Genetically modified cell production method, culture method, genetically modified cell, expression vector production method, and expression vector

Abstract

To provide methods for producing recombinant cells to enable easy generation of neural stem cells, ectoderm, mesoderm and endoderm.SOLUTION: An expression vector provided with gene sequences of seven factors including OCT4, SOX2, KLF4, c-MYC, NANOG, LIN28 and TERT is introduced into an animal cell. Thus obtained recombinant cell can be elicited to a neural stem cell state as well as to one of pluripotent stem cell state, ectoderm state, mesoderm state and endoderm state. The expression vector may further contain the gene sequences of histone H1FOO and DPPA3. The expression vector may still further contain MBD3 function inhibitor and p53 function inhibitor.SELECTED DRAWING: Figure 8

Description

[Technical Field]

[0001] The present invention particularly relates to a method for producing genetically modified cells having pluripotency and differentiation potential, a culture method, genetically modified cells, a method for producing an expression vector, and an expression vector. [Background technology]

[0002] Conventionally, referring to Patent Document 1, it has been possible to produce induced pluripotent stem cells (hereinafter referred to as "iPS cells") by introducing an expression vector encoding a DNA sequence encoding the so-called "Yamanaka four factors (OCT4, SOX2, KLF4, c-MYC)" into human or mouse somatic cells and expressing the DNA sequence. Furthermore, Patent Document 2 also describes the so-called "Thomson four factors (OCT4, SOX2, NANOG, LIN28)" as DNA sequences for producing similar induced pluripotent stem cells.

[0003] iPS cells are pluripotent stem cells that can differentiate into any cell type derived from the ectoderm, mesoderm, or endoderm that make up the animal body. Therefore, iPS cells are a cell source for regenerative medicine. Mouse iPS cells, on the other hand, are widely used in basic research on regenerative medicine. Sperm and eggs are produced from mouse iPS cells, which then produce offspring. In other words, iPS cells can be applied not only to regenerative medicine, but also to reproductive medicine. Furthermore, it is known that the efficiency of establishing human and porcine iPS cells is improved when expression vectors encoding DNA sequences encoding six factors (OCT4, SOX2, KLF4, c-MYC, NANOG, and LIN28), consisting of the four Yamanaka factors and the four Thomson factors, are introduced. Among other animal species, iPS cells from monkeys, goats, and sheep have also been produced by introducing the genes for these six factors.

[0004] On the other hand, bovine spongiform encephalopathy (BSE), a fatal neurological disease, is known to affect humans through consumption of infected cattle, causing severe neurological disorders, and has had a significant negative impact on the livestock industry. For this reason, there is a need for pathological model cells derived from differentiated neural stem cells rather than iPS cells for livestock. Furthermore, in recent years, livestock cultured meat production technology has attracted attention because it contributes to resource conservation, reduction of grain consumption as livestock feed, and reduction of methane gas emissions. Here, the above-mentioned iPS cells are capable of generating ectoderm, mesoderm, and endoderm. For this reason, iPS cells are expected to be used for the production of cultured meat. Furthermore, it is expected that germ cells will be produced from mesoderm cells generated from livestock iPS cells. Although high-quality livestock animals inevitably reach the end of their lifespan, it is expected that iPS cells produced from these animals will allow the permanent use of sperm, eggs, and fertilized eggs. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. WO2007 / 069666 [Patent Document 2] International Publication No. WO2008 / 118820 Summary of the Invention [Problem to be solved by the invention]

[0006] While various cell types derived from iPS cells, including neural stem cells, ectoderm, mesoderm, and endoderm, have been generated, this process is complex and difficult, requiring the use of multiple differentiation-inducing substances and a long culture period of more than one month. Therefore, there has been a demand for a method for producing cells that can be endowed with pluripotency like pluripotent stem cells and that can easily generate neural stem cells, ectoderm, mesoderm, and endoderm.

[0007] The present invention has been made in view of the above circumstances, and aims to solve the above-mentioned problems. [Means for solving the problem]

[0008] Book The method for producing genetically modified cells of the present invention is based on nine factors including OCT4, SOX2, KLF4, c-MYC, NANOG, LIN28, TERT, histone H1FOO, and DPPA3. Derived from fibroblasts of the Bovinae subfamily The method is characterized by producing genetically modified cells that can be induced from a neural stem cell state to any one of pluripotent stem cells, ectoderm, mesoderm, and endoderm. The method for producing genetically modified cells of the present invention is based on 11 factors including OCT4, SOX2, KLF4, c-MYC, NANOG, LIN28, TERT, histone H1FOO, DPPA3, MBD3 function inhibitor, and p53 function inhibitor. Derived from fibroblasts of the Bovinae subfamily The method is characterized by producing genetically modified cells that can be induced from a neural stem cell state to any one of pluripotent stem cells, ectoderm, mesoderm, and endoderm. The method for producing genetically modified cells of the present invention is characterized in that the cells are cultured with the addition of a LIF receptor agonist, a fibroblast growth factor receptor agonist, and a Wnt signal inhibitor, or with the addition of a LIF receptor agonist, an activin receptor agonist, a Wnt signal inhibitor, a GSK inhibitor, an Src inhibitor, and an epigenetics eraser. 。 The culture method of the present invention is characterized in that the genetically modified cells are cultured by adding an LIF receptor agonist, a fibroblast growth factor receptor agonist, and a Wnt signal inhibitor, and maintaining the genetically modified cells in a neural stem cell state. The culture method of the present invention is characterized in that the genetically modified cells are cultured by adding an LIF receptor agonist, an activin receptor agonist, a Wnt signal inhibitor, a GSK inhibitor, an Src inhibitor, and an epigenetics eraser, and the genetically modified cells are maintained in a pluripotent stem cell state. The culture method of the present invention is characterized in that the genetically modified cells are cultured in the presence of animal serum, and induced into any one of pluripotent stem cells, ectoderm, mesoderm, and endoderm. The genetically modified cells of the present invention are characterized by being produced by the genetically modified cell production method or cultured by the culture method. The method for producing an expression vector of the present invention includes the steps of: 9 factors including histone H1FOO, and DPPA3 The gene sequence of From neural stem cell state induced from bovine fibroblasts, Pluripotent stem cells, ectoderm, mesoderm, and endoderm states The Bovidae The present invention is characterized by producing an expression vector for the The expression vectors of the present invention can express OCT4, SOX2, KLF4, c-MYC, NANOG, LIN28, and TERT 9 factors including histone H1FOO, and DPPA3 The gene sequence of From neural stem cell state induced from bovine fibroblasts, They are characterized by being capable of being induced into any one of pluripotent stem cells, ectoderm, mesoderm, and endoderm states. [Effects of the Invention]

[0009] According to the present invention, by introducing an expression vector containing the gene sequences of seven factors including OCT4, SOX2, KLF4, c-MYC, NANOG, LIN28, and TERT into animal cells, genetically modified cells that can be induced to a neural stem cell state or any of a pluripotent stem cell state, an ectoderm state, a mesoderm state, and an endoderm state can be produced, thereby providing a method for producing genetically modified cells that can easily generate pluripotent stem cells, neural stem cells, ectoderm state, mesoderm state, and endoderm state. [Brief explanation of the drawings]

