TWI649467B - Kit for selecting neurological drug and uses thereof - Google Patents

Abstract

The present disclosure relates to a kit comprising several transcription factors that can be used to induce transformation of a fibroblast into an evoked embryonic neural precursor cell. The induced embryonic neural precursor cells can then differentiate into a stellate cell, an oligodendrocyte or a neuron. The present disclosure also provides for the use of the kit as a platform for screening drug candidates for the treatment of neurological diseases.

Description

Kit for screening neurological drugs and uses thereof

The present disclosure relates to the treatment of neurological diseases. More specifically, the present disclosure relates to a kit comprising a specific differentiation factor that can serve as a platform for screening drug candidates for treating neurological diseases.

At present, there is still no effective treatment for the progressive, degenerative and ultimately fatal neurological diseases such as Huntington's Disease (HD) and Alzheimer's disease (AD) in the medical field; Therefore, the pathological mechanism of these diseases and effective clinical treatment methods are still to be further clarified in subsequent studies. With pluripotency reprogramming technology, researchers can reorganize the patient's somatic cells into disease-specific pluripotent stem cells (iPSCs) and differentiate them in vitro. A variety of cell types associated with disease to establish disease patterns and develop therapeutic drugs. However, the canceration and spontaneous differentiation of iPSC may still bring follow-up related problems. In addition to iPSC, a specific transcription factor (TF) directly converts fibroblasts (FB) into induced neuron (iN), which provides additional in vitro disease patterns and drug testing. A source of nerve cells. The advantage of iN technology is that it provides a fast and simple method of preparing specific neuronal subtypes and avoids problems such as inability to control cell differentiation and tumorigenesis (associated with human iPSC). However, inducing the production of each neuron subtype requires a different combination of transcription factors, and the yield of these iNs is often much lower than the amount of cells required for clinical applications. Therefore, when a relatively large amount of specific nerve cells are prepared for different patients, the somatic cells can be directly transformed into neural stem cells/progenitors (NSC/NP) which are proliferative and have pluripotent neural differentiation potential. Technology is very important.

In the prior art, it is known that mouse somatic cells can be directly transformed into induced neural precursor cells (induced NP, iNP) by expressing a combination of different transcription factors in excess. According to previous reports, the pluripotent reforming step can be adjusted to prepare proliferative iNPs from fibroblasts, and the resulting iNPs can differentiate into neurons and glial cells. Later, several studies indicated that iNP induced by neurotrophic factors can differentiate into three major neuronal cell types in the central nervous system in the presence or absence of iPSC factors. At the same time, the report also pointed out that transcription factors can convert human somatic cells into iNP. In these studies, human iNPs were prepared primarily using a combination of transcription factors comprising at least one iPS factor, and the differentiation propensity of these iNPs was mostly restricted to central nervous system neurons.

Human embryonic stem cells (hESC) can be used as an in vitro differentiation model to generate neural phenotypes containing embryonic neural precursor cells (EMP) in different developmental states, and to explore unknown effects on neurodevelopment. Important neurogenetic factors. Based on human ESC-ENP (human ESC-ENP, hESC-ENP) with wide differentiation potential, it can produce different neuronal cell types in the central nervous system and peripheral nervous system, and can utilize transcription factors that are highly expressed in the hESC-ENP cell population. Fibroblasts were directly transformed into iNPs similar to hESC-ENP.

Here, the present disclosure confirms a series of neurotransit factors that are highly expressed in hESC-ENP (compared to the amount of fibroblasts) by comparing gene expression profiles; wherein, the excess expression of the two transcription factors can be Effectively convert human fibroblasts into pluripotent iENP. These iENPs have many similarities with hESC-ENP, including proliferative patterns, gene expression profiles, and differentiation tendencies in vitro and in vivo. More importantly, the present disclosure has found that iENP cell populations induced by different combinations of transcription factors have different proliferative properties and regional differentiation preferences. The present disclosure also demonstrates that neurons derived from AD- and HD-iENP can reproduce the major pathological features of these diseases in vitro. Overall, the present disclosure provides a strategy with potential and reproducibility to prepare iENP from somatic cells to establish disease patterns and clinically relevant applications.

