Method for producing nervous system cell

文档序号：1060775 发布日期：2020-10-13 浏览：25次中文

阅读说明：本技术 神经系统细胞的制作方法 (Method for producing nervous system cell ) 是由田边刚士井上诚朱亚峰森豊隆于 2018-11-27 设计创作，主要内容包括：一种神经系统细胞的制作方法,其包括：准备干细胞的步骤；通过感染将仙台病毒导入至干细胞,在干细胞内表达在仙台病毒中合成诱导因子的mRNA,从而将干细胞诱导为神经系统细胞的步骤。(A method of making a nervous system cell, comprising: a step of preparing stem cells; introducing Sendai virus into the stem cells by infection, and expressing mRNA for an inducer synthesized by Sendai virus in the stem cells to induce the stem cells into cells of the nervous system.)

1. A method of making a nervous system cell, comprising:

a step of preparing stem cells;

introducing Sendai virus into the stem cells by infection, and expressing mRNA for an inducer of synthesis in the Sendai virus in the stem cells, thereby inducing the stem cells into cells of the nervous system.

2. The method for producing a neural cell according to claim 1, wherein the stem cell is an induced pluripotent stem cell.

3. The method for producing the neural cell according to claim 1, wherein the stem cell is an embryonic stem cell.

4. The method for producing the neural cell according to any one of claims 1 to 3, wherein the inducer includes neurogenin (NGN).

5. The method of making nervous system cells of any one of claims 1-3, wherein the inducing factor comprises ashperican homolog (ASCL).

6. The method of any one of claims 1-3, wherein the induction factor comprises myelin transcription factor (MYT).

7. The method for producing the neural cell according to any one of claims 1 to 3, wherein the inducer includes a distal-free homeobox (DLX).

8. The method of producing the neural cell according to any one of claims 1 to 7, wherein the neural cell is at least one selected from the group consisting of an excitatory neural cell, an inhibitory neural cell, a dopaminergic neural cell, a motor neural cell, and a brain neural cell.

9. The method for producing the neural cell according to any one of claims 1 to 7, wherein the neural cell is at least one selected from the group consisting of TH-positive, TUJ 1-positive, BRN 2-positive, NGN-positive, β -III tubulin-positive, MAP 2-positive, PSA-NCAM-positive, vGLUT-positive, SATB 2-positive, chAT-positive, HB 9-positive, MUNC 13-positive, HOMER 1-positive, vGAT-positive, and GAD 65/67-positive.

10. The method for producing the neural cell according to any one of claims 1 to 9, wherein the Sendai virus expresses mRNA of a drug-resistant gene in the stem cell.

11. The method of claim 10, wherein the drug-resistant gene is at least one selected from the group consisting of a puromycin-resistant gene, a blasticidin-resistant gene, a hygromycin-resistant gene, and a neomycin-resistant gene.

12. The method for producing the neural cell according to claim 10 or 11, which comprises: and a step of selecting a cell showing drug resistance after the stem cell is infected with the Sendai virus.

Technical Field

The invention relates to a cell technology, and relates to a method for preparing nervous system cells.

Background

Induced pluripotent stem cells (iPS cells) can be transformed into any cells constituting the body. Therefore, iPS cells that can be converted into various somatic cells or tissues are expected to be used in cell transplantation therapy or drug development research. Furthermore, for example, in 2014, retinal cells made from iPS cells were used in transplantation therapy. Not only in japan but also in other countries around the world, projects have been advanced in which brain cells and cells of various organs are prepared from iPS cells and used for transplantation therapy.

Currently, there are many methods for transforming iPS cells into differentiated cells. However, in order to use iPS cells for transplantation therapy, it is important to establish a method for inducing iPS cells with good efficiency. Specifically, it is necessary to establish a technique for inducing iPS cells into differentiated cells, so that the efficiency and accuracy of induction are improved and the prepared differentiated cells can withstand transplantation therapy, for example, for functionality.

Currently, a method of inducing differentiated cells from iPS cells or embryonic stem cells (ES cells) is performed in the following manner: the resulting process is mimicked by combining hormones or growth factors, which determine the properties of the cells, and low molecular compounds, and varying their quantitative ratio or concentration over time. However, it is difficult to fully mimic the resulting process in a cuvette and is inefficient. Moreover, humans require a very long induction period compared to the induction period of mouse somatic cells, for example, at present, 3 months or more are required to produce mature nerves.

Further, the efficiency of induction varies greatly depending on the ES/iPS cell line, and there are problems such as uneven properties of the induced somatic cells. When chemicals were actually added to a plurality of ES cell clones to produce various cells, the results showed that: there are clones that are easily induced into pancreatic cells, clones that are easily induced into heart cells, and the like, and there is a difference in the ease of induction by cloning (see, for example, non-patent document 1). Furthermore, when nerve cells were produced from several tens of iPS cells by a method called serum-free aggregation suspension culture (SFEBq method), the results demonstrated that: there is cloning of iPS/ES cells that are difficult to transform into nerve cells (see, for example, non-patent document 2), and the SFEBq method described above is a method for creating nerve cells from iPS cells/ES cells by culturing iPS cells in a medium that does not contain serum or chemicals that inhibit induction into nerve cells.

Specifically, it has been confirmed that cells induced from human ES/iPS cells using a method using hormones or chemicals are somatic cells at an early stage in a fetal stage. The induction of human mature somatic cells is extremely difficult and requires long-term culture over several months. However, in drug development or transplantation medicine for developing an individual who has completed development, it is important to produce somatic cells that match the maturity of the individual.

Furthermore, there are various types of nerve cells, but these cannot be uniformly induced by ES/iPS cells in the method using hormones or chemical substances. Therefore, specific drug development and screening specific to neural subtypes cannot be performed. Therefore, the efficiency of drug development screening is low. In transplantation medicine, it is also impossible to concentrate only certain cells of a transplantation disease.

In this regard, the following methods are proposed: a gene for specifying a specific somatic cell property is directly introduced into ES/iPS cells using a deoxyribonucleic acid (DNA) virus integrated into the genome to create a target somatic cell. In contrast to the method using hormones or chemicals, the method using a DNA virus integrated into the genome can specifically produce mature nerve cells in a very short time, for example, 2 weeks. Furthermore, if a nerve cell is created by introducing a specific gene, only excitatory nerves, for example, can be uniformly obtained. Therefore, it is possible to perform development and screening of specific nerve subtype-specific drugs, and it is also possible to concentrate only certain specific cells of a transplant disease in transplant medicine.

However, in the method of inducing stem cells into somatic cells using DNA viruses integrated into the genome in order to express a specific gene, the gene is inserted into the genome of ES/iPS cells, causing damage to endogenous genes. As a result, drug development and screening cannot be performed accurately, and there is a problem that a risk of cancer is accompanied in transplantation (see, for example, non-patent documents 3 and 4).

Disclosure of Invention

Problems to be solved by the invention

The present invention addresses the problem of providing a method for producing a nervous system cell, which can efficiently produce a nervous system cell in a short time without damaging the gene of the cell.

Means for solving the problems

According to an embodiment of the present invention, there is provided a method for producing a neural cell, including: a step of preparing stem cells; introducing Sendai virus into a stem cell by infection, and expressing messenger ribonucleic acid (mRNA) that synthesizes an inducer in Sendai virus in the stem cell to induce the stem cell into a neural cell.

In the method for producing a neural cell, the stem cell may be an induced pluripotent stem cell (iPS cell).

In the method for producing a neural cell, the stem cell may be an embryonic stem cell (ES cell).

In the above method for producing a neural cell, the inducer may include neurogenin (NGN). The inducer may comprise only neurogenin (NGN).

In the above method for producing nervous system cells, the inducer may comprise achaete-scar homolog (ashl-free scala homolog).

In the above method for producing a neural cell, the inducer may include myelin transcription factor (MYT).

In the above method for producing a neural cell, the inducer may comprise a digital-less homeobox (DLX).

In the method for producing a neural cell, the neural cell may be any of a neural cell, a neural stem cell and a neuronal precursor cell. The nerve cell may be at least one selected from the group consisting of an excitatory nerve cell, an inhibitory nerve cell, a dopaminergic nerve cell, a motor nerve cell, and a brain nerve cell. The nervous system cells may be oligodendrocyte precursor cells and oligodendrocytes.

In the method for producing a neural cell, the neural cell may be at least one selected from the group consisting of NGN gene positive, ASCL gene positive, MYT gene positive, and DLX gene positive.

In the method for producing the neural cell, the neural cell may be at least one selected from the group consisting of Tyrosine Hydroxylase (TH) -positive, TUJ 1-positive, BRN 2-positive, NGN-positive, β -III tubulin-positive, MAP 2-positive, PSA-NCAM-positive, vGLUT-positive, SATB 2-positive, chAT-positive, HB 9-positive, MUNC 13-positive, HOMER 1-positive, vGAT-positive, and GAD 65/67-positive.

In the method for producing the neural cell, Sendai virus can express mRNA of a drug-resistant gene in a stem cell.

In the method for producing a neural cell, the drug-resistant gene may be at least one selected from the group consisting of a puromycin-resistant gene, a blasticidin-resistant gene, a hygromycin-resistant gene and a neomycin-resistant gene.

The method for producing a neural cell may further comprise: and a step of selecting a cell showing drug resistance after the stem cell is infected with Sendai virus.

The method for producing a neural cell may further comprise: after the stem cells are infected with Sendai virus, cells are selected that exhibit at least one drug resistance selected from the group consisting of puromycin resistance, blasticidin resistance, hygromycin resistance, and neomycin resistance.

In the above method for producing nervous system cells, the density of stem cells infected with Sendai virus may be 0.2 × 10 in the wells of a 12-well plate⁵1.0 × 10⁶Per well.

In the method for producing the neural cells, the multiplicity of infection (MOI) of Sendai virus may be 0.1 to 100.0 at a time.

In the method for producing the above-mentioned neural cells, the medium used for infecting the Stem cells with Sendai virus may be any one selected from mTeSR (registered trademark) 1, TeSR2 (registered trademark), and Stem Fit.

ADVANTAGEOUS EFFECTS OF INVENTION

According to the present invention, a method for producing a neural cell can be provided, which can efficiently produce a neural cell in a short time without damaging a gene of the cell.

Drawings

FIG. 1 is a photomicrograph of the cells of example 1.

FIG. 2 is a fluorescent micrograph of cells that were fluorescently labeled with anti-BRN 2 antibody according to example 1.