[0010] [Figure 1] 1 is a photograph of agarose gel electrophoresis of the OSKN-T2A sequence, which is the DNA sequence of an expression vector according to an example of the present invention. [Figure 2] 1 is a photograph of agarose gel electrophoresis of the T2A-CL sequence, which is the DNA sequence of an expression vector according to an example of the present invention. [Figure 3]1 is a photograph of agarose gel electrophoresis of the six-factor expression vector pCAGPB-IRed2 / OSKNCL according to an example of the present invention. [Figure 4] FIG. 1 is a conceptual diagram showing the structure of the six-factor expression vector pCAGPB-IRed2 / OSKNCL according to an example of the present invention. [Figure 5] FIG. 1 is a conceptual diagram showing the structure of a vector that expresses each factor independently according to an example of the present invention. [Figure 6] 1 is a photograph showing agarose gel electrophoresis of vectors expressing each factor alone according to an example of the present invention, which were cleaved with EcoRV. [Figure 7] 1 is a photograph of a culture dish replated in a first culture example for producing genetically modified cells according to an embodiment of the present invention. [Figure 8] 10 is a photograph of a culture dish reseeded in a second culture example for producing genetically modified cells according to an embodiment of the present invention. [Figure 9] 1 is a graph showing the expression of neural stem cell marker genes in genetically modified cells produced using seven factors according to an example of the present invention. [Figure 10] 1 is a graph showing the expression of neural stem cell marker genes in genetically modified cells produced using nine factors according to an example of the present invention. [Figure 11] 1 is a photograph of a culture dish in which genetically modified cells produced using nine factors according to an example of the present invention are cultured to maintain pluripotent stem cells. [Figure 12] 1 is a graph showing the expression levels of pluripotent stem cell marker genes when genetically modified cells produced using seven factors according to an example of the present invention were induced to a pluripotent stem cell state by adhesion culture. [Figure 13] 1 is a graph showing the expression levels of pluripotent stem cell marker genes when genetically modified cells produced using nine factors according to an example of the present invention were induced to a pluripotent stem cell state in suspension culture. [Figure 14] 1 is a graph showing the expression levels of marker genes for ectoderm, mesoderm, and endoderm when induced culture is performed to induce the generation of ectoderm, mesoderm, and endoderm according to an example of the present invention. [Figure 15] 1 is a graph showing the expression levels of ectoderm, mesoderm, and endoderm marker genes during induction culture according to an example of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0011] <Embodiment> As a result of intensive research to solve the above-mentioned problems, the present inventors have succeeded in producing genetically modified cells that can be easily induced into a neural stem cell state or any of the states of pluripotent stem cells, ectoderm, mesoderm, and endoderm by genetically introducing into animal cells an expression vector that expresses a total of seven factors: six factors (OCT4, SOX2, KLF4, c-MYC, NANOG, LIN28) consisting of the so-called Yamanaka four factors (OCT4, SOX2, KLF4, c-MYC), and the so-called Thomson four factors (OCT4, SOX2, NANOG, LIN28), plus TERT (Telomerase Reverse Transcriptase). Furthermore, we found that by combining one or more of the other four factors (histone H1FOO, DPPA3 (Developmental pluripotency-associated protein 3), p53 inhibitor, and MBD3 (Methyl-CpG-binding domain protein 3) inhibitor) with the seven factors mentioned above from an expression vector expressing these factors and then transfecting the combined vector into animal cells, we were able to improve the efficiency of producing genetically modified cells that can easily generate neural stem cell states, pluripotent stem cell states, ectoderm, mesoderm, and endoderm. Furthermore, we found that we could produce genetically modified cells with different generative abilities for ectoderm, mesoderm, and endoderm. In this regard, it was found that the genetically modified cells according to this embodiment can be produced by culturing cells into which these expression vectors have been introduced and adding an LIF receptor agonist, an FGF (fibroblast growth factor) receptor agonist, or a Wnt signal inhibitor, or by culturing cells after adding an LIF receptor agonist, an activin receptor agonist, a Wnt signal inhibitor, a GSK inhibitor, an Src inhibitor, or an epigenetics eraser. In addition, it was found that the genetically modified cells according to this embodiment can be maintained in a neural stem cell state by culturing them with the addition of an LIF receptor agonist, an FGF receptor agonist, and a Wnt signal inhibitor. Furthermore, the inventors have discovered a culture method for maintaining the genetically modified cells of this embodiment in a pluripotent stem cell state by culturing the cells with the addition of an LIF receptor agonist, an activin receptor agonist, a Wnt signal inhibitor, a GSK (Glycogen Synthase Kinase) inhibitor, an Src (Proto-oncogene tyrosine-protein kinase Src) inhibitor, and an epigenetics eraser. Furthermore, by culturing with the addition of animal serum, we succeeded in easily generating pluripotent stem cell states, ectoderm, mesoderm, and endoderm.

[0012] The expression vector and its production method according to the embodiments of the present invention, the method for producing genetically modified cells using the expression vector, the culture method, and the genetically modified cells will be described in more detail below with reference to the embodiments.

[0013] [Overview of the method for producing genetically modified cells] First, an outline of the method for producing genetically modified cells according to this embodiment will be described. The process for producing genetically modified cells according to this embodiment by genetic engineering involves producing DNA sequences encoding the seven or four factors described above, producing expression vectors incorporating these DNA sequences, introducing the genes into animal cells using the expression vectors, and applying improved iPS cell induction culture.

[0014] First, when producing DNA, the base sequences of DNA sequences encoding the seven and four factors derived from mammals such as humans, mice, and cows can be obtained from DNA sequence databases commonly known to those skilled in the art, such as GenBank. Each of these DNA sequences can be produced by known chemical synthesis methods (Thermo Fisher, GenScript, etc.). Alternatively, messenger RNA can be extracted from animal cells expressing each factor using techniques commonly known to those skilled in the art, reverse transcribed into DNA using the messenger RNA as a template, and the DNA sequence for each factor can be individually amplified by PCR.

[0015] Using recombinant DNA technology commonly known to those skilled in the art, expression vectors incorporating the DNA sequences of each factor can be produced by incorporating them into commercially available plasmid vectors, lentiviral vectors, retroviral vectors, etc. In this case, the DNA sequences of multiple factors can be incorporated together, or each factor can be incorporated individually into an expression vector. For example, a virus-derived 2A peptide-encoding DNA sequence can be used to link the DNA sequences as a single polypeptide-encoding DNA sequence, and then integrated into a single vector for production. For example, when a virus-derived 2A peptide-encoding DNA sequence commonly known to those skilled in the art is used to link the DNA sequences as a single polypeptide chain, a single messenger RNA transcribed in the transfected cell is translated into a polypeptide. In this case, the single polypeptide chain encoding each factor is split into individual factors at the 2A peptide region, enabling polycistronic expression of functional factors.

[0016] These expression vectors can be introduced into animal cells such as fibroblasts using methods commonly known to those skilled in the art, such as viral infection, calcium phosphate, lipofection, or electroporation, to produce animal cells incorporating the expression vector.

[0017] The animal cells incorporating this expression vector are cultured using a culture method (hereinafter simply referred to as "improved iPS cell induction culture") that the present inventors have adapted and improved based on iPS cell induction culture for inducing iPS cells, thereby enabling the production of genetically modified cells according to this embodiment.

[0018] These genetically modified cells are primarily in a neural stem cell state, but can be converted (switched) to a more undifferentiated pluripotent stem cell state by culturing them using other culture methods. Alternatively, ectoderm, mesoderm, and endoderm can be easily generated using other culture methods. Each of these steps is described in detail below.

[0019] (animal cells) The animals from which the animal cells of this embodiment are derived can be, for example, various mammalian animals. In particular, this embodiment describes various treatments for animal cells of the Bovinae subfamily. While the examples described below describe the generation of genetically modified cells from domestic cattle (Bos taurus), it is believed that similar results can be obtained from other animals, such as water buffalo (Bubalus) and bison (Bison), due to genetic variations in the bovine genome. Furthermore, animal cells of this embodiment can be derived from other animals, such as livestock, companion animals, laboratory animals, humans (Homo sapiens), and non-human primates. In addition to cattle, examples of domestic animals include pigs (Sus scrofa domesticus), horses (Equus caballus), sheep (Ovis aries), and rabbits (Leporinae Trouessart). Examples of companion animals include dogs (Canis lupus familiaris) and cats (Felis silvestris catus). Examples of laboratory animals include mice (Mus musculus), rats (Rattus norvegicus), and hamsters (Mesocricetus auratus). Examples of non-human primates include gorillas (Gorilla), chimpanzees (Pan troglodytes), rhesus monkeys (Macaca mulatta), and other anthropoids and prosimians. Cells of placental mammals in the subclass Eutheria, including rare mammals, can all be used to produce genetically modified cells according to this embodiment.

[0020] In addition, the genetically modified cells of this embodiment may be cells of a disease model animal, transgenic cells into which genes have been introduced by genetic recombination techniques using various vectors, or mammalian cells whose genetic information has been processed by techniques such as gene knockout or conditional knockout.

[0021] In this embodiment, cells obtained from the various animal bodies described above can be obtained by methods commonly known to those skilled in the art. These various cells may be cells that have nuclei and can be cultured in adherent or suspension culture. For example, fibroblasts and the like can be easily obtained as such cells. Specifically, in the case of bovine (Bos taurus), for example, bovine fibroblasts can be obtained by finely slicing tissue pieces obtained from the bovine ear, seeding them on a culture dish, and culturing them in Dulbecco's MEM medium supplemented with nutrients such as fetal calf serum (hereinafter referred to as "FCS").