SUMMARY OF THE INVENTION The Summary of the Disclosure is intended to provide a basic understanding of the present disclosure. This Summary is not an extensive overview of the disclosure, and is not intended to be an

A first aspect of the present disclosure is directed to a kit (hereinafter referred to as a first kit) that can be used to screen a candidate drug for treating a neurological disorder. According to an embodiment of the present disclosure, the first set comprises six polynucleotides comprising CBX2 (SEQ ID NO: 1), HES1 (SEQ ID NO: 2), ID1 (SEQ ID NO: 3), TFAP2A (SEQ ID NO: :4), ZFP42 (SEQ ID NO: 5) and ZNF423 (SEQ ID NO: 6) genes.

In accordance with an optional embodiment of the present disclosure, in addition to the first to sixth polynucleotides, the first set further comprises additional polynucleotides. In one embodiment, the first set further comprises 9 polynucleotides comprising DACH1 (SEQ ID NO: 7), FOXG1 (SEQ ID NO: 8), MYCN (SEQ ID NO: 9), NR2F2 (SEQ ID NO: :10), NR6A1 (SEQ ID NO: 11), SOX2 (SEQ ID NO: 12), SOX11 (SEQ ID NO: 13), ZIC2 (SEQ ID NO: 14), and ZIC3 (SEQ ID NO: 15). In another embodiment, the first set further comprises 19 polynucleotides comprising DACH1 (SEQ ID NO: 7), FOXG1 (SEQ ID NO: 8), MYCN (SEQ ID NO: 9), NR2F2 (sequence), respectively. No.: 10), NR6A1 (sequence number: 11), SOX2 (sequence number: 12), SOX11 (sequence number: 13), ZIC2 (sequence number: 14), ZIC3 (sequence number: 15), GATA3 (sequence number: 16), PAX6 (sequence number: 17), SALL2 (sequence number: 18), LHX2 (sequence number: 19), MBD2 (sequence number: 20), DEPDC 1 (sequence number: 21), MYEF2 (sequence number: 22) ), OTX2a (SEQ ID NO: 23), SIX3 (SEQ ID NO: 24), and SOX1 (SEQ ID NO: 25) genes.

Optionally, the first set may further comprise a reporter polynucleotide comprising the sequence of SEQ ID NO: 26.

A second aspect of the present disclosure is directed to another kit (hereinafter referred to as a second set) that can be used to screen a candidate drug for treating a neurological disorder. According to an embodiment of the present disclosure, the second set comprises seven polynucleotides comprising TFAP2A (SEQ ID NO: 4), ZFP42 (SEQ ID NO: 5), FOXG1 (SEQ ID NO: 8), NR2F2 (SEQ ID NO: :10), GATA3 (SEQ ID NO: 16), PAX6 (SEQ ID NO: 17) and SALL2 (SEQ ID NO: 18) genes.

In addition to the seven polynucleotides described above, the second set may further comprise other polynucleotides. In one embodiment, the second set further comprises 6 polynucleotides comprising CBX2 (SEQ ID NO: 1), HES1 (SEQ ID NO: 2), NR6A1 (SEQ ID NO: 11), SOX11 (SEQ ID NO: :13), ZIC2 (SEQ ID NO: 14) and LHX2 (SEQ ID NO: 19) genes. In another embodiment, the second set further comprises 18 polynucleotides comprising CBX2 (SEQ ID NO: 1), HES1 (SEQ ID NO: 2), ID1 (SEQ ID NO: 3), ZNF423 (sequence), respectively. No.: 6), DACH1 (sequence number: 7), MYCN (sequence number: 9), NR6A1 (sequence number: 11), SOX2 (sequence number: 12), SOX11 (sequence number: 13), ZIC2 (sequence number: 14), ZIC3 (sequence number: 15), LHX2 (sequence number: 19), MBD2 (sequence number: 20), DEPDC1 (sequence number: 21), MYEF2 (sequence number: 22), OTX2a (sequence number: 23) , SIX3 (sequence number: 24) and SOX1 (sequence number: 25) genes.