FIG. 3 is a fluorescent micrograph of cells that were fluorescently labeled using the anti-HOMER 1 antibody of example 2.

FIG. 4 is a fluorescent micrograph of cells fluorescently labeled with anti-MAP 2 antibody according to example 3.

FIG. 5 is a fluorescent micrograph of cells that were fluorescently labeled with the anti-MUNC 13-1 antibody of example 4.

FIG. 6 is a fluorescent micrograph of cells labeled with an anti-SATB 2 antibody in example 5.

Fig. 7 is a fluorescent micrograph of cells fluorescently labeled with the anti-TUJ 1 antibody of example 6.

Fig. 8 is a fluorescent micrograph of cells that were fluorescently labeled using anti-vGLUT antibody of example 7.

FIG. 9 is a fluorescent micrograph of cells that were fluorescently labeled with an anti-chAT antibody according to example 8.

FIG. 10 is a fluorescent micrograph of cells labeled with anti-HB 9 antibody in example 9.

FIG. 11 is a fluorescent micrograph of cells labeled with an anti-TH antibody in example 10.

FIG. 12 (a) and FIG. 12 (b) are photomicrographs of the cells of example 11. Fig. 12 (c) is a fluorescence micrograph of cells that were fluorescently labeled with the anti-TUJ 1 antibody in example 11.

FIG. 13 is a fluorescent micrograph of cells that were fluorescently labeled with anti-BRN 2 antibody according to example 12.

FIG. 14 is a fluorescent micrograph of cells fluorescently labeled with the anti-GAD 65/67 antibody of example 13.

FIG. 15 is a fluorescent micrograph of cells that were fluorescently labeled with anti-MAP 2 antibody according to example 14.

Fig. 16 is a fluorescent micrograph of cells that were fluorescently labeled with the anti-vGAT antibody of example 15.

Fig. 17 is a fluorescent micrograph of cells fluorescently labeled with the anti-TUJ 1 antibody of example 16.

FIG. 18 is a fluorescent micrograph of cells that were fluorescently labeled with anti-MAP 2 antibody according to example 17.

FIG. 19 is a fluorescent micrograph of cells that were fluorescently labeled with anti-BRN 2 antibody according to example 18.

Fig. 20 is a fluorescent micrograph of cells that were fluorescently labeled with the anti-vGAT antibody of example 19.

FIG. 21 is a fluorescent micrograph of cells fluorescently labeled with the anti-GAD 65/67 antibody of example 20.

FIG. 22 is a fluorescent micrograph of cells labeled with an anti-TH antibody in example 21.

FIG. 23 is a fluorescent micrograph of cells that were fluorescently labeled with an anti-chAT antibody according to example 22.

Fig. 24 is a fluorescent micrograph of cells that were fluorescently labeled using an anti-vGLUT antibody according to example 23.

FIG. 25 is a fluorescent micrograph of cells labeled with fluorescence using an anti-SATB 2 antibody in example 24.

FIG. 26 is a graph showing the results of flow cytometry in example 25.

Detailed Description

Hereinafter, embodiments of the present invention will be described in detail. The embodiments described below are intended to exemplify an apparatus or a method for embodying the technical idea of the present invention, and the technical idea of the present invention is not intended to specify a combination of constituent members and the like as follows. The technical idea of the present invention can be variously modified within the scope of the claims.

The method for producing somatic cells from stem cells according to the embodiment of the present invention comprises: a step of preparing stem cells; introducing Sendai virus into the stem cells by infection, and expressing mRNA for an inducer synthesized by Sendai virus in the stem cells to induce the stem cells into neural cells.

Both induced pluripotent stem cells (iPS cells) and embryonic stem cells (ES cells) can be used as the stem cells. The stem cells may be human stem cells or non-human animal stem cells.

The induction includes reprogramming, priming, transformation, differentiation transformation (differentiation or transformation), differentiation induction, Cell fate transformation (Cell fate transformation), and the like.

Examples of the induced nervous system cells include nerve cells, neural stem cells, and neuronal precursor cells. Examples of the nerve cells include excitatory nerve cells, inhibitory nerve cells, dopaminergic nerve cells, motor nerve cells, and brain nerve cells. Alternatively, the nervous system cells may be oligodendrocyte precursor cells, oligodendrocytes, and the like.

Sendai virus (SeV) introduced into stem cells has an RNA genome that expresses the following mRNA: mRNA of an induction factor inducing stem cells into nerve cells is synthesized. Further, a recombinant Sendai virus vector capable of expressing an arbitrary mRNA can be prepared, for example, by requesting a patent technique from ID Pharma. Examples of the mRNA of the synthesis-inducing factor include mRNA of an NGN gene such as NGN2, mRNA of an ASCL gene such as ASCL1, mRNA of a MYT gene such as MYT1L, and mRNA of a DLX gene such as DLX 2. NGN2, ASCL1, MYT1L, and DLX2 are switch proteins required for the production of nerve cells. NGN2 can produce excitatory nerve cells. ASCL1, MYT1L, DLX2, respectively, can produce inhibitory nerve cells. Combinations of ASCL1 and MYT1L can produce excitatory, inhibitory, and dopaminergic nerve cells.

For example, Sendai virus introduced into stem cells expresses mRNA as a synthesis-inducing factor selected from at least one of mRNA of NGN gene such as NGN2, mRNA of ASCL gene such as ASCL1, mRNA of MYT gene such as MYT1L, and mRNA of DLX gene such as DLX 2.

Sendai virus may also have an RNA genome that expresses mRNA of a drug resistance gene. Examples of the drug include puromycin, blasticidin, hygromycin, neomycin, G418, and bleomycin. For example, Sendai virus introduced into stem cells expresses mRNA of a drug-resistant gene. Cells expressing the mRNA of the drug resistance gene show drug resistance.

When Sendai virus expresses mRNA of a drug-resistant gene, cells showing drug resistance can be selected after infection. For example, when at least any one of mrnas of puromycin-resistant gene, blasticidin-resistant gene, hygromycin-resistant gene, neomycin-resistant gene, G418-resistant gene, and bleomycin-resistant (Zeocin, registered trademark) gene is expressed by sendai virus, cells other than sendai virus-introduced cells can be killed by exposing infected cells to the corresponding antibiotic, thereby selecting sendai virus-introduced cells.

Sendai virus, for example, can have RNA that can express the following mRNA: mRNA containing the NGN2 gene and mRNA containing the puromycin-resistant gene (hereinafter referred to as "NGN 2-Puro mRNA"). The sequence of mRNA of the NGN2 gene corresponds to the DNA sequence shown in SEQ ID No. 1 (mouse) or SEQ ID No. 2 (human), for example. Cells expressing NGN2-Puro mRNA produced NGN2 and showed resistance to puromycin.

Sendai virus, for example, can have RNA that can express the following mRNA: mRNA containing mRNA of ASCL1 gene, mRNA of MYT1L gene, and mRNA of puromycin-resistant gene (hereinafter referred to as "ASCL 1-MYT1L-Puro mRNA"). The sequence of the mRNA of the ASCL1 gene corresponds to the sequence of the DNA shown in SEQ ID No. 3 (mouse) or SEQ ID No. 4 (human), for example. The sequence of mRNA of the MYT1L gene corresponds to the sequence of DNA shown in, for example, seq id No. 5 (mouse), seq id No. 6 (mouse), seq id No. 7 (mouse), or seq id No. 8 (human). Cells expressing ASCL1-MYT1L-Puro mRNA produced ASCL1 and MYT1L and showed resistance to puromycin.

Sendai virus, for example, can have RNA that can express the following mRNA: mRNA containing the DLX2 gene and mRNA containing the blasticidin-resistant gene (hereinafter referred to as "DLX 2-Blast mRNA"). The sequence of the mRNA of the DLX2 gene corresponds to the sequence of the DNA shown in SEQ ID No. 9 (mouse) or SEQ ID No. 10 (human), for example. Cells expressing DLX2-Blast mRNA produced DLX2 and showed blasticidin resistance.

Sendai virus is suspended in a cell culture medium and introduced into stem cells. Sendai virus recognizes cell surface antigens and infects stem cells.

The density of stem cells infected with Sendai virus in the wells of a 12-well plate is, for example, 0.2 × 10⁵1.0 × 10⁶0.5 × 10 per hole⁵8.0 × 10⁵Per hole, or 1.0 × 10⁵One/hole-4 × 10⁵Per well.

The effective value of the Sendai virus used is, for example, 1 × 10¹²CIU/mL～1×10⁵CIU/mL、1×10¹⁰CIU/mL～1×10⁶CIU/mL, or 1 × 10⁹CIU/mL～1×10⁷CIU/mL. The number of infection (MOI) of Sendai virus is, for example, 0.1 to 100.0, 1.0 to 50.0, or 1.0 to 20.0 at a time.

Examples of the medium used for infecting Sendai virus include Stem cell media such as mTeSR1, TeSR2 (registered trademark, Stem cell technology Co., Ltd.), and Stem Fit (ReProCELL Co., Ltd.). After infection with Sendai virus, the medium may be replaced with a medium suitable for induction of nervous system cells from the medium (hereinafter referred to as "neural induction medium"). Examples of the nerve-inducing medium include NDiff 227 (registered trademark, TAKARA BIO INC.), Neurobasal (registered trademark, seimer feishal technology), human ES/iPS neuron differentiation medium (Merck KGaA), and N3 medium. N3 medium can be prepared, for example, by adding 10mL of B27, 5mL of N2 supplement (Sammerfell technology) and 1.6mL of insulin at a concentration of 6.25mg/mL to 500mL of DMEMF 12.

The culture medium used for infection with Sendai virus, before and after infection with Sendai virus, may further contain B18R protein. The B18R protein moderates the innate anti-viral response of cells. The B18R protein is useful for inhibiting cell death caused by immune responses associated with viral infection. However, the method according to the embodiment can produce nervous system cells from stem cells in a short time, and the culture medium may contain no B18R protein or B18R protein at a low concentration of 0.01% to 1%.

The stem cells are induced to be nervous system cells within 25 days, within 20 days, within 10 days, within 9 days, within 8 days, within 7 days, within 6 days, within 5 days or within 4 days of infection with Sendai virus. Whether or not a stem cell can be induced into a nervous system cell can be confirmed by, for example, determining whether or not at least one selected from MAP2, TUJ1 (. beta. -III tubulin), NGN such as BRN2, HOMER1 and NGN2, PSA-NCAM, MUNC13-1, SATB2, vGLUT, chAT, HB9, LHX3, GAD65, GAD67, TH, ASCL1(MASH1), ASCL, vGAT, SOX1, SOX2, CD133, Nestin (Nestin), HB9, ISL1, O4, PLP1, MOG, and MBP is positive.