[0022] (Method for producing expression vector) First, we will explain the seven factors for producing genetically modified cells that easily generate neural stem cell states, pluripotent stem cell states, ectoderm, mesoderm, and endoderm, as well as the method for producing an expression vector using these four additional factors, and the structure of this expression vector. In this embodiment, an example of producing an expression vector containing six of the seven factors excluding TERT, and an example of producing an expression vector that expresses TERT and each of the remaining four factors independently will be described.

[0023] First, we will explain how to produce the coding DNA sequences for six factors, including OCT4, SOX2, KLF4, NANOG, c-MYC, and LIN28. For example, DNA sequences encoding animal proteins such as OCT4, SOX2, KLF4, NANOG, c-MYC, and LIN28 are produced by chemical synthesis (Thermo Fisher, GenScript, etc.), a method commonly used by those skilled in the art. During chemical synthesis, the codons, AT / CG ratio, number of repeats, etc. may be optimized taking into consideration the animal species of cells to be transfected, ease of synthesis, etc. Alternatively, the six factors can be produced by extracting messenger RNA from animal cells expressing the six factors using techniques commonly known to those skilled in the art, reverse-trancribing the messenger RNA into DNA using the messenger RNA as a template, and amplifying the DNA sequences encoding the six factors individually using PCR. In this case, the primers used in PCR may incorporate various sequences for improving translation efficiency, such as Kozak sequences. Furthermore, to link the DNA sequences encoding the individual factors, the primers may incorporate DNA encoding a viral 2A peptide sequence (E2A, F2A, P2A, etc.), and may be designed to generate an overlapping DNA sequence of approximately 20 to 25 bases with the DNA sequence encoding the downstream factor. Primers may be chemically synthesized using techniques commonly known to those skilled in the art. A thermostable DNA polymerase may be used to amplify the DNA sequences by PCR. Here, since the six factors of various animal species share homology in nucleotide sequence, amino acid sequence, protein three-dimensional structure, etc., six factors derived from various vertebrates such as mammals, birds, and amphibians may be produced. Furthermore, family genes showing homology in nucleotide sequence, amino acid sequence, protein three-dimensional structure, etc., such as L-MYC instead of c-MYC and KLF2 instead of KLF4, may be used.

[0024] Next, a method for producing expression vectors for the six factors from the DNA sequences encoding the six factors thus produced will be described. In this embodiment, a six-factor expression vector can be produced by cleaving the cloning site of an expression vector for various animal cells with a restriction enzyme or the like, introducing the DNA sequence of each factor, blunting the ends, and ligating. Expression vectors for the other four factors can also be produced in a similar manner. Here, the expression vector may be a commercially available plasmid vector (PB531A-2, SBI), lentiviral vector (pLVSIN-CMV Neo Vector, Takara Bio), retroviral vector (pGP Vector, Takara Bio), or the like, as long as it can express a foreign gene introduced into animal cells.

[0025] The six factors may be expressed from a single (single, individual) expression vector, as in the expression vectors for TERT and four factors described below, or they may be divided into four, three, or two factors to produce a polycistronic expression vector. In other words, it is equally feasible to produce two DNA sequences encoding four factors (OCT4, SOX2, KLF4, NANOG) and two factors (c-MYC, LIN28), or to produce a DNA sequence encoding two factors (c-MYC, LIN28). That is, in this embodiment, the DNA sequences of the factors can be introduced into one or more expression vectors in any combination, that is, the expression vector of this embodiment may be one or more.

[0026] Next, a method for producing an expression vector that expresses each of the above-mentioned TERT and four factors independently will be described. Of these factors, TERT is essential for the genetically modified cells of this embodiment. The other four factors have the effect of facilitating the production of genetically modified cells and improving production efficiency.

[0027] Specifically, the efficiency of producing the genetically modified cells according to this embodiment is improved by introducing an expression vector comprising the gene sequences of histones H1FOO and DPPA3 out of the four factors.

[0028] Furthermore, the efficiency of producing the genetically modified cells according to this embodiment can be improved by introducing an expression vector carrying the gene sequences of an MBD3 function inhibitor and a p53 function inhibitor.

[0029] Among these, antisense MBD3 RNA can be used as an MBD3 function inhibitor. Antisense MBD3 RNA can be produced by incorporating an MBD3-encoding DNA sequence in the reverse orientation into an expression vector. This allows expression of a single-stranded RNA as an antisense RNA that complementarily binds to MBD3 mRNA, thereby inhibiting MBD3 protein expression. Alternatively, MBD3 function inhibitors may be short hairpin MBD3 RNA, antisense MBD3 synthetic oligo DNA, or other polypeptides, proteins, DNA, RNA, PNA (peptide nucleic acid), etc. that inhibit the function of the MBD3 gene and MBD3 protein.

[0030] As a p53 function inhibitor, p53DD can be used. p53DD is a polypeptide corresponding to positions 231 to 324 from the N-terminus of the tumor suppressor gene product p53 protein in bovines, and has the activity of binding to p53 protein and inhibiting its function. p53DD can also be produced by extracting messenger RNA from animal cells expressing these factors using techniques commonly known to those skilled in the art, reverse transcribing the messenger RNA into DNA using the messenger RNA as a template, and amplifying the DNA sequences encoding these factors individually by PCR. Alternatively, other polypeptides can be used as p53 function inhibitors as long as they substantially inhibit the function of p53 protein. For example, short hairpin p53 RNA, antisense p53 RNA, antisense p53 synthetic oligo DNA, and the like that inhibit the expression of the p53 gene, as well as polypeptides, proteins, DNA, RNA, PNA, and the like that inhibit the function of the p53 gene and p53 protein, can also be used as p53 function inhibitors.

[0031] The DNA sequences of the above-mentioned TERT and four factors can also be produced by chemical synthesis. During chemical synthesis, codons may be optimized to match the animal species of the cells to be transfected. Furthermore, because factors from various animal species share homology in nucleotide sequence, amino acid sequence, protein three-dimensional structure, etc., factors derived from various vertebrates, such as mammals, birds, and amphibians, may also be used. In this embodiment, an example is described in which the above-mentioned TERT and four factors are each introduced into one expression vector. As described above, vectors that express two or more of these factors polycistronically may be produced in any combination.

[0032] (Introduction of expression vector into cells) Next, a method for introducing the above-mentioned expression vector into animal cells will be described. Gene transfer into animal cells using an expression vector can be carried out by, for example, the lipofection method commonly known to those skilled in the art, or by methods commonly known to those skilled in the art, such as viral infection, calcium phosphate, or electroporation. In the case of the bovine fibroblasts mentioned above, gene transfer is possible by adding a complex of the expression vector and ViaFect (Promega), a lipofection reagent, and incubating for approximately 6 hours.

[0033] In this embodiment, the expression vectors used to produce genetically modified cells include a TERT expression vector in addition to the six factor expression vectors, thereby introducing a total of seven factors. In addition, to improve the efficiency of producing genetically modified cells, expression vectors for one or more factors selected from the group consisting of an MBD3 function inhibitor, a p53 function inhibitor, histone H1FOO, and DPPA3 may be simultaneously introduced.

[0034] Furthermore, when producing genetically modified cells that efficiently generate ectoderm and endoderm, as described below, it is preferable to use a combination of expression vectors for histones H1FOO and DPPA3 in addition to the seven factors mentioned above. That is, the combination of factors for producing genetically modified cells according to this embodiment that are suitable for generating ectoderm and endoderm is nine factors: OCT4, SOX2, KLF4, c-MYC, NANOG, LIN28, TERT, histone H1FOO, and DPPA3.

[0035] Alternatively, when producing genetically modified cells that efficiently generate ectoderm and mesoderm, as described below, it is preferable to use a combination of expression vectors for histone H1FOO, DPPA3, a p53 function inhibitor, and an MBD3 function inhibitor in addition to the seven factors. In other words, the combination of factors used to produce genetically modified cells according to this embodiment that are suitable for generating ectoderm and mesoderm is 11 factors: OCT4, SOX2, KLF4, c-MYC, NANOG, LIN28, TERT, histone H1FOO, DPPA3, an MBD3 function inhibitor, and a p53 function inhibitor.