Optionally, the second set may further comprise a reporter polynucleotide comprising the sequence of SEQ ID NO:27.

In accordance with an embodiment of the present disclosure, the first or second kit of the present invention further comprises an enhancer selected from the group consisting of RepSox, PP242, DZNep, Vitamin C, and combinations thereof.

A third aspect of the present disclosure is directed to a method for screening for a drug candidate suitable for treating a neurological disorder, such as a neurodevelopmental disease, a neurodegenerative disease, or a motor neuron disease. According to some embodiments of the present disclosure, the method comprises: (a) transferring a first or a second set of polynucleotides of the present disclosure into a fibroblast to induce the transformation of the fibroblast into an induction (i) an embryonic neural progenitor cell (iENP cell); (b) culturing the iENP cell of step (a) in a differentiation culture medium, thereby inducing transformation of the iENP cell into an astrocyte, An oligodendrocyte or a neuron; (c) contacting the stellate cell of step (b), the oligodendrocyte or the neuron with one or more drugs to be tested; d) screening the candidate drug from the one or more test drugs, wherein the candidate drug changes the phenotype, gene expression of the stellate cell, the oligodendrocyte or the neuron.

Basically, the fibroblast can be derived from a healthy individual or an individual suffering from a neurodegenerative disease. According to a preferred embodiment, the fibroblast is derived from an individual suffering from a neurodegenerative disease.

Another aspect of the present disclosure is directed to a method of treating an individual suffering from or suspected of suffering from a neurodegenerative disease. The method comprises: (a) isolating a fibroblast from the individual; (b) placing the first to sixth polynucleotides of the first set of the disclosure or the first to seventh of the second set Nucleotide is transferred into the fibroblast to induce the transformation of the fibroblast into an iENP cell; (c) optionally, the iENP cell of step (b) is cultured in a differentiation medium to induce the The iENP cell is transformed into a stellate cell, an oligodendrocyte or a neuron; (d) the subject is administered an effective amount of the iENP cell of step (b), or an effective amount of the star of step (c) Cells, oligodendrocytes or neurons to slow or reduce the symptoms associated with this neurological disease.

Generally, the neurological disease can be a neurodevelopmental disease, a neurodegenerative disease, or a motor neuron disease.

The disclosure also provides cells prepared from the kit of the invention comprising an iENP cell, a stellate cell, an oligodendrocyte, and a neuron. The cells can be used to treat a neurological disorder; for example, a neurodevelopmental disease, a neurodegenerative disease, or a motor neuron disease.

The basic spirit and other objects of the present invention, as well as the technical means and implementations of the present invention, will be readily apparent to those skilled in the art of the invention.

The description of the embodiments of the present invention is intended to be illustrative and not restrictive. The features of various specific embodiments, as well as the method steps and sequences thereof, are constructed and manipulated in the embodiments. However, other specific embodiments may be utilized to achieve the same or equivalent function and sequence of steps.

Although numerical ranges and parameters are used to define a broad range of values for the present invention, the relevant values in the specific embodiments have been presented as precisely as possible. However, any numerical value inherently inevitably contains standard deviations due to individual test methods. As used herein, "about" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a particular value or range. Alternatively, the term "about" means that the actual value falls within the acceptable standard error of the average, depending on the considerations of those of ordinary skill in the art to which the invention pertains. Except for the experimental examples, or unless otherwise explicitly stated, all ranges, quantities, values, and percentages used herein are understood (eg, to describe the amount of material used, the length of time, the temperature, the operating conditions, the quantity ratio, and the like. Are all modified by "about". Therefore, unless otherwise indicated to the contrary, the numerical parameters disclosed in the specification and the appended claims are intended to be At a minimum, these numerical parameters should be understood as the number of significant digits indicated and the values obtained by applying the general carry method. Ranges of values are expressed herein as being from one endpoint to another or between two endpoints; unless otherwise stated, the numerical ranges recited herein are inclusive.