NGN2 is a switch protein required for the generation of nerve cells, and is a marker for excitatory nerve cells. TUJ1(β -III tubulin), MAP2, BRN2, ASCL1(MASH1) and PSA-NCAM are markers for nerve cells. HOMER1 and MUNC13-1 are markers for mature neuronal cells that form synapses. SATB2 is a marker for cerebral cortical neurons. vGLUT is a marker for excitatory nerve cells such as glutamatergic nerve cells. chAT is a marker for motor nerve cells such as cholinergic nerve cells. HB9 is a marker for motor nerve cells. GAD65 and GAD67 are markers of inhibitory nerve cells such as GABAergic nerve cells. TH is a marker for dopaminergic neurons. vGAT is a marker for inhibitory nerve cells. SOX1, SOX2, CD133, and Nestin (Nestin) are markers for neural stem cells. LHX3, HB9 and ISL1 are markers for motor nerve cells. O4, PLP1, MOG, and MBP are markers for oligodendrocyte precursor. Furthermore, GFAP and CD44 can be used as markers for astrocyte precursors and astrocytes.

According to the method of the embodiment of the present invention described above, by expressing mRNA of a synthesis-inducing factor in stem cells, it is possible to efficiently produce nervous system cells without integration without damaging genes of the stem cells.

In the method of producing a neural cell from a stem cell using only a hormone or a chemical substance, a very long time is required for producing the neural cell. In contrast, according to the method of the embodiment of the present invention, the nervous system cell can be produced in a short time.

In addition, in the method of producing a neural cell from a stem cell using only a hormone or a chemical substance, only a part of the stem cell becomes a target neural cell. In contrast, according to the method of the embodiment of the present invention, the cells that have been infected with Sendai virus can basically become target nervous system cells.

In addition, in the method of producing a neural cell from a stem cell using only a hormone or a chemical substance, even if the same protocol is followed, there are clones that become target neural cells and clones that do not become target neural cells, and a difference occurs between the clones.

In contrast, according to the method of the present embodiment, a higher induction efficiency can be obtained in a plurality of clones.

In addition, when a transplantation cell is produced by inducing an undifferentiated cell population with a cytokine or the like, there is a possibility that cells that are not induced remain in the transplantation cell. There is a risk that: the remaining non-induced cells are independently cell-divided and proliferated at the transplantation site to form teratomas and the like. In contrast, according to the method of the present embodiment, since mRNA of the drug resistance gene can be simultaneously expressed, it is possible to select a drug for cells into which sendai virus has been introduced. Therefore, the risk of contamination with uninduced cells or formation of teratoma can be avoided, and the method is suitable for transplantation medical treatment.

In addition, according to the method of the embodiment of the present invention, Sendai virus can be used without using a virus in which a gene is inserted into a genome. Therefore, it is non-integrated, does not damage the gene of stem cells, and does not cause the risk of canceration in the produced nervous system cells, and thus it is clinically useful.

In addition, for example, when iPS cells are produced from blood cells in a clean environment of a completely closed system and then neural cells are produced from iPS cells in a clean environment of a completely closed system by the method according to the embodiment of the present invention, safe neural cells can be produced more cleanly.

In addition, according to the method of the embodiment of the present invention, since the neural cells can be produced in a short time, B18R or the like can be omitted, and the concentration can be made very low even when used.

(example 1)

Sendai virus expressing NGN2-Puro mRNA was prepared by ID Pharma corporation, and the potent value of the prepared Sendai virus was 1 × 10⁹CIU/mL。

A 12-well dish coated with a soluble base film preparation (Matrigel, Corning) was prepared, and a feeder-free medium (mTeSR (registered trademark) 1, dry cell technology) containing ROCK (Rho-associated coiled coil-forming kinase)/Rho-associated kinase) inhibitor (seleck) was placed in each well at a concentration of 10 nmol/mL. ROCK inhibitors inhibit cell death.

Exfoliation of iPS cells dispersed in tissue/cultured cellsIn an/isolation/Dispersion solution (Accutase, Innovative cell Technologies), which was seeded into 12-well dishes at 2 × 10 per 1 well⁵iPS cells infected with Sendai virus were inoculated at a density of 2.0 × 10 per 1 well⁵Control iPS cells not infected with sendai virus were inoculated at individual densities. Thereafter, iPS cells were cultured in a feeder-free medium under gas conditions of a carbon dioxide concentration of 5% and an oxygen concentration of 20% for 24 hours.

iPS cells were infected with Sendai virus expressing NGN2-Puro mRNA at an MOI of 20. On day 2 after Sendai virus infection, the medium in the wells was replaced with a neural induction medium (N3 medium) containing puromycin at a concentration of 2. mu.g/mL, and uninfected cells were killed.

N3 medium was prepared by adding 10mL of B27, 5mL of N2, and 1.6mL of insulin at a concentration of 6.25mg/mL to 500mL of DMEMF 12.

The microphotographs of the cells on days 2, 3, 5, and 7 after the infection with Sendai virus are shown in FIG. 1 (a). A photomicrograph of the cells on day 24 after infection with Sendai virus is shown in FIG. 1 (b). As shown in FIG. 1, it was morphologically confirmed that the cells were induced into nervous system cells after infection with Sendai virus.

On day 28 after Sendai virus infection, the medium was removed from the plate and the cells were washed with PBS. Then, 4% Paraformaldehyde (PFA) was put into a dish, and reacted at 4 ℃ for 15 minutes to fix the cells. After washing the cells 2 times with PBS, the primary antibody was diluted with PBS medium containing 5% CCS and 0.1% Triton and added to the plate. Goat monoclonal antibody (Santa cruz biotechnology) to the marker BRN2 of nerve cells was used as the primary antibody.

After one hour reaction at room temperature, PBS was added to the dish, and after sufficient fusion on the dish, PBS was discarded. PBS was added again, and then discarded, after which a solution containing a fluorescently labeled gorilla anti-goat IgG (H + L) secondary antibody (AlexaFluor, registered trademark, 555, conjugate, sermer feishol) was added to the plate, and allowed to react at room temperature for 30 minutes. Thereafter, the cells were washed 2 times with PBS and observed with a fluorescence microscope. As a result, as shown in fig. 2, it was confirmed that the induced nervous system cells expressed BRN 2.

(example 2)

iPS cells were infected with sendai virus expressing NGN2-Puro mRNA in the same manner as in example 1, and cells were fluorescently labeled using a rabbit monoclonal antibody (SYNAPTIC SYSTEMS) to the marker HOMER1 of synaptogenic mature nerve cells as a primary antibody and observed with a fluorescence microscope. As a result, as shown in fig. 3, it was confirmed that the induced nervous system cells expressed homo 1.

(example 3)

iPS cells were infected with sendai virus expressing NGN2-Puro mRNA in the same manner as in example 1, and cells were fluorescently labeled using a mouse monoclonal antibody (Sigma Aldrich) for the marker MAP2 of nerve cells as a primary antibody and observed with a fluorescence microscope. As a result, as shown in fig. 4, it was confirmed that the induced nervous system cells expressed MAP 2.

(example 4)

iPS cells were infected with sendai virus expressing NGN2-Puro mRNA in the same manner as in example 1, and cells were fluorescently labeled using a rabbit monoclonal antibody (SYNAPTIC SYSTEMS) against a synaptic mature nerve cell marker MUNC13-1 as a primary antibody and observed with a fluorescence microscope. As a result, as shown in FIG. 5, it was confirmed that the induced nervous system cells expressed MUNC 13-1.

(example 5)

iPS cells were infected with sendai virus expressing NGN2-Puro mRNA in the same manner as in example 1, and cells were fluorescently labeled using a mouse monoclonal antibody (Abcam) of a marker for cranial nerve cells, SATB2, as a primary antibody, and observed with a fluorescence microscope. As a result, as shown in FIG. 6, it was confirmed that the induced neural cells expressed SATB 2.

(example 6)

iPS cells were infected with sendai virus expressing NGN2-Puro mRNA in the same manner as in example 1, and cells were fluorescently labeled using a mouse monoclonal antibody (Biolegend) against a neural cell marker TUJ1 as a primary antibody and observed with a fluorescence microscope. As a result, as shown in fig. 7, it was confirmed that the induced nervous system cells expressed TUJ 1.

(example 7)

iPS cells were infected with sendai virus expressing NGN2-Puro mRNA in the same manner as in example 1, and cells were fluorescently labeled using a rabbit monoclonal antibody (SYNAPTICSYSTEMS) to vgut, a marker for excitatory nerve cells such as glutamatergic nerve cells, as a primary antibody, and observed with a fluorescence microscope. As a result, it was confirmed that the induced nervous system cells expressed vGLUT as shown in fig. 8.

(example 8)

iPS cells were infected with sendai virus expressing NGN2-Puro mRNA in the same manner as in example 1, and cells were fluorescently labeled using a mouse monoclonal antibody (Millipore) against the marker chAT of motor nerve cells as a primary antibody, and observed with a fluorescence microscope. As a result, as shown in fig. 9, it was confirmed that the induced nervous system cells expressed chAT.

(example 9)

iPS cells were infected with sendai virus expressing NGN2-Puro mRNA in the same manner as in example 1, and cells were fluorescently labeled using a mouse monoclonal antibody (Thermo) against a marker HB9 of motor nerve cells as a primary antibody and observed with a fluorescence microscope. As a result, as shown in fig. 10, it was confirmed that the induced nervous system cells expressed HB 9.

(example 10)

iPS cells were infected with sendai virus expressing NGN2-Puro mRNA in the same manner as in example 1, and cells were fluorescently labeled using a sheep monoclonal antibody (Pel Freez) for the marker TH of dopaminergic nerve cells as a primary antibody and observed with a fluorescence microscope. As a result, as shown in fig. 11, it was confirmed that the induced nervous system cells expressed TH.

(example 11)

iPS cells were infected with Sendai virus in the same manner as in example 1, except that Sendai virus expressing ASCL1-MYT1L-Puro mRNA and DLX2-Blast mRNA was used. The microphotographs of the cells on days 2, 5, 11, and 21 after the infection with Sendai virus are shown in FIG. 12 (a). A photomicrograph of the cells on day 24 after infection with Sendai virus is shown in FIG. 12 (b). As shown in fig. 12 (a) and 12 (b), it was morphologically confirmed that cells were induced into nervous system cells after infection with sendai virus.