[0036] (Cultivation method for producing genetically modified cells) Next, the genetically modified cells according to this embodiment are produced by applying improved iPS induction culture to the animal cells into which the expression vector has been introduced. Specifically, in the improved iPS cell induction culture for generating genetically modified cells according to this embodiment, animal cells incorporating an expression vector are cultured by adding to a basal culture medium for animal cells a leukemia inhibitory factor receptor agonist (hereinafter simply referred to as "LIF"), an FGF receptor agonist such as fibroblast growth factor (hereinafter referred to as "FGF"), an activin receptor agonist, a small molecule enzyme inhibitor, an Src inhibitor, and an epigenetics eraser. In this embodiment, the small molecule enzyme inhibitors include at least a Wnt signal inhibitor and a GSK inhibitor.

[0037] More specifically, the improved iPS induction culture according to this embodiment can be performed by adding a LIF receptor agonist, a fibroblast growth factor receptor agonist, and a Wnt signal inhibitor to a basal culture medium, or by using a production culture medium prepared by adding a LIF receptor agonist, an activin receptor agonist, a Wnt signal inhibitor, a GSK inhibitor, an Src inhibitor, and an epigenetics eraser to a basal culture medium. This makes it possible to easily produce the genetically modified cells according to this embodiment.

[0038] In this embodiment, the basal culture medium for animal cells may be α-MEM or the like containing nutrients such as FCS. In this case, the nutrients are not limited to FCS, but may be animal serum such as bovine serum, horse serum, or human serum, or serum substitutes such as albumin, Knockout Serum Replacement (Gibco), or StemSure Replacement (Wako Pure Chemical Industries, Ltd.).

[0039] The LIF (LIF receptor agonist) may be, for example, an LIF family protein such as IL6, or a synthetic LIF receptor agonist, as long as it has the effect of stimulating the LIF receptor. Alternatively, 2i or 3i culture may be used in combination with an ERK inhibitor, MEK inhibitor, GSK inhibitor, or FGF receptor inhibitor, which provides an effect similar to that achieved by adding LIF.

[0040] The fibroblast growth factor receptor agonist may be, for example, FGF2. The FGF2 may be any FGF, such as FGF1 or FGF4, or any FGF receptor agonist, as long as it has the effect of stimulating the FGF receptor, or may be a synthetic FGF receptor agonist.

[0041] The Wnt signal inhibitor may be, for example, IWR1, or an inhibitor such as XAV939.

[0042] The activin receptor agonist can be activin A. Alternatively, various inhibitors that can stimulate the activin receptor can be used as the activin receptor agonist. For example, activin B, activin E, BMP, TGFβ superfamily members such as TGF, and activin receptor agonists can be used. Furthermore, synthetic activin receptor agonists can also be used.

[0043] The GSK inhibitor may be a GSK3β inhibitor. More specifically, CHIR99021 may be used as the GSK3β inhibitor. Alternatively, 6Bio or the like may be used as the GSK3β inhibitor. Furthermore, other GSK inhibitors other than GSK3β may also be used.

[0044] WH-4-023 can be used as the Src inhibitor. Other Src inhibitors that can be used include AZM475271.

[0045] Vitamin C (L-ascorbic acid) can be used as an epigenetic eraser. Other epigenetic erasers that can be used include 5-aza-cytidine and HDAC inhibitors (sodium butyrate, trichostatin A, valproic acid, SAHA, etc.).

[0046] The concentrations of these LIF receptor agonists, fibroblast growth factor receptor agonists, Wnt signal inhibitors, activin receptor agonists, Wnt signal inhibitors, GSK inhibitors, Src inhibitors, and epigenetics erasers added to the basal culture medium may be any concentrations that are substantially effective.

[0047] The low molecular enzyme inhibitors may also include MAPK inhibitors, ROCK inhibitors, HDAC inhibitors, and the like. The culture medium for producing improved iPS cells according to this embodiment can be an optimal culture medium for each type of animal cell. Furthermore, amino acids, vitamins, antioxidants, antibiotics, collagen precursors, trace metal ions or complexes, various salts, and the like may be added to the culture medium. Other components may also be added at concentrations set according to the type of animal or cell being cultured.

[0048] In the improved iPS cell induction culture according to this embodiment, animal cells incorporating an expression vector are cultured for 3 to 4 weeks in a production culture medium containing the above-mentioned components. The cells are then replated onto a collagen (gelatin)-coated culture dish or the like. After 6 to 8 weeks of iPS cell induction culture, cell colonies formed on the culture dish are harvested and replated. Once the cell number has increased, they are subcultured using techniques commonly known to those skilled in the art, thereby producing genetically modified cells that can easily generate neural stem cells, pluripotent stem cells, ectoderm, mesoderm, and endoderm. In other words, based on 11 factors, including seven factors including OCT4, SOX2, KLF4, c-MYC, NANOG, LIN28, and TERT, and also histone H1FOO, DPPA3, an MBD3 function inhibitor, and a p53 function inhibitor, it is possible to easily change (switch) between mainly neural stem cell states and pluripotent stem cell states, and to produce genetically modified cells that can be easily induced to differentiate into either ectoderm, mesoderm, or endoderm.

[0049] Next, a culture method for maintaining the neural stem cell state or inducing the cells to any one of pluripotent stem cells, ectoderm, mesoderm, and endoderm states will be described.

[0050] (Culture method to maintain neural stem cell state) Next, a maintenance culture method for maintaining genetically modified cells in a neural stem cell state according to this embodiment will be described. By culturing the above-mentioned genetically modified cells in a maintenance culture medium supplemented with an LIF receptor agonist, a fibroblast growth factor receptor agonist, and a Wnt signal inhibitor, it is possible to maintain the genetically modified cells produced by the above-mentioned improved iPS induction culture in a neural stem cell state. In the case of the bovine fibroblasts mentioned above, for example, the genetically modified cells can be cultured (37°C, 5% CO2) using α-MEM as the basal culture medium, supplemented with FCS as a nutrient, LIF (10 ng / ml), FGF2 (20 ng / ml), and IWR1 (2.5 μM). Once the cell number has increased, they can be subcultured using methods commonly known to those skilled in the art, and maintained in a neural stem cell state. As with the above-mentioned production culture medium, the components and concentrations can be appropriately changed within the range of optimization by those skilled in the art, and may be any concentration that substantially achieves the desired effect.

[0051] (Culture method for maintaining pluripotent stem cells) Next, a switch culture method for maintaining genetically modified cells according to this embodiment in a pluripotent stem cell state will be described. Genetically modified cells cultured using the improved iPS induction culture method described above, or genetically modified cells cultured using the maintenance culture method described above and maintained in a neural stem cell state, can be maintained in a pluripotent stem cell state similar to iPS cells by culturing them with the addition of an LIF receptor agonist, an activin receptor agonist, a Wnt signal inhibitor, a GSK inhibitor, an Src inhibitor, and an epigenetic eraser. In the case of the bovine fibroblasts mentioned above, for example, α-MEM is used as the basal culture medium, and FCS is added as a nutrient, and LIF (10 ng / ml), activin A (20 ng / ml), IWR1 (5 μM), WH-4-023 (0.3 μM), CHIR99021 (1 μM), and vitamin C (50 μg / ml) are added to culture the genetically modified cells (37°C, 5% CO2).Once the cell number has increased, the cells can be subcultured using methods commonly known to those skilled in the art, and maintained in a pluripotent stem cell state. As with the above-mentioned production culture medium, the components and concentrations can be appropriately changed within the range of optimization by those skilled in the art, and may be any concentration that substantially achieves the desired effect.

[0052] (Culture method for easily generating pluripotent stem cells, ectoderm, mesoderm, and endoderm) Next, a method for inducing and culturing the genetically modified cells according to this embodiment, which allows for the simple generation of pluripotent stem cell states, ectoderm, mesoderm, and endoderm, will be described. It is now possible to easily generate pluripotent stem cell states, ectoderm, mesoderm, and endoderm from genetically modified cells cultured using the improved iPS induction culture method described above, genetically modified cells cultured using the maintenance culture method described above and maintained in a neural stem cell state, and genetically modified cells maintained in a pluripotent stem cell state using the switch culture method described above. In the case of the bovine fibroblasts mentioned above, for example, α-MEM is used as the basal culture medium, and FCS is added as a nutrient. Genetically modified cells are cultured in suspension (37°C, 5% CO2) using low-adhesion culture dishes such as LipidureCoat Plates (NOF Corporation). Alternatively, adherent culture (37°C, 5% CO2) can be performed using adherent culture dishes (TrueLine, etc.). This allows for the simple generation of pluripotent stem cells, ectoderm, mesoderm, and endoderm within 6 days in adherent culture and within 16 days in suspension culture. As with the above-mentioned production culture medium, the components and concentrations can be appropriately changed within the range of optimization by those skilled in the art, and may be any concentration that substantially achieves the desired effect.

