT GCG (SEQ ID NO: 28);

H5-Klf4 / 2A-H6 forward primer: tat ctc gct tta gag cag ctc gag atg agg cag cca (SEQ ID NO: 29);

H5-Klf4 / 2A-H6 reverse primer: ttg acg ctg tat agg tct ggg acc tgg gtt gct ctc (SEQ ID NO: 30);

H6-SigPen-H7 forward primer: aga cct ata cag cgt caa ATG CTG CTG CTG CTG CTG (SEQ ID NO: 31);

H6-SigPen-H7 Reverse primer: gag tga agg gta atc aga CTT CTT CCA CTT CAT GCG (SEQ ID NO: 32);

H7-Cmyc / 2A-XbaI forward primer: tct gat tac cct tca ctc Ggc gcg cca ctg gat ttc (SEQ ID NO: 33); And

H7-Cmyc / 2A-XbaI reverse primer: aga ata ggg ccc tct aga tta tgc acc aga gtt tcg (SEQ ID NO: 34).

<1-4> Green fluorescence  smoking SecPen - OSKM Construct  Produce

The SecPen-OSKM gene cassette prepared in Example < 1-1 > was amplified using an internal ribosomal entry site (IRES) in order to allow observation of the cell into which the SecPen-OSKM gene was introduced in real time (EGFP) gene was inserted into the gene cassette, and the TK gene was also inserted into the gene cassette to selectively kill only the cells containing the gene cassette or inserted into the chromosome. The SecPen-OSKM-EGFP -TK gene cassette was prepared (Fig. 6).

<1-5> Green fluorescence  Drug-induced release SecPen - OSKM Construct  Produce

The EGFP gene showing green fluorescence was inserted into the pREV-TRE-SecPen-OSKM gene cassette prepared in Example <1-2> using an internal ribosomal entry site (IRES), and a gene cassette The TK gene was also inserted into the gene cassette in order to selectively kill only the cells inserted into the chromosome (Fig. 9). The SecPen-OSKM-EGFP-TK-pREV_TRE retrovirus gene cassette was prepared.

<1-6> Green fluorescence  smoking SigPen - OSKM Construct  Produce

The SigPen-OSKM gene cassette prepared in Example < 1-3 > was amplified using an internal ribosomal entry site (IRES) in order to observe the cells into which the SigPen-OSKM gene was introduced in real time The TK gene was also inserted into the gene cassette in order to selectively kill only the cells containing the gene cassette or inserted into the chromosome. Thus, SigPen-OSKM-EGFP (EGFP) -TK gene cassette was prepared (Fig. 6).

< Example  2> SecPen - OSKM  Expression of gene cassette

<2-1> Western Blot  through SecPen - OSKM  Expression of gene cassette

The gene fusion construct of the homeodomain and the iPSC inducible protein prepared in the present invention is expressed by the intracellular protein cleavage enzyme recognizing the 2A region in animal cells, and the SecPen-Oct4, SecPen-Sox2, SecPen-Klf4 and SecPen- The pcDNA3-SecPen-OSKM vector prepared in Example <1-1> was transfected into mouse embryonic fibroblast (MEF) cells and then assayed by Western blotting.

Specifically, 5 × 10 ⁶ cells / ml of MEF cells were divided into 100 mm culture dishes and cultured in DMEM medium containing 10% bovine serum for 1 day. The cultured cells were transformed with GenJet plus DNA in vitro transfection reagent (Fermentas) and pcDNA3-SecPen-OSKM. After 6 hours of transformation, the medium was replaced and after 48 hours cells or medium were obtained and Western blot was performed.

As a result, as shown in FIG. 10, it was confirmed that SecPen-Oct4, SecPen-Sox2, SecPen-Klf4 and SecPen-cMyc proteins were finally isolated from the cell lysate and secreted into a cell culture medium (FIG.

<2-2> Immunocytochemistry  through SecPen - OSKM  Expression of gene cassette

The gene fusion construct of the homeodomain and the iPSC inducible protein prepared in the present invention is expressed by the intracellular protein cleavage enzyme recognizing the 2A region in animal cells, and the SecPen-Oct4, SecPen-Sox2, SecPen-Klf4 and SecPen- And the pcDNA3-SecPen-OSKM vector and pcDNA3-SigPen-OSKM and the control vector FUW-OSKM lacking the secretion sequence were transfected into MEF cells using GenJet plus DNA invitro transfection reagent (Fermentas) Respectively. After 6 hours of transformation, the medium was changed. After 48 hours, the cells were detached and mixed with EGFP-expressing MEF cells at a ratio of 2: 1. Forty-eight hours after co-culture, cells were fixed and immunostained with Oct4 and C-myc primary antibodies. Cy3 was used as a secondary antibody to detect Oct4, Cy5 was used as a secondary antibody to detect C-myc, and fluorescence was confirmed using a confocal microscope (200X).