On day 28 after Sendai virus infection, cells were fluorescently labeled using a mouse monoclonal antibody (Biolegend) against TUJ1, a marker for general nerve cells, as a primary antibody, and observed with a fluorescence microscope. As a result, as shown in fig. 12 (c), it was confirmed that the induced nervous system cells expressed TUJ 1.

(example 12)

iPS cells were infected with sendai virus expressing ASCL1-MYT1L-Puro mRNA and DLX2-BlastmRNA in the same manner as in example 11, and cells were fluorescently labeled using a goat monoclonal antibody (Santa cruz biotechnology) to the marker BRN2 of nerve cells as a primary antibody and observed with a fluorescence microscope. As a result, as shown in fig. 13, it was confirmed that the induced nervous system cells expressed BRN 2.

(example 13)

iPS cells were infected with sendai virus expressing ASCL1-MYT1L-Puro mRNA and DLX2-BlastmRNA in the same manner as in example 11, and cells were fluorescently labeled using a mouse monoclonal antibody (Millipore) against a marker GAD65/67 for inhibitory (gabaergic) nerve cells as a primary antibody and observed with a fluorescence microscope. As a result, as shown in FIG. 14, it was confirmed that the induced nervous system cells expressed GAD 65/67.

(example 14)

iPS cells were infected with sendai virus expressing ASCL1-MYT1L-Puro mRNA and DLX2-BlastmRNA in the same manner as in example 11, and cells were fluorescently labeled using a mouse monoclonal antibody (Sigma Aldrich) for a marker MAP2 of nerve cells as a primary antibody and observed with a fluorescence microscope. As a result, as shown in fig. 15, it was confirmed that the induced nervous system cells expressed MAP 2.

(example 15)

iPS cells were infected with sendai virus expressing ASCL1-MYT1L-Puro mRNA and DLX2-BlastmRNA in the same manner as in example 11, and cells were fluorescently labeled using a rabbit monoclonal antibody (SYNAPTICSYSTEMS) to a marker vGAT of inhibitory nerve cells as a primary antibody and observed with a fluorescence microscope. As a result, it was confirmed that the induced nervous system cells expressed vGAT as shown in fig. 16.

(example 16)

iPS cells were infected with Sendai virus in the same manner as in example 1, except that Sendai virus expressing ASCL1-MYT1L-Puro mRNA was used. On day 21 after Sendai virus infection, cells were fluorescently labeled using a mouse monoclonal antibody (Biolegend) against TUJ1, a marker for general nerve cells, as a primary antibody, and observed with a fluorescence microscope. As a result, as shown in fig. 17, it was confirmed that the induced nervous system cells expressed TUJ 1.

(example 17)

iPS cells were infected with sendai virus expressing ASCL1-MYT1L-Puro mRNA in the same manner as in example 22, and cells were fluorescently labeled using a mouse monoclonal antibody (Sigma Aldrich) for the marker MAP2 of nerve cells as a primary antibody and observed with a fluorescence microscope. As a result, as shown in fig. 18, it was confirmed that the induced nervous system cells expressed MAP 2.

(example 18)

iPS cells were infected with sendai virus expressing ASCL1-MYT1L-Puro mRNA in the same manner as in example 22, and cells were fluorescently labeled using a goat monoclonal antibody (Santa Cruz Biotechnology) to the marker BRN2 of nerve cells as a primary antibody and observed with a fluorescence microscope. As a result, as shown in fig. 19, it was confirmed that the induced nervous system cells expressed BRN 2.

(example 19)

iPS cells were infected with sendai virus expressing ASCL1-MYT1L-Puro mRNA in the same manner as in example 22, and cells were fluorescently labeled using a rabbit monoclonal antibody (SYNAPTIC SYSTEMS) to a marker vGAT of inhibitory nerve cells as a primary antibody, and observed with a fluorescence microscope. As a result, it was confirmed that the induced nervous system cells expressed vGAT as shown in fig. 20.

(example 20)

iPS cells were infected with sendai virus expressing ASCL1-MYT1L-Puro mRNA in the same manner as in example 22, and cells were fluorescently labeled using a mouse monoclonal antibody (Millipore) of the marker GAD65/67 for inhibitory nerve cells (gabanergic nerve cells) as a primary antibody and observed with a fluorescence microscope. As a result, as shown in FIG. 21, it was confirmed that the induced nervous system cells expressed GAD 65/67.

(example 21)

iPS cells were infected with sendai virus expressing ASCL1-MYT1L-Puro mRNA in the same manner as in example 22, and cells were fluorescently labeled using a sheep monoclonal antibody (Pel Freez) for the marker TH of dopaminergic nerve cells as a primary antibody and observed with a fluorescence microscope. As a result, as shown in fig. 22, it was confirmed that the induced nervous system cells expressed TH.

(example 22)

iPS cells were infected with sendai virus expressing ASCL1-MYT1L-Puro mRNA in the same manner as in example 22, and cells were fluorescently labeled using a goat monoclonal antibody (Millipore) to the marker chAT of cholinergic nerve cells as a primary antibody, and observed with a fluorescence microscope. As a result, as shown in fig. 23, it was confirmed that the induced nervous system cells expressed chAT.

(example 23)

iPS cells were infected with sendai virus expressing ASCL1-MYT1L-Puro mRNA in the same manner as in example 22, and cells were fluorescently labeled using a rabbit monoclonal antibody (SYNAPTICSYSTEMS) to a marker vgut of excitatory nerves (glutamatergic nerve cells) as a primary antibody, and observed with a fluorescence microscope. As a result, it was confirmed that the induced nervous system cells expressed vGLUT as shown in fig. 24.

(example 24)

iPS cells were infected with sendai virus expressing ASCL1-MYT1L-Puro mRNA in the same manner as in example 22, and cells were fluorescently labeled using a mouse monoclonal antibody (Abcam) of marker SATB2 for cranial nerve cells as a primary antibody and observed with a fluorescence microscope. As a result, as shown in FIG. 25, it was confirmed that the induced neural cells expressed SATB 2.

(example 25)

The control cells prepared in example 1 and not infected with Sendai virus expressing NGN2-Puro mRNA were analyzed by flow cytometry, and the marker PSA-NCAM of neural cells was negative as shown in FIG. 26 (a). The Sendai virus-infected cells expressing NGN2-Puro mRNA prepared in example 1 were analyzed by flow cytometry at day 7 after infection, and as a result, the neural cell marker PSA-NCAM was positive, as shown in FIG. 26 (b). In addition, flow cytometry analysis was performed on day 4 post-infection, and the marker PSA-NCAM, which is also a neural cell, was positive.

The cells infected with Sendai virus expressing ASCL1-MYT1L-Puro mRNA and DLX2-BlastmRNA prepared in example 11 were analyzed by flow cytometry at day 7 after infection, and the result was positive for the neural cell marker PSA-NCAM as shown in FIG. 26 (c). In addition, flow cytometry analysis was performed on day 4 post-infection, and the marker PSA-NCAM, which is also a neural cell, was positive. The Sendai virus cells infected with ASCL1-MYT 1L-PurormRNA prepared in example 22 were analyzed by flow cytometry at day 7 after infection, and as a result, the neural cell marker PSA-NCAM was positive, as shown in FIG. 26 (d). In addition, flow cytometry analysis was performed on day 4 post-infection, and the marker PSA-NCAM, which is also a neural cell, was positive.