[0053] Cultured cells produced by the method for producing genetically modified cells according to this embodiment can be distinguished from other cells by confirming, by PCR or other methods, that each factor has been incorporated into the genome or as an external gene. Furthermore, even when the genetically modified cells according to this embodiment are cultured using the above-mentioned culture methods, they can be distinguished from cells in other states by observing, measuring, etc., various markers and traits common to those skilled in the art. Furthermore, those skilled in the art will understand that because cells in such states change state as a result of complex biochemical processes, there are special circumstances that make it impossible or impractical to directly identify them by their structure or characteristics.

[0054] The above configuration can provide the following effects. Cattle and pigs are important livestock for industry, and their reproduction is controlled by humans. When breeding, important economic traits such as milk production and meat quality are continuously improved through genetic breeding. In addition, the sperm, eggs, and fertilized eggs of superior livestock are semi-permanently frozen and stored to produce offspring. Meanwhile, livestock cultured meat production technology, which involves decomposing organic matter such as biomass and effectively utilizing it as a culture medium component, is attracting attention because it contributes to resource conservation, reducing the amount of grain consumed as livestock feed, and reducing methane gas emissions. Furthermore, it is possible to produce germ cells through mesoderm generated from livestock iPS cells. While high-quality livestock inevitably reach the end of their lifespan, iPS cells produced from these individuals can be used to permanently utilize sperm, eggs, and fertilized eggs. Therefore, it has been desired to provide a method for producing genetically modified cells that can easily generate neural stem cell states, pluripotent stem cell states, ectoderm, mesoderm, and endoderm.

[0055] iPS cells can be used to generate various cell types derived from the ectoderm, mesoderm, and endoderm (e.g., hematopoietic stem cells, erythrocytes, cardiomyocytes, skeletal muscle cells, primordial germ cells, etc.). However, this process is complicated because it requires the combination of many different differentiation-inducing substances, and also requires a long culture period of more than one month. It was also possible to generate ectoderm, mesoderm, and endoderm by transplanting iPS cells into nude mice, but this required rearing the mice in a sterile environment, which was tedious. Furthermore, although it is possible to generate neural stem cells from iPS cells, it takes two months to generate mouse neural stem cells and six months to generate human neural stem cells. In addition, cytokines such as Noggin, FGF2, Shh, Wnt3A, BMP4, and retinoic acid must be used in combination depending on the differentiation induction stage, requiring complex, multi-step cell culture methods, making the process both cumbersome and difficult.

[0056] In contrast, the method for producing genetically modified cells according to this embodiment makes it possible to produce genetically modified cells that can be induced into a neural stem cell state or any of the following states: pluripotent stem cell, ectoderm, mesoderm, and endoderm, by introducing expression vectors for seven factors: OCT4, SOX2, KLF4, c-MYC, NANOG, LIN28, and TERT into animal cells. Thus, the genetically modified cells produced by the above-described method for producing genetically modified cells can be easily induced to a neural stem cell state or any of a pluripotent stem cell state, an ectoderm state, a mesoderm state, and an endoderm state by a simple culture method. In other words, it is possible to provide genetically modified cells that can easily generate neural stem cells, ectoderm state, mesoderm state, and endoderm state.

[0057] On the other hand, bovine spongiform encephalopathy (BSE), a fatal neurological disease, is known to affect humans through consumption of infected cattle, causing serious neurological disorders, and has had a significant negative impact on the livestock industry.Since no preventive or therapeutic drugs have been developed to prevent the onset of BSE, pathological tests are carried out at meat processing facilities to detect infected cattle at the border and prevent them from being distributed to the market. To overcome this situation, neural stem cells, particularly from cattle, were sought as a pathological model cell for BSE. However, there were problems such as the cost of consistently obtaining neural stem cells from living cattle, the difficulty of obtaining neural stem cells with high proliferation capacity and high purity, and the difficulty of obtaining highly reproducible experimental results due to individual differences in physiological functions of neural stem cells obtained from living cattle. To overcome this situation, bovine iPS cells, in particular, were sought as a pathological model cell for BSE. However, those skilled in the art know that the difficulty of generating pluripotent stem cells such as iPS cells varies greatly depending on the mammal, and it has been known that generating iPS cells from the Bovinae subfamily has been extremely difficult. In fact, although several researchers in Japan and overseas have reported the generation of iPS cells from bovines (Bos taurus), the reproducibility of the results has been extremely poor. Furthermore, it has been difficult to differentiate these cells into neural stem cells.

[0058] In contrast, the genetically modified cells according to this embodiment can be easily switched to either a neural stem cell state or a pluripotent stem cell state. The genetically modified cells according to this embodiment, when in a neural stem cell state, can be used as pathological model cells for bovine spongiform encephalopathy (BSE), a neurodegenerative disease. These pathological model cells are useful for BSE research, and allow for in vitro study of the relationship between neuronal formation and maturation and the onset of BSE, which will greatly contribute to the development of BSE-related pharmaceuticals.

[0059] Furthermore, when the genetically modified cells according to this embodiment are in a pluripotent stem cell state, they can be used to produce cultured steak meat, cultured liver, sperm, eggs, etc. from high-quality cattle. When induced to differentiate into mesoderm, skeletal muscle and adipocytes can be produced. Furthermore, since liver (liver) can be produced from endoderm and epidermis (leather) can be produced from ectoderm, it becomes possible to easily produce cultured meat. Additionally, venture companies are being established both in Japan and overseas to develop cultured meat production technology. Furthermore, bovine iPS cells can be used to produce high-quality bovine sperm and eggs, making it a viable way to permanently utilize high-quality genetic resources. Furthermore, if it is used in livestock embryos, it will contribute to improving the productivity of high-quality livestock, and if it is used in laboratory animal or wild animal embryos, it will contribute to the preservation of valuable genetic resources and the protection of rare animals.

[0060] Furthermore, the method for producing genetically modified mammalian cells of this embodiment does not require any special operations or techniques, and can efficiently produce high-quality genetically modified cells. This eliminates the need to manufacture special equipment and eliminates the need for training to operate it, reducing costs. In addition, the ability to obtain high-quality cells reduces the burden on all parties involved.

[0061] It goes without saying that the configurations and operations of the above-described embodiments are merely examples, and can be modified as appropriate within the scope of the present invention. [Example]

[0062] Next, the present invention will be described in more detail based on examples, but the following specific examples are not intended to limit the present invention.

[0063] (Method of producing DNA sequences encoding each factor) First, Table 1 below shows the NCBI accession numbers of bovine OCT4, SOX2, KLF4, NANOG, c-MYC, LIN28, and TERT, which are factors for easily generating neural stem cell states and pluripotent stem cell states.

[0064] [Table 1]

[0065] Here, we present an example of producing two DNA sequences encoding four factors (OCT4, SOX2, KLF4, NANOG) and two factors (c-MYC, LIN28) to produce genetically engineered cells that can easily generate neural stem cell and pluripotent stem cell states. In this example, a PCR product was obtained from bovine cells using synthetic DNA as a template and the primers in Table 2 below, based on techniques commonly known to those skilled in the art, and was used to prepare an expression vector.

[0066] [Table 2]

[0067] Each primer was designed to incorporate a DNA sequence encoding the 2A peptide and to generate an overlapping DNA sequence of approximately 20-25 bases with the DNA sequence encoding the downstream factor in order to link the DNA sequences encoding the individual factors by PCR. The designed primers were then chemically synthesized (Fasmac Co., Ltd., Hokkaido System Science Co., Ltd.). PCR amplification was performed using the thermostable DNA polymerase KOD plus (Toyobo Co., Ltd.) according to the attached protocol.

[0068] For NANOG, the E2A peptide-encoding DNA sequence was incorporated into the forward primer (bNANOG E2A F) of the NANOG sequence, and PCR amplification was performed using the reverse primer (GAbNANOG NotI stop R) and the chemically synthesized NANOG-encoding DNA sequence as a template.