As a result, it was confirmed that the cells were infiltrated into the dedifferentiation-inducing transcription factor EGFP-MEF cells secreted from the cells transformed with the pcDNA3-SecPen-OSKM vector and the pcDNA3-SigPen-OSKM vector as shown in FIG. 12 (FIG. 12) .

< Example  3> Gene cassette De-differentiation  Stem Cells Induction ability  Confirm

To determine whether the differentiation was induced by the expression of the gene cassette prepared in Example 1, 5 × 10 ⁶ cells / ml MEF cells were added to a 100 mm culture dish and DMEM containing 10% bovine serum And cultured in medium for 1 day. The cultured cells were transformed with GenJet plus DNA in vitro transfection reagent (Fermentas) and pcDNA3-SecPen-OSKM or pcDNA3-SigPen-OSKM. The transformed cells were incubated for 3 days, and the transformation procedure was repeated two times and then cultured in iPSC culture medium. After 14 days of culture, cells were observed under an optical microscope.

As a result, it was confirmed that all of pcDNA3-SecPen-OSKM (Fig. 11A) and pcDNA3-SigPen-OSKM (Fig. 11B) were induced into dedifferentiated stem cells as shown in Fig. 11 (Fig. 11).

<110> Korea Advanced Institute of Science and Technology (KAIST) <120> Method for producing induced pluripotent stem cell using          intercellular protein delivery <130> 11p-08-58 <160> 34 <170> Kopatentin 1.71 <210> 1 <211> 81 <212> DNA <213> Homo sapiens <400> 1 cagagcctgc ggcaggagct cggcctcaac gagcgccaga tcaagatttg gttccagaat 60 cgtcgcatga agtggaagaa g 81 <210> 2 <211> 99 <212> DNA <213> Homo sapiens <400> 2 atgctgctgc tgctgctgct gctgggcctg aggctacagc tctccctggg ccgccagatc 60 aagatttggt tccagaatcg tcgcatgaag tggaagaag 99 <210> 3 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Hind III-SecPen-H1 forward primer <400> 3 gggagaccca agcttatgca gagcctgcgg caggag 36 <210> 4 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Hind III-SecPen-H1 reverse primer <400> 4 agtgcctaaa accatacgct tcttccactt catgcg 36 <210> 5 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> H1-Oct4 / 2A-H2 forward primer <400> 5 cgtatggttt taggcactgc tggacacctg gcttca 36 <210> 6 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> H1-Oct4 / 2A-H2 reverse primer <400> 6 ctgacttgag ccgtatatag gcccggggtt ttcttc 36 <210> 7 <211> 36 <212> DNA <213> Artificial Sequence <220> H2-SecPen-H3 forward primer <400> 7 atatacggct caagtcagca gagcctgcgg caggag 36 <210> 8 <211> 36 <212> DNA <213> Artificial Sequence <220> H2-SecPen-H3 reverse primer <400> 8 caccgctctg tcaaaattct tcttccactt catgcg 36 <210> 9 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> H3-Sox2 / 2A-H4 forward primer <400> 9 aattttgaca gagcggtggc atgcatgtat aacatg 36 <210> 10 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> H3-Sox2 / 2A-H4 reverse primer <400> 10 gctataattg gaaccgcttg ggccgggatt ttcctc 36 <210> 11 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> H4-Secpen-H5 forward primer <400> 11 agcggttcca attatagcca gagcctgcgg caggag 36 <210> 12 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> H4-Secpen-H5 reverse primer <400> 12 ctgctctaaa gcgagatact tcttccactt catgcg 36 <210> 13 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> H5-Klf4 / 2A-H6 forward primer <400> 13 tatctcgctt tagagcagct cgagatgagg cagcca 36 <210> 14 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> H5-Klf4 / 2A-H6 reverse primer <400> 14 ttgacgctgt ataggtctgg gacctgggtt gctctc 36 <210> 15 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> H6-Secpen-H7 forward primer <400> 15 agacctatac agcgtcaaca gagcctgcgg caggag 36 <210> 16 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> H6-Secpen-H7 reverse primer <400> 16 gagtgaaggg taatcagact tcttccactt catgcg 36 <210> 17 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> H7-Cmyc / 2A-XbaI forward primer <400> 17 tctgattacc cttcactcgg cgcgccactg gatttc 36 <210> 18 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> H7-Cmyc / 2A-XbaI reverse primer <400> 18 agaatagggc cctctagatt atgcaccaga gtttcg 36 <210> 19 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Hind III-SigPen-H1 forward primer <400> 19 gggagaccca agcttatgat gctgctgctg ctgctg 36 <210> 20 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Hind III-SigPen-H1 reverse primer <400> 20 agtgcctaaa accatacgct tcttccactt catgcg 36 <210> 21 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> H1-Oct4 / 2A-H2 forward primer <400> 21 cgtatggttt taggcactgc tggacacctg gcttca 36 <210> 22 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> H1-Oct4 / 2A-H2 reverse primer <400> 22 ctgacttgag ccgtatatag gcccggggtt ttcttc 36 <210> 23 <211> 36 <212> DNA <213> Artificial Sequence <220> H2-SigPen-H3 forward primer <400> 23 atatacggct caagtcagat gctgctgctg ctgctg 36 <210> 24 <211> 36 <212> DNA <213> Artificial Sequence <220> H2-SigPen-H3 reverse primer <400> 24 caccgctctg tcaaaattct tcttccactt catgcg 36 <210> 25 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> H3-Sox2 / 2A-H4 reverse primer <400> 25 aattttgaca gagcggtggc atgcatgtat aacatg 36 <210> 26 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> H3-Sox2 / 2A-H4 reverse primer <400> 26 gctataattg gaaccgcttg ggccgggatt ttcctc 36 <210> 27 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> H4-SigPen-H5 forward primer <400> 27 agcggttcca attatagcat gctgctgctg ctgctg 36 <210> 28 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> H4-SigPen-H5 reverse primer <400> 28 ctgctctaaa gcgagatact tcttccactt catgcg 36 <210> 29 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> H5-Klf4 / 2A-H6 forward primer <400> 29 tatctcgctt tagagcagct cgagatgagg cagcca 36 <210> 30 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> H5-Klf4 / 2A-H6 reverse primer <400> 30 ttgacgctgt ataggtctgg gacctgggtt gctctc 36 <210> 31 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> H6-SigPen-H7 forward primer <400> 31 agacctatac agcgtcaaat gctgctgctg ctgctg 36 <210> 32 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> H6-SigPen-H7 reverse primer <400> 32 gagtgaaggg taatcagact tcttccactt catgcg 36 <210> 33 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> H7-Cmyc / 2A-XbaI forward primer <400> 33 tctgattacc cttcactcgg cgcgccactg gatttc 36 <210> 34 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> H7-Cmyc / 2A-XbaI reverse primer <400> 34 agaatagggc cctctagatt atgcaccaga gtttcg 36