Sequence listing

<110> Anoerious Greenwich Ltd

<120> method for producing nervous system cell

<130>XT374AJP0007-WO

<150>US 62/592,629

<151>2017-11-30

<160>10

<170>PatentIn version 3.5.1

<210>1

<211>2244

<212>DNA

<213> Artificial sequence

<220>

<223> Induction factor

<400>1

gcagccactg aaccacaagc agctcggctt taactggagt gccttggagt cgcgtgccag 60

cagccacacg gccagggact gactgacaga caaccacgca cgagaacgac aacacacgag 120

actcgggcga gctgccgcgg tcgtccgggc tcttggcaaa gtcgcccagc cgagaggccc 180

ccccgcggag gtgcgcctag gaagcgccaa gcccgcggcg cggaggacac cgtgctcggt 240

tccgggctgc ggggacattc ccggacacac accggagcag cagctgcgcc gcgacacatc 300

tggagccgcg taggatgttc gtcaaatctg agactctgga gttgaaggag gaagaggagg 360

tactgatgct gctgggctcg gcttccccgg cctcggcgac cctgaccccg atgtcctcca 420

gcgcggacga ggaggaggac gaggagctgc gccggccggg ctccgcgcgt gggcagcgtg 480

gagcggaagc cgggcagggg gtgcagggca gtccggcgtc gggtgccggg ggttgccggc 540

cagggcggct gctgggcctg atgcacgagt gcaagcgtcg cccgtcgcgc tcacgggccg 600

tctcccgagg tgccaagacg gcggagacgg tgcagcgcat caagaagacc cgcaggctca 660

aggccaacaa ccgcgagcgc aaccgcatgc acaacctaaa cgccgcgctg gacgcgctgc 720

gcgaggtgct gcccaccttc cccgaggatg ccaagctcac gaagatcgag acgctgcgct 780

tcgcccacaa ttacatctgg gcgctcaccg agactctgcg cctggcggac cactgcgccg 840

gcgccggtgg cctccagggg gcgctcttca cggaggcggt gctcctgagc ccgggagctg 900

cgctcggcgc cagcggggac agcccttctc caccttcctc ctggagctgc accaacagcc 960

cggcgtcatc ctccaactcc acgtccccat acagctgcac tttatcgccc gctagccccg 1020

ggtcagacgt ggactactgg cagcccccac ctccggagaa gcatcgttat gcgcctcacc 1080

tgcccctcgc cagggactgt atctagagct gcgggtctcc ctctctcgtc ctctacccgg 1140

ccctcttccc atccttctcc cgcccctcac cctccacgcc ccggactcca cttcacagag 1200

cagaggtggc ccttgcaatc ccctcggcgg ctggtgcatt cgggggtgga gaccagctct 1260

ggtttattga agatgtgagg atttatggtc aaagaggact atggcgtgtg ggagtggggg 1320

ctggcgtggg gaacctcgta agactgtaaa agacactgag aaaaagtacc ataactaacg 1380

agtgtgcaga gcagactgac gctcctcccc tctctcagag ctgctggagg agaactccgg 1440

gcaggcagtt cgtgtgaatc tctcagaggg aatgcaactg gtccctgtga tcttttcacc 1500

ttcgtttcta catagagatg ttaatgtcag tcgaaagaaa tgtattttag catctgaatg 1560

aatttactgg taataatatt atccacacat ttgcaatggc tggcatctgc tctattccca 1620

ttgctgtctg caggctgtgg gaatttcacc tgtcaaacca aactttccct ctctgatgtg 1680

cactttgttt ttttcccaga ttcgtcacaa tgcctattgt cccgcccttc tttttgcttt 1740

ttttctccat tttgccatct gtctcttatg atttataagg gggaaaaact tgttttgtta 1800

gagggccagg ttagaagtca ttgtataatt tgtaggcttt tgtaagggtt gaatgcaagc 1860

gtggaaattt aggctgaatt ctctatcaaa agaaaaaatg tgaaggaaaa aggaaaaatc 1920

aggagggagg attgcttcat gcattattta tctcgacctt ttaggggaga aggaactccc 1980

ccatcctttc aagagattaa aaataaatca acagtctgaa aacctaagca gacacggggc 2040

attgccagga tcagccacac acgtgtttcc ttctatttat tttgaagaaa aatttcatgg 2100

gaaagtatgt atttttttgt atattctaca gagtttattc tagtatgtat ttacatcccg 2160

aagaataaga aaattgtttt gtgattaagc tataaataaa gtatctaatt ttcataaaaa 2220

aaaaaaaaaa aaaaaaaaaa aaaa 2244

<210>2

<211>2370

<212>DNA

<213> Artificial sequence

<220>

<223> Induction factor

<400>2

cgcagccact gaaccacaag cagcttcgcg ttaactggag tgcctgggag tcgcgtgcca 60

ggagccgcac ggccagggac tgactgacag acagacacgc accaccacca caacacacga 120

gacccgggcg ggccgccgcc gccgccgccg gggctcttgg caaactcgcc ggtcgcagag 180

gtcccccgcg gagctgcgcc acagtagcgc cgggcttgca gctttcacgc cgggcgaagg 240

acccggcgct gcgctcgcag ctgcgcggag attcccggca caggccaaag tcacagcaac 300

gctgaggcac agttagagcc aactaagatg ttcgtcaaat ccgagacctt ggagttgaag 360

gaggaagagg acgtgttagt gctgctcgga tcggcctccc ccgccttggc ggccctgacc 420

ccgctgtcat ccagcgccga cgaagaagag gaggaggagc cgggcgcgtc aggcggggcg 480

cgtcggcagc gcggggctga ggccgggcag ggggcgcggg gcggcgtggc tgcgggtgcg 540

gagggctgcc ggcccgcacg gctgctgggt ctggtacacg attgcaaacg gcgcccttcc 600

cgggcgcggg ccgtctcccg aggcgccaag acggccgaga cggtgcagcg catcaagaag 660

acccgtagac tgaaggccaa caaccgcgag cgaaaccgca tgcacaacct caacgcggca 720

ctggacgcgc tgcgcgaggt gctccccacg ttccccgagg acgccaagct caccaagatc 780

gagaccctgc gcttcgccca caactacatc tgggcactca ccgagaccct gcgcctggcg 840

gatcactgcg ggggcggcgg cgggggcctg ccgggggcgc tcttctccga ggcagtgttg 900

ctgagcccgg gaggagccag cgccgccctg agcagcagcg gagacagccc ctcgcccgcc 960

tccacgtgga gttgcaccaa cagccccgcg ccgtcctcct ccgtgtcctc caattccacc 1020

tccccctaca gctgcacttt atcgcccgcc agcccggccg ggtcagacat ggactattgg 1080

cagcccccac ctcccgacaa gcaccgctat gcacctcacc tccccatagc cagggattgt 1140

atctagagct gccatttctg ctacccacgc caggccttag tgggttccct ttcctgtccc 1200

cagtcgagcc ctcctccctt cccctgcccc tcctttccac gccctggaaa ccatctcact 1260

tcacagggca ggtgtagcct ttctgattcc tcggttgttt cttgcatttc ttggctttgg 1320

gtatccttca ttcagacggg ctctgattta ctgaaggtgt gatggagctt attgtcaaag 1380

ccaagggtgg cgttttgggg gcgcttcttg agacgaaaaa gaccctggga agagatgatg 1440

gtggcatatc taaagagttt gcagagcgga ctgacgctcc tcccctttct ctttaacgcc 1500

gaaggacttg gtgcagttcg tgtgaatctc acagggggaa tgcaactggt tcctgtgatc 1560

tcttcacctt tgcttctaca tagagatgtt aatgtcgagt agaaagaaat gtatcttagc 1620

atctgaatga ttttgctggt aataatatta tccacagatt tgcaatggct ggcatctgct 1680

ttattcccat tgctgtctgc aggctgtggg aatttcacct gtcaaaccaa acttccctct 1740

ctgatgtgca ctttgttctg tttcccagat tcgtcacaat gcctattgtc ctgtccttct 1800

ctttcctttt tcttccccat tttgccatct gtctcttatg atttataagg ggaaaaaaac 1860

ttgttttgtt agaggggcag gttagaagtc attgtataat ttgtaggctt tgtaatgatt 1920

gaatgcaagc gtggaaattt aggctgaact ctctatcaaa aggaaaaatg tggaggaaaa 1980

gggaaaaatc aggagggagg attgcctcat gtattattta tttcgacctt ttaggggaga 2040

aggaactccc ccattctttc aagagattaa aaataaatca acagtctgaa aacctaagca 2100

gacacggagc attatccgga tcagccacac acgtgttccc ttctatttat tataaagaaa 2160

tttttcatgg gaaaatatgt attttttgta tattctacag agtttattct agtatgtatt 2220

tacatcttga agaacaagaa agttgttctt gtgattaaac tataaataaa ctatctaatt 2280

ttcataaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2340

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2370

<210>3

<211>2259

<212>DNA

<213> Artificial sequence

<220>

<223> Induction factor

<400>3

agcagtctct cacttctggc cagggaacgt ggaaggcgta ccggctggga gccggttagg 60

gagggcgaat tggggggaac gagagagcaa ttagaaagaa agggggttca accaaataat 120

cccagaagca ggctcaagcc caggctggag caagggagag cgggcgcaag aaagcgcagc 180

cccggagcag ctccacctgg cagagtgcgc tcggcactga cttttgcggc tgctttcctt 240

ttccctttcc tcttttaaaa ccgagaaggc gccggcggcg gccgcacacg cgagcgccac 300

gcgaggctcc cgaagccaac cgcggcggga ggaggggagg gaggaggcgg cgcagaggga 360

agacgatcgc ccaggcacct tcctccgctg cagcctgaca actctgcctc cttctgcgcg 420

tttcttcccc tttaactttc ctccggggct cgtttctccc ctctcctttt tcttcgtccc 480

cctttgatcg tgcttcgcag ccccgcttcc ttcaagggct ctgcgcaccc tgcgtcccca 540

actcgttctc ccccgcgaca gtttggcccg gcatggagag ctctggcaag atggagagtg 600

gagccggcca gcagccgcag cccccgcagc ccttcctgcc tcccgcagcc tgcttctttg 660

cgaccgcggc ggcggcggca gcggcggcgg ccgcggcagc tcagagcgcg cagcagcaac 720

agccgcaggc gccgccgcag caggcgccgc agctgagccc ggtggccgac agccagccct 780

cagggggcgg tcacaagtca gcggccaagc aggtcaagcg ccagcgctcg tcctctccgg 840

aactgatgcg ctgcaaacgc cggctcaact tcagcggctt cggctacagc ctgccacagc 900

agcagccggc cgccgtggcg cgccgcaacg agcgcgagcg caaccgggtc aagttggtca 960

acctgggttt tgccaccctc cgggagcatg tccccaacgg cgcggccaac aagaagatga 1020

gcaaggtgga gacgctgcgc tcggcggtcg agtacatccg cgcgctgcag cagctgctgg 1080

acgagcacga cgcggtgagc gctgcctttc aggcgggcgt cctgtcgccc accatctccc 1140

ccaactactc caacgacttg aactctatgg cgggttctcc ggtctcgtcc tactcctccg 1200