[0069] The PCR-amplified DNA sequences encoding OCT4, SOX2, KLF4, and NANOG were mixed, and overlapping DNA sequences were amplified using a forward primer (IFbOCT4 XbaI pCAGPB startF) and a reverse primer (IFbNANOG T2A R) to obtain a single OCT4-F2A-SOX2-P2A-KLF4-E2A-NANOG-T2A sequence (OSKN-T2A sequence).

[0070] More specifically, for OCT4, a Kozak sequence was incorporated into the forward primer (GAbOCT4 NheI Kozak start F) to improve translation efficiency, and the F2A peptide-encoding DNA sequence was incorporated into the reverse primer (bOCT4 F2A R). PCR amplification was performed using the chemically synthesized OCT4-encoding DNA sequence as a template with KOD-plus DNA polymerase (Toyobo Co., Ltd.). For SOX2, the F2A peptide-encoding DNA sequence was incorporated into the SOX2 forward primer (bSOX2 F2A start F), the P2A peptide-encoding DNA sequence was incorporated into the reverse primer (bSOX2 P2A R), and PCR amplification was performed using a chemically synthesized SOX2-encoding DNA sequence as a template. For KLF4, the P2A peptide-encoding DNA sequence was incorporated into the KLF4 forward primer (bKLF4 P2A F), the E2A peptide-encoding DNA sequence was incorporated into the reverse primer (bKLF4 E2A R), and PCR amplification was performed using a chemically synthesized KLF4-encoding DNA sequence as a template. For NANOG, the E2A peptide-encoding DNA sequence was incorporated into the NANOG sequence forward primer (bNANOG E2A F), and PCR amplification was performed using a chemically synthesized NANOG-encoding DNA sequence as a template with a reverse primer (GAbNANOG NotI stop R). The PCR-amplified OCT4, SOX2, KLF4, and NANOG-encoding DNA sequences were mixed, and overlapping DNA sequences were used with a forward primer (IFbOCT4 XbaI pCAGPB startF) and a reverse primer (IFbNANOG T2A R) to obtain a single OCT4-F2A-SOX2-P2A-KLF4-E2A-NANOG-T2A sequence (hereafter referred to as the "OSKN-T2A sequence").

[0071] Figure 1 shows an agarose gel electrophoresis image of the OSKN-T2A sequence produced by PCR. The 4.6 kb band in the right lane represents the OSKN-T2A sequence. The left lane is a DNA size marker, representing 23.13 kb, 9.42 kb, 6.56 kb, 2.32 kb, and 2.02 kb, respectively, from top to bottom.

[0072] Next, the CL sequences encoding the two factors (c-MYC and LIN28) were amplified by PCR using the primers shown in Table 2. For c-MYC, the forward primer (IFbc-MYC T2A F) contained the DNA sequence encoding the T2A peptide, and the reverse primer (bc-MYC P2A R) contained the DNA sequence encoding the P2A peptide. PCR amplification was performed using a chemically synthesized c-MYC coding DNA sequence as a template. For LIN28, the P2A peptide-encoding DNA sequence was incorporated into the forward primer (bLIN28 P2A F), and PCR amplification was performed using the reverse primer (IFbLIN28 BamHI stopR) and a chemically synthesized LIN28-encoding DNA sequence as a template. These PCR-amplified c-MYC and LIN28-encoding DNAs were then mixed. PCR was performed using a forward primer (IFbc-MYC T2A F) and a reverse primer (IFbLIN28 BamHI stopR). By utilizing overlapping DNA sequences, these were amplified as a single DNA sequence encoding the linked T2A-c-MYC-P2A-LIN28 (hereafter referred to as the "T2A-CL sequence").

[0073] Figure 2 shows an agarose gel electrophoresis image of the T2A-CL sequence produced by PCR. The 2.1 kb band in the left lane represents the T2A-CL sequence. The right lane is a DNA size marker, which, from top to bottom, represents 6.18 kb, 3.63 kb, 2.94 kb, 2.03 kb, and 1.44 kb, respectively.

[0074] (Method for producing six factor expression vectors) Next, a method for producing an expression vector for six factors will be described. The expression vector 531A-2 (SBI) was digested with restriction enzymes StuI and XbaI (NEB) to remove the EF1α promoter. The expression vector pMK10 (Kobayashi et al., Biotechniques, vol. 21, pp. 398-402, 1996) was digested with restriction enzymes NruI and XbaI (NEB), and the CAG promoter was excised and inserted into the StuI and XbaI sites of 531A-2 to produce pCAGPB-IRed2. The cloning site of this pCAGPB-IRed2 (SBI) was digested with restriction enzymes XbaI and BamHI (NEB). This cloning site is shown in Figure 4, described below. The digested pCAGPB-IRed2, OSKN-T2A sequence, and T2A-CL sequence were mixed together, and then the six-factor expression vector pCAGPB-IRed2 / OSKNCL was prepared using the In-Fusion HD Cloning Kit (Takara Bio Inc.), incorporating a DNA sequence encoding OCT4-F2A-SOX2-P2A-KLF4-E2A-NANOG-T2A-c-MYC-P2A-LIN28 (hereinafter referred to as the "OSKNCL sequence") between the XbaI and BamHI sites.

[0075] Figure 3 shows an agarose gel electrophoresis image of the constructed six-factor expression vector pCAGPB-IRed2 / OSKNCL. The left lane shows the bands of the 9.7 kb and 5.0 kb sequences obtained by cleaving the 14.7 kb pCAGPB-IRed2 / OSKNCL with the restriction enzyme XhoI. The right lane shows DNA size markers, which, from top to bottom, represent 23.13 kb, 9.42 kb, 6.56 kb, 2.32 kb, and 2.02 kb, respectively.

[0076] FIG. 4 shows the structure of this six-factor expression vector pCAGPB-IRed2 / OSKNCL.

[0077] (Method for producing vectors that promote the production of genetically modified cells) Next, a method for producing an expression vector that expresses a factor that promotes the production of genetically modified cells will be described. Figures 5(a) to 5(e) show the structures of vectors that express human TERT, bovine antisense MBD3, bovine histone H1FOO, bovine p53DD, and bovine DPPA3 alone, respectively, as factors that promote the production of these genetically modified cells. In addition, the NCBI accession numbers of the DNA sequences encoding these factors, TERT, antisense MBD3, histone H1FOO, p53DD, and DPPA3, are shown in Table 3 below.

[0078] [Table 3]

[0079] The primers listed in Table 2 above were used to amplify the DNA sequences encoding these factors by PCR. Specifically, the expression vector PBQM812A-1 (SBI) was cleaved with restriction enzymes NheI and NotI and the DNA sequences encoding each factor were mixed and inserted between the NheI and NotI sites using the In-Fusion HD Cloning Kit to produce the expression vectors PBQM812A-1 / TERT, PBQM812A-1 / antisense MBD3, PBQM812A-1 / histone H1FOO, and PBQM812A-1 / p53DD, which express these factors individually. The DNA sequence encoding DPPA3 was cleaved with restriction enzymes NheI and NotI and inserted into PBQM812A-1 cleaved with NheI and NotI using the Rapid DNA Ligation Kit (Promega) to produce PBQM812A-1 / DPPA3. These expression vectors were further cleaved with the restriction enzymes MfeI and HpaI, blunted using a Blunting High Kit (Toyobo Co., Ltd.), and subjected to a self-ligation reaction using a Rapid DNA Ligation Kit. This resulted in the construction of pCMV5PB / TERT, pCMV5PB / antisense MBD3, pCMV5PB / histone H1FOO, pCMV5PB / p53DD, and pCMV5PB / DPPA3, which express these factors individually.

[0080] Figure 6 shows the results of agarose gel electrophoresis of the constructed vectors expressing TERT, p53DD, antisense MBD3, histone H1FOO, and DPPA3, each digested with the restriction enzyme EcoRV. TERT (lane 1) separated into 7.5 kb and 3.4 kb bands. p53DD (lane 3) gave a 7.8 kb band. Antisense MBD3 (lane 4) gave an 8.3 kb band. Histone H1FOO (lane 5) gave an 8.6 kb band. DPPA3 (lane 7) gave an 8.0 kb band. Lanes 2, 6, and 8 are DNA size markers, showing bands at 23.13 kb, 9.42 kb, 6.56 kb, 2.32 kb, and 2.02 kb, respectively.