The scientific and technical terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention pertains, unless otherwise defined herein. In addition, the singular noun used in this specification covers the plural of the noun in the case of no conflict with the context; the plural noun of the noun is also included in the plural noun used.

In the present disclosure, the term "introduction" refers to the transfer of a polynucleotide (eg, a polynucleotide of the kit of the invention) into a cell or organism. The nucleic acid of the polynucleotide may be naked DNA or RNA, DNA or RNA linked to a different protein, or DNA or RNA inserted into a vector. The term "introduce" is used in the broadest scope of this disclosure; for example, transfer to a transfer effect that includes the following methods: transfection (transfection, physical and/or chemical treatment will be used) Polynucleotide transfer to a eukaryotic cell), transformation method (transformation method using a physical and / or chemical treatment to transfer a polynucleotide into a prokaryotic cell), viral method / viral transfection method (viral method / Viral transduction method, using a viral or viral vector to transfer a polynucleotide into a eukaryotic and/or prokaryotic cell), conjugation method, by cell-cell direct contact or intercellular cytoplasmic bridge, The nucleotide is transferred from one cell to another) and the fusion method (fusion of two cells, including homotypic cell fusion and heterotypic cell fusion).

In the present disclosure, the term "neurological disease" refers to a disease or condition caused by dysplasia, disease, genetic defect, injury or toxin which causes structural or functional abnormalities of the nervous system. Such diseases or conditions affect the central nervous system (eg, brain, brainstem, and cerebellum), peripheral nervous systems (eg, cranial nerves, spinal nerves, and sympathetic and parasympathetic nervous systems) and/or autonomic nervous systems (eg, regulation of involuntary movements) The nervous system and the nervous system that can be divided into the sympathetic and parasympathetic nervous systems). Exemplary neurological diseases include, but are not limited to, neurodevelopmental diseases, neurodegenerative diseases, and motor neuron diseases.

The term "subject" refers to an animal comprising humans which is acceptable for the treatment of the methods of the invention. Unless specifically stated otherwise, the term "subject" means both male and female and includes any age, such as a child or an adult.

The present disclosure provides three kits, each of which can be used to induce the conversion of a fibroblast into an iENP cell, thereby screening for treatment of a neurodegenerative disease (eg, a neurodevelopmental disease, a neurodegenerative disease, or a motor nerve). Candidate drugs for meta-diseases.

The first kit contains 6 nucleotides (ie, first to sixth nucleotides), which contain CBX2 (SEQ ID NO: 1), HES1 (SEQ ID NO: 2), and ID1 (SEQ ID NO: 3), respectively. , TFAP2A (SEQ ID NO: 4), ZFP42 (SEQ ID NO: 5), and ZNF423 (SEQ ID NO: 6); the present disclosure refers to this set as 6TF (6-transcription factor).

Depending on the needs of use, the first set may further comprise at least one gene selected from DACH1 (SEQ ID NO: 7), FOXG1 (SEQ ID NO: 8), MYCN (SEQ ID NO: 9), NR2F2 (SEQ ID NO: :10), NR6A1 (sequence number: 11), SOX2 (sequence number: 12), SOX11 (sequence number: 13), ZIC2 (sequence number: 14), ZIC3 (sequence number: 15), GATA3 (sequence number: 16) ), PAX6 (sequence number: 17), SALL2 (sequence number: 18), LHX2 (sequence number: 19), MBD2 (sequence number: 20), DEPDC1 (sequence number: 21), MYEF 2 (sequence number: 22) , OTX2a (sequence number: 23), SIX3 (sequence number: 24), and SOX 1 (sequence number: 25).

According to some embodiments of the present disclosure, the first set further comprises 9 polynucleotides (ie, seventh to fifteenth polynucleotides) comprising DACH1 (SEQ ID NO: 7), FOXG1 (sequence, respectively ) No.: 8), MYCN (sequence number: 9), NR2F2 (sequence number: 10), NR6A1 (sequence number: 11), SOX2 (sequence number: 12), SOX11 (sequence number: 13), ZIC2 (sequence number: 14) and ZIC3 (sequence number: 15) genes. The present disclosure refers to a set comprising 15 polynucleotide nucleuses (ie, first to fifteenth polynucleotides) as 15TF.