acgagggatc ctacgaccct cttagcccag aggaacaaga gctgctggac tttaccaact 1260

ggttctgagg acctgccagg ctctcctggg aatggacttt ggaagcagga tggcagcaga 1320

tcctgcatct ttagtgtttc tcgccaacga cgtcaaatgg ggaggcagaa aaacaagggg 1380

aaaaaagaag aagaaatgaa acaaacaaac cagacagcca acctacaggg gcaccttcac 1440

taagatgcaa tgttctcagc aaacaggggt gggctccaac agtgtctctg cattccaaca 1500

tcatttccag acacgagaag agtgactggt gtctgaacct aagcccgaat cacagatggg 1560

ttcctttcct ggagcaagag cgtcacacac acacacacac acacagacag acactatatt 1620

aactcccaac cactaacagg cagggctgga agcgcgcatg tgcaagtgcc ttcacctccc 1680

actctctgtc agagctgtct tagccccctg aaactgggtt gatgtctttc ctcagtcacc 1740

cccattccag cgatctatgg acatttgcct ccattgaagc aacgtcagtt ctcggacagc 1800

ctttccctct cctggtggcc tcctccccaa accccacatc gccctcccac ggtctttgct 1860

tctgttttct tcatagaatg cttccaatct ttgtgaattt ttttattata agaaaaaaat 1920

ctatttgtat ctatcctaac cagtttgggg atatattaag atatttttgt acataagaaa 1980

aagagagaga aaaaatttat agaagttttg tacaaatggt ttaaaaatgt gtatatcttg 2040

atactttaac atgtaatgag attacctctg cgtactttag atatgtagtt catcttacaa 2100

ctgccatccc cacccccatc cccagtgtgg ttttggaaag aactctcctc ataggtgaga 2160

tctaaatgcc accagaatga cttcagcacc aatgtgtctt acttcacaga aacgtggtta 2220

atgtattaat gatgttatta aaaaaaactg ttcaagaag 2259

<210>4

<211>2490

<212>DNA

<213> Artificial sequence

<220>

<223> Induction factor

<400>4

agcactctct cacttctggc cagggaacgt ggaaggcgca ccgacaggga tccggccagg 60

gagggcgagt gaaagaagga aatcagaaag gaagggagtt aacaaaataa taaaaacagc 120

ctgagccacg gctggagaga ccgagacccg gcgcaagaga gcgcagcctt agtaggagag 180

gaacgcgaga cgcggcagag cgcgttcagc actgactttt gctgctgctt ctgctttttt 240

ttttcttaga aacaagaagg cgccagcggc agcctcacac gcgagcgcca cgcgaggctc 300

ccgaagccaa cccgcgaagg gaggagggga gggaggagga ggcggcgtgc agggaggaga 360

aaaagcattt tcactttttt tgctcccact ctaagaagtc tcccggggat tttgtatata 420

ttttttaact tccgtcaggg ctcccgcttc atatttcctt ttctttccct ctctgttcct 480

gcacccaagt tctctctgtg tccccctcgc gggccccgca cctcgcgtcc cggatcgctc 540

tgattccgcg actccttggc cgccgctgcg catggaaagc tctgccaaga tggagagcgg 600

cggcgccggc cagcagcccc agccgcagcc ccagcagccc ttcctgccgc ccgcagcctg 660

tttctttgcc acggccgcag ccgcggcggc cgcagccgcc gcagcggcag cgcagagcgc 720

gcagcagcag cagcagcagc agcagcagca gcagcaggcg ccgcagctga gaccggcggc 780

cgacggccag ccctcagggg gcggtcacaa gtcagcgccc aagcaagtca agcgacagcg 840

ctcgtcttcg cccgaactga tgcgctgcaa acgccggctc aacttcagcg gctttggcta 900

cagcctgccg cagcagcagc cggccgccgt ggcgcgccgc aacgagcgcg agcgcaaccg 960

cgtcaagttg gtcaacctgg gctttgccac ccttcgggag cacgtcccca acggcgcggc 1020

caacaagaag atgagtaagg tggagacact gcgctcggcg gtcgagtaca tccgcgcgct1080

gcagcagctg ctggacgagc atgacgcggt gagcgccgcc ttccaggcag gcgtcctgtc 1140

gcccaccatc tcccccaact actccaacga cttgaactcc atggccggct cgccggtctc 1200

atcctactcg tcggacgagg gctcttacga cccgctcagc cccgaggagc aggagcttct 1260

cgacttcacc aactggttct gaggggctcg gcctggtcag gccctggtgc gaatggactt 1320

tggaagcagg gtgatcgcac aacctgcatc tttagtgctt tcttgtcagt ggcgttggga 1380

gggggagaaa aggaaaagaa aaaaaaaaga agaagaagaa gaaaagagaa gaagaaaaaa 1440

acgaaaacag tcaaccaacc ccatcgccaa ctaagcgagg catgcctgag agacatggct 1500

ttcagaaaac gggaagcgct cagaacagta tctttgcact ccaatcattc acggagatat 1560

gaagagcaac tgggacctga gtcaatgcgc aaaatgcagc ttgtgtgcaa aagcagtggg 1620

ctcctggcag aagggagcag cacacgcgtt atagtaactc ccatcacctc taacacgcac 1680

agctgaaagt tcttgctcgg gtcccttcac ctcctcgccc tttcttaaag tgcagttctt 1740

agccctctag aaacgagttg gtgtctttcg tctcagtagc ccccacccca ataagctgta 1800

gacattggtt tacagtgaaa ctatgctatt ctcagccctt tgaaactctg cttctcctcc 1860

agggcccgat tcccaaaccc catggcttcc ctcacactgt cttttctacc attttcatta 1920

tagaatgctt ccaatctttt gtgaattttt tattataaaa aatctatttg tatctatcct 1980

aaccagttcg gggatatatt aagatatttt tgtacataag agagaaagag agagaaaaat 2040

ttatagaagt tttgtacaaa tggtttaaaa tgtgtatatc ttgatacttt aacatgtaat 2100

gctattacct ctgcatattt tagatgtgta gttcacctta caactgcaat tttccctatg 2160

tggttttgta aagaactctc ctcataggtg agatcaagag gccaccagtt gtacttcagc 2220

accaatgtgt cttactttat agaaatgttg ttaatgtatt aatgatgtta ttaaatactg 2280

ttcaagaaga acaaagttta tgcagctact gtccaaactc aaagtggcag ccagttggtt 2340

ttgataggtt gccttttgga gatttctatt actgcctttt tttttcttac tgttttatta 2400

caaacttaca aaaatatgta taaccctgtt ttatacaaac tagtttcgta ataaaacttt 2460

ttcctttttt taaaatgaaa ataaaaaaaa 2490

<210>5

<211>7198

<212>DNA

<213> Artificial sequence

<220>

<223> Induction factor

<400>5

gtggagctgc gtgcattagg ggctggctgt ggaggtaaag cagatctctt aggcccgcta 60

gcctctcctg ctgcatgtgt cattcctgcc cgcgcgacat tttcccccta agctactgca 120

cacaaaacag agcgagaaag cttctccctg cagtcttctt ggaggcctcc tggtttctca 180

cccattgttg gtgggtgtat ttcaattttt tgattccctg gactgtgggt tatgaaatca 240

atgctgctga agacaaaagc aaccttccct gcctctcggt gctatgcgtg gtcctcctcg 300

gcctcccact tgtgggggaa agggttttct ctttctttct gtgtgttttg agccagcaca 360

gttaccaaaa ttgaacttgc cgtcacttgt gagcggtgtg gtcatggtgt gaggggtccc 420

acagaggctg cagctgaggt ctgggtgtgtgcaattctca gctgggcttt gccctaccca 480

ggttgacgac tgaagaatca cagagtgtgg aaaagaacac aaggagaaat tggtgagaac 540

atctgcctag catctccaag tcctgtggag ggagccagca gtgctggtcc aaagaggacc 600

cagagagtga actcagagtg accacatctg atagaagagg ggaagatgta gtttctgagt 660

ccagtccagt gtttgtgtct ctcacattgt caacaaaaga aaggctccag ctgtccccac 720

agacatatgg atattccagg agccacgtaa agatggagaa atggaggcac agagaaatta 780

agtgacttgg ccacagtcac aagctgggga ggaccaggga aagcctagag agagctggct 840

ctgggcctgc atcctgccca cggagtcacc ctgcctccgt cctcaggaga gaaggcttcc 900

tacaagatgg acgtggactc tgaggagaag cgccatcgca cacggtccaa aggggttcga 960

gttcctgtgg agccagccat acaagagctg ttcagctgtc ccactccagg ctgcgacggc 1020

agtggtcacg tcagtggcaa atatgcacga cacagaagtg tatatggttg tcccttggct 1080

aaaaaaagaa aaacgcaaga taaacagccc caagaacctg ctcccaagcg aaaaccattt 1140

gcagtaaaag cagatagttc ctcagtagac gaatgttatg agagtgatgg tactgaagac 1200

atggatgata aggaggaaga tgatgatgag gagttctctg aagacaatga tgagcaaggg 1260

gatgatgacg acgaagatga ggtggatcgg gaagacgagg aggagatcga ggaggaagat 1320

gatgaagaag atgatgatga tgaagatggt gacgatgtag aagaggaaga agaggatgat 1380

gatgaagagg aggaagaaga ggaagaggaa gaagaaaatg aagaccatca aatgagttgt 1440

actcgaataa tgcaggacac agacaaggat gataacaaca atgatgagta tgataactat 1500

gatgaactgg tagctaagtc gctattaaat cttggcaaaa ttgctgagga tgcagcatac 1560

cgagccagga ctgaatcaga gatgaacagc aatacctcca atagtctgga ggacgatagt 1620

gacaaaaacg aaaacctcgg tcggaaaagc gaactgagtc tagacttaga cagtgatgtt 1680

gttagagaaa cagtggactc ccttaagctg ttagcacaag gacatggtgt tgtgctatca 1740

gagaatatca gtgacagaag ttatgctgag gggatgtcac agcaggacag tagaaatatg 1800

aactatgtca tgctagggaa gcccatgaac aatggactca tggagaagat ggtggaggag 1860

agtgatgagg aagtgtgtct aagtagtcta gagtgcctga ggaaccagtg ctttgacctg 1920

gccaggaaac tcagcgagac caacccacag gacaggagtc agccacccaa catgagtgtg 1980

cgccaacatg tccggcaaga ggacgacttc cctgggagga cgccagacag gagctactcg 2040

gatatgatga accttatgcg gctggaggag cagctcagtc ccaggtctag aacgttctcc 2100

agctgtgcca aggaggatgg gtgtcatgag agggatgatg acaccacctc agtgaactca 2160

gacaggtctg aggaagtgtt tgacatgacc aagggcaacc tgactctgct agagaaagcc 2220

attgccttgg agacagagag agccaaggcc atgcgggaga agatggccat ggatgctggg 2280

agaagggata acctgagatc ctatgaggac cagtctccaa gacagctggc tggggaagac 2340

agaaaatcca aatccagtga cagccatgtc aaaaagccat actatggtaa agatccctca 2400

agaacagaaa agagagagag caagtgtcca acccccgggt gtgatggaac cggccacgta 2460

actgggcttt acccgcatca ccgcagtctg tctggatgcc cgcacaaaga tagggtccct 2520