[0081] (Culture method for producing genetically modified cells) Next, a culture method for producing genetically modified cells that can easily generate neural stem cells, pluripotent stem cells, and ectoderm, mesoderm, or endoderm will be described. As a first culture example, tissue pieces obtained from a bovine ear were cut into small pieces, seeded on a culture dish, and cultured (37°C, 5% CO2) in Dulbecco's MEM medium (Sigma) supplemented with nutrients such as fetal calf serum (hereinafter referred to as "FCS") to obtain bovine fibroblasts. Bovine fibroblasts were seeded (1.0 × 10 6 The cells were then transfected into bovine fibroblasts at 37°C for 16 hours at 5% CO2. The expression vector (2 μg) was mixed with the lipofection reagent ViaFect (10 μl, Promega). The expression vectors used to produce the genetically modified cells included the TERT expression vector in addition to the six-factor expression vector, resulting in a total of seven factors being introduced. The gene was transfected into the bovine fibroblasts by adding the complex of the expression vector and ViaFect and incubating for approximately 6 hours. After transfection of the expression vector into bovine fibroblasts, iPS cell induction culture was initiated (37°C, 5% CO2). The basal culture medium was α-MEM (Wako Pure Chemical Industries), supplemented with 10% FCS (Biological Industries), LIF (10 ng / ml, Merck Millipore), fibroblast growth factor FGF2 (20 ng / ml, abm), and the Wnt signal inhibitor IWR1 (2.5 μM, Cayman). After 3-4 weeks of iPS cell induction culture, the cells were replated onto a 0.1% gelatin-coated culture dish (φ6cm, TrueLine). After 6-8 weeks of iPS cell induction culture, cell colonies formed on the culture dish were harvested and replated onto a 0.1% gelatin-coated 12-well culture dish (TrueLine).

[0082] Figure 7 shows the state of cells on the replated culture dish in the first culture example described above. Specifically, Figure 7 shows genetically modified cells generated by culturing with the addition of an LIF receptor agonist, a fibroblast growth factor receptor agonist, and a Wnt signal inhibitor. These cells are genetically modified cells produced using nine factors (OCT4, SOX2, KLF4, c-MYC, NANOG, LIN28, TERT, histone H1FOO, and DPPA3). Within the black oval frame, flattened cell colonies formed by the genetically modified cells can be seen.

[0083] In the second culture example, bovine fibroblasts were prepared as in the first culture example and transfected with an expression vector. Then, iPS cell induction culture was initiated (37°C, 5% CO2). The basal culture medium was α-MEM (Wako Pure Chemical Industries), supplemented with 10% FCS (Biological Industries), LIF (10 ng / ml), activin A (20 ng / ml, Wako Pure Chemical Industries), the Wnt signal inhibitor IWR1 (5 μM), the Src inhibitor WH-4-023 (0.3 μM, Enzo), the GSK inhibitor CHIR99021 (1 μM, Focus), and the epigenetic eraser vitamin C (50 μg / ml, Sigma). After 3-4 weeks of iPS cell induction culture, the cells were replated onto a 0.1% gelatin-coated culture dish (φ6 cm, TrueLine). After 6-8 weeks of iPS cell induction culture, cell colonies formed on the culture dish were harvested and replated onto a 0.1% gelatin-coated 12-well culture dish (TrueLine).

[0084] Figure 8 shows the state of cells on the replated culture dish in the second culture example described above. Specifically, Figure 8 shows genetically modified cells generated by culturing with the addition of an LIF receptor agonist, an activin receptor agonist, a Wnt signal inhibitor, a GSK inhibitor, an Src inhibitor, and vitamin C. These cells are genetically modified cells produced using nine factors (OCT4, SOX2, KLF4, c-MYC, NANOG, LIN28, TERT, histone H1FOO, and DPPA3). Dome-shaped cell colonies formed by the genetically modified cells can be seen within the black oval frame.

[0085] In both the first and second culture examples, once the cell numbers have increased, they are subcultured using techniques commonly known to those skilled in the art, thereby producing genetically modified cells that can easily generate neural stem cell states, pluripotent stem cell states, ectoderm, mesoderm, and endoderm. The results of this genetically engineered cell production are shown in Table 4 below:

[0086] [Table 4]

[0087] The six factors (OCT4, SOX2, KLF4, c-MYC, NANOG, and LIN28) consisting of the four Yamanaka factors and the four Thomson factors previously described could not generate pluripotent genetically modified cells. However, genetically modified cells according to this embodiment can be efficiently produced by introducing seven factors (OCT4, SOX2, KLF4, c-MYC, NANOG, LIN28, TERT), nine factors (OCT4, SOX2, KLF4, c-MYC, NANOG, LIN28, TERT, histone H1FOO, DPPA3), or eleven factors (OCT4, SOX2, KLF4, c-MYC, NANOG, LIN28, TERT, histone H1FOO, DPPA3, antisense MBD3, p53DD).

[0088] (Method for maintaining neural stem cell state) Next, a culture method for maintaining genetically modified cells in a neural stem cell state according to this embodiment will be described. The genetically modified cells produced based on the above-mentioned seven and nine factors were subcultured in α-MEM as the basal culture medium, supplemented with 10% FCS as nutrients, LIF (10 ng / ml), FGF2 (20 ng / ml), and IWR1 (2.5 μM) at 37°C and 5% CO2. The genetically modified cells in this state maintained the same morphology as shown in Figures 7 and 8. Each colony was isolated, RNA was extracted using an RNeasy-mini kit (Qiagen), and cDNA was synthesized using ReverTra Ace (Toyobo) with random primers and oligo dT primers. Gene expression levels were quantified by real-time PCR using a QuantiTect RT-PCR kit (Qiagen).

[0089] The primers for this real-time PCR method are shown in Table 5 below.

[0090] [Table 5]

[0091] Figure 9 shows the expression levels of the neural stem cell marker gene NESTIN when genetically modified cells produced with seven factors (OCT4, SOX2, KLF4, c-MYC, NANOG, LIN28, and TERT) were cultured to maintain the neural stem cell state. The relative expression levels of NESTIN in genetically modified cells and fibroblasts are shown.

[0092] Figure 10 shows the expression levels of neural stem cell marker genes SOX2 and NESTIN under similar conditions in genetically modified cells produced with nine factors (OCT4, SOX2, KLF4, c-MYC, NANOG, LIN28, TERT, histone H1FOO, and DPPA3). The relative expression levels of NESTIN in genetically modified cells and fibroblasts are shown. Thus, neural stem cell marker genes were strongly expressed in the genetically modified cells produced using the nine factors. Furthermore, once the cell numbers increased, they could be maintained in a neural stem cell state by subculturing using techniques commonly known to those skilled in the art.

[0093] (Culture method for maintaining pluripotent stem cells) Next, we will show a culture method for maintaining the produced genetically modified cells in a pluripotent stem cell state. The genetically modified cells were subcultured in α-MEM as the basal culture medium, supplemented with 10% FCS as nutrients, LIF (10 ng / ml), activin A (20 ng / ml), IWR1 (5 μM), WH-4-023 (0.3 μM), CHIR99021 (1 μM), and vitamin C (50 μg / ml) at 37°C and 5% CO2.

[0094] Figure 11 shows the morphology of cell colonies obtained by this method of pluripotent stem cell maintenance culture. This figure shows a culture dish in which genetically modified cells produced using nine factors were cultured. Within the black oval frame, dome-shaped cell colonies specific to the pluripotent stem cell state can be seen, formed by the genetically modified cells. In this way, once the number of cells has increased, they can be maintained in a pluripotent stem cell state by subculturing them using a method commonly known to those skilled in the art.

[0095] (A culture method for easily generating pluripotent stem cells, ectoderm, mesoderm, and endoderm) Next, a culture method (hereinafter referred to as "induction culture") for easily generating pluripotent stem cell states, ectoderm, mesoderm, and endoderm will be described. Using α-MEM as the basal culture medium and supplemented with FCS as a nutrient, the genetically modified cells were cultured in suspension (37°C, 5% CO2) on low-adhesion culture dishes such as LipidureCoat Plates (NOF Corporation), or in adherent culture dishes (TrueLine, etc.) at 37°C, 5% CO2. This enabled the simple generation of pluripotent stem cells, ectoderm, mesoderm, and endoderm within 6 days in adherent culture and within 16 days in suspension culture.