Claims (16)

A genetic construct comprising a gene consisting of a nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2 encoding a secretion-infiltration site and a gene encoding a dedifferentiation factor linked thereto;
The gene construct according to claim 1, wherein the gene construct comprises two or more kinds of fusion genes to which a gene consisting of a nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2 encoding a secretion- And the fusion genes are linked via a gene fragment encoding the cleavage site of the protease.
2. The genetic construct of claim 1, wherein the dedifferentiation inducing factor is any one selected from the group consisting of Oct4, Sox2, Klf4, and c-myc.
The genetic construct according to claim 1, wherein the genetic construct is a genetic construct disclosed in Fig. 1 or 2.
A recombinant expression vector operably linked to a genetic construct according to any one of claims 1 to 4.
6. The recombinant expression vector of claim 5, wherein the recombinant expression vector further comprises an operably linked reporter gene.
Claim 7 has been abandoned due to the setting registration fee. The reporter gene according to claim 6, wherein the reporter gene is selected from the group consisting of enhanced green fluorescent protein (EGFP), green fluorescent protein (GFP), enhanced red fluorescent protein (ERFP) (RFP), Enhanced Blue Fluorescent Protein (EBFP), Blue Fluorescent Protein (BFP), Enhanced Yellow Fluorescent Protein (EYFP), Yellow Fluorescent Protein (EYFP), Enhanced Blue Fluorescent Protein Wherein the recombinant expression vector is selected from the group consisting of luciferase, gaussia luciferase,? -Galactosidase, and alkaline phosphatase.
Claim 8 has been abandoned due to the setting registration fee. 8. The recombinant expression vector of claim 7, wherein the recombinant expression vector further comprises an antibiotic resistance gene operably linked as a selection marker.
6. The recombinant expression vector according to claim 5, wherein the expression vector is a retrovirus vector.
A transformant obtained by introducing the recombinant expression vector of claim 5 into a host cell.
11. The transformant according to claim 10, wherein the host cell is mouse embryonic fibroblast (MEF).
A method for inducing undifferentiated cells from differentiated cells, comprising culturing and co-culturing the transformant of claim 10 or a culture thereof and target cells differentiated.
1) co-culturing the transformant of claim 10 or its culture with target cells differentiated and co-culturing; And
2) isolating the embryonic stem cell-like colony from the target cell differentiated from the co-culture of step 1).
14. The method of claim 13, wherein the transformant of step 1) secretes a dedifferentiation-inducing factor.
14. The method of claim 13, wherein the differentiated cells of step 1) are selected from the group consisting of adipocytes, bone marrow cells, osteoblasts, chondrocytes, fibroblasts, fibroblasts, neurons, skeletal muscle cells, myocardial cells, vascular endothelial cells, , Hepatocytes, and kidney cells. &Lt; RTI ID = 0.0 &gt; 11. &lt; / RTI &gt;
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