ccagaaattc ttgccatgca tgaaaatgtt ctcaagtgtc ccactccagg ctgcacaggg2580

cgagggcatg tgaatagcaa caggaactcg cacagaagcc tctctggatg ccccattgct 2640

gctgcagaaa aactggcaaa ggcccaagag aaacaccaga gctgtgatgt gtccaaatcc 2700

aaccaggcct cagaccgagt cctcaggcca atgtgctttg tcaaacagct tgagattcct 2760

cagtatggct acagaaacaa tgttcccaca accacaccac gctccaacct ggccaaggag 2820

cttgagaaat actccaagac ttcgtttgag tacaacagtt acgacaacca tacttatggc 2880

aaaagagcca tagctcccaa ggtgcaaacc agggacatat cccccaaagg atatgacgat 2940

gccaagcggt actgcaagaa tgccagcccc agcagcagca ccaccagcag ctatgcacct 3000

agcagcagca gcaacctcag ctgtggtggt ggcagcagcg ccagtagcac gtgtagcaag 3060

agcagctttg actacacaca tgacatggag gccgcacaca tggcagccac agccattctc 3120

aacctgtcca cacgttgtcg tgaaatgcca cagaacctgt ccaccaagcc acaggacctg 3180

tgtactgccc ggaacccaga catggaggtg gatgagaatg gcaccctgga cctgagcatg 3240

aacaagcaga ggcctcgaga cagctgctgc ccagtcctga cacccctgga acccatgtct 3300

ccgcagcagc aggccgtgat gagcagccga tgcttccagc tgagcgaggg ggattgctgg 3360

gacttgcctg tagactacac caaaatgaag cctcggaggg tagatgagga tgagcccaaa 3420

gagattaccc cagaagactt ggacccattc caggaggctc tggaagaaag acggtatcca 3480

ggggaggtga ccatcccaag ccccaaaccc aagtaccctc agtgcaagga aagcaaaaag 3540

gacttaataa ctctgtctgg ctgccccctg gcggacaaaa gcattcgaag tatgctggcc 3600

accagttccc aagagctcaa gtgccccacc cctggctgtg acggttctgg acacatcact 3660

ggcaattacg cttctcatcg aagcctttct gggtgcccga gagcaaagaa gagtggcatc 3720

cggatagcac agagcaaaga ggacaaggaa gaccaggagc caatcaggtg tccggtacct 3780

ggctgtgacg gtcagggaca catcactggg aagtatgcat cccaccgcag cgcctccggg 3840

tgtcccttgg cagccaagag gcagaaagat gggtacctta atggctccca gttctcctgg 3900

aagtcggtca agacggaggg catgtcctgc cctacccccg ggtgtgatgg gtcaggacac 3960

gtcagtggca gcttcctcac acaccgcagc ttgtcaggat gtccaagagc cacatcagca 4020

atgaagaaag caaagctgtc tggagaacag atgttgacta tcaagcagcg agccagcaac 4080

ggtatagaaa atgatgaaga aatcaagcag ttagatgaag agatcaagga gcttaatgag 4140

tccaattccc agatggaggc tgacatgatc aaactcagaa ctcagatcac cacaatggag 4200

agcaacctga agacgattga ggaggagaac aaagtcattg aacagcagaa tgagtcgctc 4260

ttgcacgagt tggccaacct gagccagtcc ctgatccaca gcctcgccaa catccagctg 4320

cctcacatgg atccaatcaa tgaacaaaat tttgatgctt acgtgactac tttgacggaa 4380

atgtatacaa atcaagatcg ttatcagagt ccagaaaata aagccctact ggaaaatata 4440

aagcaggctg tgagaggaat tcaggtctga acagctgctc tagtggtgac tcatgcttaa 4500

aaaggatgcc tcttgtttct tgctgctgta acttaccaga aagtgttata tatttatttc 4560

tgtcggaaca gtgttatgct acaagacttc ataatggttt tgtgtgctct cgagagagta 4620

cctgcagact agttttggat acattcacat tttgtacgtt ttcatataag ctgacatagt 4680

gtgatttgcc atgtaatgtt tatagctgct gctgtctgca catttggggg tctctatatt 4740

tctgaagagg taagctgatg aaaataaata gagtgtaaat tctttttaat gctttagtga 4800

ttaaatgttt tagtattttg aactgaaatg gacaaaaaaa gaaaaaggaa aaaaaaagca 4860

ggtttgaacg atcactttgt ggcctcctgg ccacttttag acattatcat tttgcctcag 4920

gttggaggac tttgtggaat ttaagaaata cattttgtgt gcatattgtt tcatagcaag 4980

aattggttgc aaaaaaatgc tttatttttg aacaatgctt ggaaatatta tgtgactttt 5040

ttgtttgttt gttttaggag gatggtgtat ggtgggggca ataaatgagg ttttttgcat 5100

tccaaggaaa tggcatatgg attaactata agaaatgaaa taagtaattt attgtaagac 5160

aacatcaagc catggaaact tggcagaaga ttcaaagcag cttaaacagc acttttaatt 5220

aactcctaaa cattacatgg tgggactgtg gagactccgt taagacagga gcttgtcaga 5280

ggtggacaac acgaagattt cctttgcatt ttcagtaacc tttggagcac acttctctta 5340

tttctcctag agccccgtgc ccctctgaag tgttaccaca atctagctta cctagcagcc 5400

gttgattgtt ttttttccat ctgagcaaac aggtaattta agcatttatc tcccctttca 5460

ctttcaaaaa gaatcatcat tgattattct tgtttcacat ctgaggatga aggctaacgg 5520

ctgtgcttgc cctggttact ggatgggatt ctctgctggg cgggtgggga agcagctcta 5580

cccttcccta cccctccctg ccccaactct gacttcgaat aagccttggt cctatccaga 5640

atacactgga caccaaggcc aaggacgttc acgttctgcc ctcgctgggt ccagctgact 5700

ctagcgtctg cacagccttg tgtgactcgt ggtgacctaa cctgagaaag agtgcaatca 5760

gatgttaaag taactaaata gactgcggca cttttttgct ttaaactaga tcatcttaga 5820

tttgtcgata ccttcgaaat ttgatggttt catcccaaat gactgcacta tatgtatgca 5880

tacggccact tttgattgct gcgccccttc tgagtagtct ttgacaatgt gttgtgttcc 5940

ccgatgtcga cttgatttcc ttttagtagc atctctctct tccatgtctt gatgttatgc 6000

aggaagtaca aaagtacttt aaaattttgt tatgaaataa aaaaaaaaag gatgggtttt 6060

gtaaaaataa taaaaaaaat atttttagca gaacaggact tacagggtca ttgtccccac 6120

aatgtgccag tcggctcttt gcactcgcct tgtcctatat atccgtacgg aggtgtgcaa 6180

tcctgtgtca gtcgccttgt gacactgaag tggatgagtt atagaggagg ccctcgaggc 6240

tgacccaata cggttactaa gggagactac agggatctca cgacaaacat tctgatacaa 6300

tactcaacct cggtatatat atatatatat atatatatat acatatatat atatgtataa 6360

atataagaat atcccagcgg cactttatac tgttcactgt acaaaagctt acagttttcc 6420

acaaggactt taataactag ctggggaaaa gattatgtaa ttacttgggg ctctgcagga 6480

ccttctctgt ccagcgcccc ctttctgttg tgcgattagt tgtagctgcc atgctcagaa 6540

ttgccttttg agagctgaag caaggtgctt actgtcacct gatgccatac acatggtccc 6600

aggcccacac ccggggggcc tctgttcata gcggcacatg catttcccca ccgcgtcttg 6660

tctgcagctt cttggccaat gtagtaatgc ttttagtaga gtaataggta gtatcagttt 6720

ggattcttat tgttatcacc tatgtacaat ggagaggggt tctaagcaca aatctgctgc 6780

tcatgtaacg gtggtacataatatcaaatc aaaagttatc tgtgactata tatagggatc 6840

acaaagtgtc acatgttaga atgctgacct tccacatggg gttattgtga gtcatcagag 6900

catatttatt ataacttatt gttcatattc atttctaagt taatttaagt aatcatttat 6960

taagacagaa ttttgtataa actatttatt gtgctctctg tggaactgaa gtttgattta 7020

tttttgtact acacggcatg ggtttgttga cactttaatt ttgctataaa tgtgtggaat 7080

cacaagttgc tgtgatactt catttttaaa ttgtgaactt tgtacaaact ttgtcatgct 7140

gatgtgaaca catcttactc tgaataaaaa ggtgttgcca cgtttgtagc acgaagga 7198

<210>6

<211>7104

<212>DNA

<213> Artificial sequence

<220>

<223> Induction factor

<400>6

gtctgctgcc tgctgatgga ggctgcgccg catccaccag gcagggctct gcacgcatgc 60

ccgtcttccc cggcgtttag tggtgtcaac ttattaattt ccatcttcga cttcaggcct 120

gttagtttcc tgtctgaaag cattattact tttgtccatg tggcaaaata attagcatac 180

agatgagaag tggccccgtg tctgcgcgcc gatagagaga agatgtgcgg ttcgaagttg 240

attggatgaa tggagaacaa atggtgggcg ttcaaaagca ttagaagagt tttgtagtcg 300

cggagcaggc tggagaattt tctgggggtc ccacagaggc tgcagctgag gtctgggtgt 360

gtgcaattct cagctgggct ttgccctacc caggttgacg actgaagaat cacagagtgt 420

ggaaaagaac acaaggagaa attggtgaga acatctgcct agcatctcca agtcctgtgg 480

agggagccag cagtgctggt ccaaagagga cccagagagt gaactcagag tgaccacatc 540

tgatagaaga ggggaagatg tagtttctga gtccagtcca gtgtttgtgt ctctcacatt 600

gtcaacaaaa gaaaggctcc agctgtcccc acagacatat ggatattcca ggagccacgt 660

aaagatggag aaatggaggc acagagaaat taagtgactt ggccacagtc acaagctggg 720

gaggaccagg gaaagcctag agagagctgg ctctgggcct gcatcctgcc cacggagtca 780

ccctgcctcc gtcctcagga gagaaggctt cctacaagat ggacgtggac tctgaggaga 840

agcgccatcg cacacggtcc aaaggggttc gagttcctgt ggagccagcc atacaagagc 900

tgttcagctg tcccactcca ggctgcgacg gcagtggtca cgtcagtggc aaatatgcac 960

gacacagaag tgtatatggt tgtcccttgg ctaaaaaaag aaaaacgcaa gataaacagc 1020

cccaagaacc tgctcccaag cgaaaaccat ttgcagtaaa agcagatagt tcctcagtag 1080

acgaatgtta tgagagtgat ggtactgaag acatggatga taaggaggaa gatgatgatg 1140

aggagttctc tgaagacaat gatgagcaag gggatgatga cgacgaagat gaggtggatc 1200

gggaagacga ggaggagatc gaggaggaag atgatgaaga agatgatgat gatgaagatg 1260

gtgacgatgt agaagaggaa gaagaggatg atgatgaaga ggaggaagaa gaggaagagg 1320

aagaagaaaa tgaagaccat caaatgagtt gtactcgaat aatgcaggac acagacaagg 1380

atgataacaa caatgatgag tatgataact atgatgaact ggtagctaag tcgctattaa 1440