[0096] First, we will explain the results of generating a pluripotent stem cell state using the above-mentioned induction culture. Genetically modified cells produced using seven factors (OCT4, SOX2, KLF4, c-MYC, NANOG, LIN28, and TERT) were cultured in α-MEM supplemented with 10% FCS on an adhesive culture dish (7 × 10 4 Cells were cultured at 37°C in 5% CO2 using cells / φ10cm, TrueLine, etc. On day 6 of culture, colonies were isolated from each sample, and RNA was extracted in the same manner as in the previous experiments. The mRNA expression levels of each marker were then examined by real-time PCR.

[0097] Figure 12 shows the expression levels of pluripotent stem cell marker genes on day 6 of adherent culture when genetically modified cells produced with seven factors were induced to a pluripotent stem cell state. Figure 12(a) shows the relative mRNA expression levels of OCT4 and Figure 12(b) shows NANAG between genetically modified cells and fibroblasts. The genetically modified cells of this example expressed these markers of the pluripotent stem cell state.

[0098] Next, the genetically modified cells produced with nine factors (OCT4, SOX2, KLF4, c-MYC, NANOG, LIN28, TERT, histone H1FOO, and DPPA3) were cultured to reach a more undifferentiated pluripotent stem cell state. In this case, α-MEM supplemented with 10% FCS was used, and the nine genetically modified cells were cultured in suspension (5–10 × 10 3 After 6 days, the recombinant cells were transferred to a 6-well plate (2-4 cell clumps / well, TrueLine) and cultured under adhesion conditions (37°C, 5% CO).

[0099] FIG. 13 shows the expression levels of pluripotent stem cell marker genes on day 16 from the start of suspension culture when genetically modified cells produced with nine factors were cultured to induce a pluripotent stem cell state. Compared to the genetically modified cells produced using the seven factors in Figure 12, the expression levels of pluripotent stem cell markers were higher.

[0100] Next, induction culture was carried out to induce the generation of ectoderm, mesoderm, and endoderm. First, genetically modified cells produced using seven factors (OCT4, SOX2, KLF4, c-MYC, NANOG, LIN28, and TERT) were cultured on adherent culture dishes (7 × 10 4 Adherent culture (37°C, 5% CO2) was initiated using cells (10cm diameter, TrueLine, etc.).

[0101] Figure 14 shows the expression levels of marker genes on day 6 of adherent culture after induction culture to induce the generation of ectoderm, mesoderm, and endoderm. Expression of the marker genes TUBB3 for ectoderm, BMP4 for mesoderm, and GATA6 for endoderm was measured for both the induced and cultured genetically modified cells and the control fibroblasts. As a result, the induced and cultured genetically modified cells of this example expressed more of each marker than fibroblasts.

[0102] Next, we performed differentiation induction culture to induce the generation of ectoderm, mesoderm, and endoderm for genetically engineered cells produced with 11 factors (OCT4, SOX2, KLF4, c-MYC, NANOG, LIN28, TERT, histone H1FOO, DPPA3, antisense MBD3, and p53DD) and genetically engineered cells produced with 9 factors (OCT4, SOX2, KLF4, c-MYC, NANOG, LIN28, TERT, histone H1FOO, and DPPA3). For each genetically engineered cell, we used α-MEM supplemented with 10% FCS and cultured the genetically engineered cells in suspension culture (5–10 × 10 cells) on low-adhesion culture dishes, Lipidure-coated plates (96 wells, NOF Corporation). 3 After 6 days, the recombinant cells were transferred to a 6-well plate (2-4 cell clumps / well, TrueLine) and cultured under adhesion conditions (37°C, 5% CO).

[0103] FIG. 15 shows the expression levels of marker genes on day 16 of suspension culture after differentiation-inducing culture of these genetically modified cells to induce the generation of ectoderm, mesoderm, and endoderm. Figure 15(a), (b), and (c) show examples of measuring the expression of FGF5 in the ectoderm, T in the mesoderm, and GATA6 in the endoderm in genetically modified bovine cells produced using 11 factors. The figures show the measured values ​​of the markers in genetically modified cells cultured to maintain a neural stem cell state, the measured values ​​after induction culture, and the measured values ​​in control fibroblasts, respectively. The bovine genetically modified cells produced using these 11 factors could be induced to differentiate in the same way as iPS cells, and were capable of generating ectoderm and mesoderm. In addition, when cells were produced using these 11 factors, the expression levels of mesodermal markers after differentiation induction were particularly high compared to fibroblasts.

[0104] Figure 15(d), (e), and (f) show similar measurements of marker gene expression in genetically modified bovine cells produced using nine factors. These figures show the measured values ​​of markers in genetically modified cells cultured to maintain a neural stem cell state, the measured values ​​in induction culture, and the measured values ​​in control fibroblasts, respectively. The genetically modified bovine cells produced using these nine factors could also be induced to differentiate, similar to iPS cells, and were able to generate ectoderm and endoderm. In addition, when cells were produced using the nine factors, the expression levels of endoderm markers after differentiation induction were particularly high compared to fibroblasts.

[0105] In addition, the genetically modified cells produced using these 9 and 11 factors all showed higher expression levels of ectodermal, mesodermal, and endodermal marker genes than the genetically modified cells produced using 7 factors. [Industrial Applicability]

[0106] The present invention can be used in the livestock industry for livestock production and artificial meat production, and is therefore industrially applicable.

Claims

1. Based on nine factors including OCT4, SOX2, KLF4, c-MYC, NANOG, LIN28, TERT, histone H1FOO, and DPPA3, From neural stem cells induced from bovine fibroblasts, genetically modified cells that can be induced to pluripotent stem cells, ectoderm, mesoderm, or endoderm are produced. A method for producing genetically modified cells, comprising:

2. Based on 11 factors including OCT4, SOX2, KLF4, c-MYC, NANOG, LIN28, TERT, histone H1FOO, DPPA3, MBD3 function inhibitor, and p53 function inhibitor, From neural stem cells induced from bovine fibroblasts, genetically modified cells that can be induced to pluripotent stem cells, ectoderm, mesoderm, or endoderm are produced. A method for producing genetically modified cells, comprising:

3. Furthermore, the cells are cultured after adding an LIF receptor agonist, a fibroblast growth factor receptor agonist, and a Wnt signal inhibitor, or after adding an LIF receptor agonist, an activin receptor agonist, a Wnt signal inhibitor, a GSK3β inhibitor, an Src inhibitor, and an epigenetics eraser.

3. The method for producing genetically modified cells according to claim 1 or 2.

4. The genetically modified cell according to any one of claims 1 to 3, The cells are cultured in the presence of an LIF receptor agonist, a fibroblast growth factor receptor agonist, and a Wnt signal inhibitor. Maintaining the genetically modified cells in a neural stem cell state A culture method characterized by:

5. The genetically modified cell according to any one of claims 1 to 3, Adding an LIF receptor agonist, an activin receptor agonist, a Wnt signal inhibitor, a GSK inhibitor, an Src inhibitor, and an epigenetics eraser, and culturing the cells; Maintaining the genetically modified cells in a pluripotent stem cell state. A culture method characterized by:

6. The genetically modified cell according to any one of claims 1 to 3, Culture with the addition of animal serum to induce pluripotent stem cells into either ectoderm, mesoderm, or endoderm. A culture method characterized by:

7. Produced by the method for producing a genetically modified cell according to any one of claims 1 to 3, or Cultured by the culture method according to any one of claims 4 to 6 A genetically modified cell characterized by:

8. The gene sequences of nine factors including OCT4, SOX2, KLF4, c-MYC, NANOG, LIN28, TERT, histone H1FOO, and DPPA3 are introduced to produce an expression vector for Bovine subfamily (Bovinae) that can induce neural stem cells induced from fibroblasts of the Bovine subfamily (Bovinae) into any one of pluripotent stem cells, ectoderm, mesoderm, and endoderm states. A method for producing an expression vector.

9. By introducing gene sequences of nine factors including OCT4, SOX2, KLF4, c-MYC, NANOG, LIN28, TERT, histone H1FOO, and DPPA3, neural stem cells derived from bovine fibroblasts can be induced to pluripotent stem cells, ectoderm, mesoderm, or endoderm states. An expression vector characterized by:

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