atcttggcaa aattgctgag gatgcagcat accgagccag gactgaatca gagatgaaca 1500

gcaatacctc caatagtctg gaggacgata gtgacaaaaa cgaaaacctc ggtcggaaaa 1560

gcgaactgag tctagactta gacagtgatg ttgttagaga aacagtggac tcccttaagc 1620

tgttagcaca aggacatggt gttgtgctat cagagaatat cagtgacaga agttatgctg 1680

aggggatgtc acagcaggac agtagaaata tgaactatgt catgctaggg aagcccatga 1740

acaatggact catggagaag atggtggagg agagtgatga ggaagtgtgt ctaagtagtc 1800

tagagtgcct gaggaaccag tgctttgacc tggccaggaa actcagcgag accaacccac 1860

aggacaggag tcagccaccc aacatgagtg tgcgccaaca tgtccggcaa gaggacgact 1920

tccctgggag gacgccagac aggagctact cggatatgat gaaccttatg cggctggagg 1980

agcagctcag tcccaggtct agaacgttct ccagctgtgc caaggaggat gggtgtcatg 2040

agagggatga tgacaccacc tcagtgaact cagacaggtc tgaggaagtg tttgacatga 2100

ccaagggcaa cctgactctg ctagagaaag ccattgcctt ggagacagag agagccaagg 2160

ccatgcggga gaagatggcc atggatgctg ggagaaggga taacctgaga tcctatgagg 2220

accagtctcc aagacagctg gctggggaag acagaaaatc caaatccagt gacagccatg 2280

tcaaaaagcc atactatgat ccctcaagaa cagaaaagag agagagcaag tgtccaaccc 2340

ccgggtgtga tggaaccggc cacgtaactg ggctttaccc gcatcaccgc agtctgtctg 2400

gatgcccgca caaagatagg gtccctccag aaattcttgc catgcatgaa aatgttctca 2460

agtgtcccac tccaggctgc acagggcgag ggcatgtgaa tagcaacagg aactcgcaca 2520

gaagcctctc tggatgcccc attgctgctg cagaaaaact ggcaaaggcc caagagaaac 2580

accagagctg tgatgtgtcc aaatccaacc aggcctcaga ccgagtcctc aggccaatgt 2640

gctttgtcaa acagcttgag attcctcagt atggctacag aaacaatgtt cccacaacca 2700

caccacgctc caacctggcc aaggagcttg agaaatactc caagacttcg tttgagtaca 2760

acagttacga caaccatact tatggcaaaa gagccatagc tcccaaggtg caaaccaggg 2820

acatatcccc caaaggatat gacgatgcca agcggtactg caagaatgcc agccccagca 2880

gcagcaccac cagcagctat gcacctagca gcagcagcaa cctcagctgt ggtggtggca 2940

gcagcgccag tagcacgtgt agcaagagca gctttgacta cacacatgac atggaggccg 3000

cacacatggc agccacagcc attctcaacc tgtccacacg ttgtcgtgaa atgccacaga 3060

acctgtccac caagccacag gacctgtgta ctgcccggaa cccagacatg gaggtggatg 3120

agaatggcac cctggacctg agcatgaaca agcagaggcc tcgagacagc tgctgcccag 3180

tcctgacacc cctggaaccc atgtctccgc agcagcaggc cgtgatgagc agccgatgct 3240

tccagctgag cgagggggat tgctgggact tgcctgtaga ctacaccaaa atgaagcctc 3300

ggagggtaga tgaggatgag cccaaagaga ttaccccaga agacttggac ccattccagg 3360

aggctctgga agaaagacgg tatccagggg aggtgaccat cccaagcccc aaacccaagt 3420

accctcagtg caaggaaagc aaaaaggact taataactct gtctggctgc cccctggcgg 3480

acaaaagcat tcgaagtatg ctggccacca gttcccaaga gctcaagtgc cccacccctg 3540

gctgtgacgg ttctggacac atcactggca attacgcttc tcatcgaagc ctttctgggt 3600

gcccgagagc aaagaagagt ggcatccgga tagcacagag caaagaggac aaggaagacc 3660

aggagccaat caggtgtccg gtacctggct gtgacggtca gggacacatc actgggaagt 3720

atgcatccca ccgcagcgcc tccgggtgtc ccttggcagc caagaggcag aaagatgggt 3780

accttaatgg ctcccagttc tcctggaagt cggtcaagac ggagggcatg tcctgcccta 3840

cccccgggtg tgatgggtca ggacacgtca gtggcagctt cctcacacac cgcagcttgt 3900

caggatgtcc aagagccaca tcagcaatga agaaagcaaa gctgtctgga gaacagatgt 3960

tgactatcaa gcagcgagcc agcaacggta tagaaaatga tgaagaaatc aagcagttag 4020

atgaagagat caaggagctt aatgagtcca attcccagat ggaggctgac atgatcaaac 4080

tcagaactca gatcaccaca atggagagca acctgaagac gattgaggag gagaacaaag 4140

tcattgaaca gcagaatgag tcgctcttgc acgagttggc caacctgagc cagtccctga 4200

tccacagcct cgccaacatc cagctgcctc acatggatcc aatcaatgaa caaaattttg 4260

atgcttacgt gactactttg acggaaatgt atacaaatca agatcgttat cagagtccag 4320

aaaataaagc cctactggaa aatataaagc aggctgtgag aggaattcag gtctgaacag 4380

ctgctctagt ggtgactcat gcttaaaaag gatgcctctt gtttcttgct gctgtaactt 4440

accagaaagt gttatatatt tatttctgtc ggaacagtgt tatgctacaa gacttcataa 4500

tggttttgtg tgctctcgag agagtacctg cagactagtt ttggatacat tcacattttg 4560

tacgttttca tataagctga catagtgtga tttgccatgt aatgtttata gctgctgctg 4620

tctgcacatt tgggggtctc tatatttctg aagaggtaag ctgatgaaaa taaatagagt 4680

gtaaattctt tttaatgctt tagtgattaa atgttttagt attttgaact gaaatggaca 4740

aaaaaagaaa aaggaaaaaa aaagcaggtt tgaacgatca ctttgtggcc tcctggccac 4800

ttttagacat tatcattttg cctcaggttg gaggactttg tggaatttaa gaaatacatt 4860

ttgtgtgcat attgtttcat agcaagaatt ggttgcaaaa aaatgcttta tttttgaaca 4920

atgcttggaa atattatgtg acttttttgt ttgtttgttt taggaggatg gtgtatggtg 4980

ggggcaataa atgaggtttt ttgcattcca aggaaatggc atatggatta actataagaa 5040

atgaaataag taatttattg taagacaaca tcaagccatg gaaacttggc agaagattca 5100

aagcagctta aacagcactt ttaattaact cctaaacatt acatggtggg actgtggaga 5160

ctccgttaag acaggagctt gtcagaggtg gacaacacga agatttcctt tgcattttca 5220

gtaacctttg gagcacactt ctcttatttc tcctagagcc ccgtgcccct ctgaagtgtt 5280

accacaatct agcttaccta gcagccgttg attgtttttt ttccatctga gcaaacaggt 5340

aatttaagca tttatctccc ctttcacttt caaaaagaat catcattgat tattcttgtt 5400

tcacatctga ggatgaaggc taacggctgt gcttgccctg gttactggat gggattctct 5460

gctgggcggg tggggaagca gctctaccct tccctacccc tccctgcccc aactctgact 5520

tcgaataagc cttggtccta tccagaatac actggacacc aaggccaagg acgttcacgt 5580

tctgccctcg ctgggtccag ctgactctag cgtctgcaca gccttgtgtg actcgtggtg 5640

acctaacctg agaaagagtg caatcagatg ttaaagtaac taaatagact gcggcacttt 5700

tttgctttaa actagatcat cttagatttg tcgatacctt cgaaatttga tggtttcatc 5760

ccaaatgact gcactatatg tatgcatacg gccacttttg attgctgcgc cccttctgag 5820

tagtctttga caatgtgttg tgttccccga tgtcgacttg atttcctttt agtagcatct 5880

ctctcttcca tgtcttgatg ttatgcagga agtacaaaag tactttaaaa ttttgttatg 5940

aaataaaaaa aaaaaggatg ggttttgtaa aaataataaa aaaaatattt ttagcagaac 6000

aggacttaca gggtcattgt ccccacaatg tgccagtcgg ctctttgcac tcgccttgtc 6060

ctatatatcc gtacggaggt gtgcaatcct gtgtcagtcg ccttgtgaca ctgaagtgga 6120

tgagttatag aggaggccct cgaggctgac ccaatacggt tactaaggga gactacaggg 6180

atctcacgac aaacattctg atacaatact caacctcggt atatatatat atatatatat 6240

atatatacat atatatatat gtataaatat aagaatatcc cagcggcact ttatactgtt 6300

cactgtacaa aagcttacag ttttccacaa ggactttaat aactagctgg ggaaaagatt 6360

atgtaattac ttggggctct gcaggacctt ctctgtccag cgcccccttt ctgttgtgcg 6420

attagttgta gctgccatgc tcagaattgc cttttgagag ctgaagcaag gtgcttactg 6480

tcacctgatg ccatacacat ggtcccaggc ccacacccgg ggggcctctg ttcatagcgg 6540

cacatgcatt tccccaccgc gtcttgtctg cagcttcttg gccaatgtag taatgctttt 6600

agtagagtaa taggtagtat cagtttggat tcttattgtt atcacctatg tacaatggag 6660

aggggttcta agcacaaatc tgctgctcat gtaacggtgg tacataatat caaatcaaaa 6720

gttatctgtg actatatata gggatcacaa agtgtcacat gttagaatgc tgaccttcca 6780

catggggtta ttgtgagtca tcagagcata tttattataa cttattgttc atattcattt 6840

ctaagttaat ttaagtaatc atttattaag acagaatttt gtataaacta tttattgtgc 6900

tctctgtgga actgaagttt gatttatttt tgtactacac ggcatgggtt tgttgacact 6960

ttaattttgc tataaatgtg tggaatcaca agttgctgtg atacttcatt tttaaattgt 7020

gaactttgta caaactttgt catgctgatg tgaacacatc ttactctgaa taaaaaggtg 7080

ttgccacgtt tgtagcacga agga 7104

<210>7

<211>7189

<212>DNA

<213> Artificial sequence

<220>

<223> Induction factor

<400>7

gtggagctgc gtgcattagg ggctggctgt ggaggtaaag cagatctctt aggcccgcta 60

gcctctcctg ctgcatgtgt cattcctgcc cgcgcgacat tttcccccta agctactgca 120

cacaaaacag agcgagaaag cttctccctg cagtcttctt ggaggcctcc tggtttctca 180

cccattgttg gtgggtgtat ttcaattttt tgattccctg gactgtgggt tatgaaatca 240

atgctgctga agacaaaagc aaccttccct gcctctcggt gctatgcgtg gtcctcctcg 300

gcctcccact tgtgggggaa agggttttct ctttctttct gtgtgttttg agccagcaca 360

gttaccaaaa ttgaacttgc cgtcacttgt gagcggtgtg gtcatggtgt gaggggtccc 420

acagaggctg cagctgaggt ctgggtgtgt gcaattctca gctgggcttt gccctaccca 480