The second kit contains 7 polynucleotides (ie, first to seventh polynucleotides) comprising TFAP2A (SEQ ID NO: 4), ZFP42 (SEQ ID NO: 5), and FOXG1 (SEQ ID NO: 8) ), NR2F2 (SEQ ID NO: 10), GATA3 (SEQ ID NO: 16), PAX6 (SEQ ID NO: 17), and SALL2 (SEQ ID NO: 18); the present disclosure refers to this set as 7TF.

In general, the second set may further comprise at least one gene selected from CBX2 (SEQ ID NO: 1), HES1 (SEQ ID NO: 2), ID1 (SEQ ID NO: 3), ZNF423 (SEQ ID NO: 6 ), DACH1 (sequence number: 7), MYCN (sequence number: 9), NR6A1 (sequence number: 11), SOX2 (sequence number: 12), SOX11 (sequence number: 13), ZIC2 (sequence number: 14), ZIC3 (sequence number: 15), LHX2 (sequence number: 19), MBD2 (sequence number: 20), DEPDC1 (sequence number: 21), MYEF2 (sequence number: 22), OTX2a (sequence number: 23), SIX 3 A group consisting of (sequence number: 24) and SOX1 (sequence number: 25).

According to some embodiments of the present disclosure, the second set further comprises 6 polynucleotides (ie, eighth to thirteenth polynucleotides) comprising CBX2 (SEQ ID NO: 1), HES1 (sequence, respectively ) No.: 2), NR6A1 (sequence number: 11), SOX11 (sequence number: 13), ZIC2 (sequence number: 14), and LHX2 (sequence number: 19). The present disclosure refers to a set comprising 13 polynucleotides (ie, first to thirteenth polynucleotides) as 13TF.

The third kit contains 25 polynucleotides containing CBX2 (SEQ ID NO: 1), HES1 (SEQ ID NO: 2), ID1 (SEQ ID NO: 3), TFAP2A (SEQ ID NO: 4), and ZFP42 ( SEQ ID NO: 5), ZNF423 (sequence number: 6), DACH1 (sequence number: 7), FOXG1 (sequence number: 8), MYCN (sequence number: 9), NR2F2 (sequence number: 10), NR6A1 (sequence number) :11), SOX2 (sequence number: 12), SOX11 (sequence number: 13), ZIC2 (sequence number: 14), ZIC3 (sequence number: 15), GATA3 (sequence number: 16), PAX6 (sequence number: 17) ), SALL2 (sequence number: 18), LHX2 (sequence number: 19), MBD2 (sequence number: 20), DEPDC1 (sequence number: 21), MYEF2 (sequence number: 22), OTX2a (sequence number: 23), SIX3 (SEQ ID NO: 24) and SOX1 (SEQ ID NO: 25) genes. The present disclosure refers to a set comprising 25 polynucleotides (ie, first to twenty-fifth polynucleotides) as 25TF.

In accordance with certain non-essential embodiments of the present disclosure, the kit of the invention (i.e., 6TF, 7TF, 13TF, 15TF or 25TF) may further comprise a reporter polynucleotide; for example, PAX6: EGFP or SOX1: EGFP . In one embodiment, the kit 6TF or 15TF further comprises PAX6: EGFP , which comprises the sequence of SEQ ID NO: 26. In another embodiment, the kit 7TF or 13TF further comprises SOX1: EGFP , which comprises the sequence of SEQ ID NO: 27.

In accordance with embodiments of the present disclosure, a kit of the invention (ie, 6TF, 7TF, 13TF, 15TF or 25TF) can be used to induce the conversion of fibroblasts into iENP cells, wherein the iENP cells can be re-differentiated into stellate under appropriate conditions. Cells, oligodendrocytes, and/or neurons (including neuronal subtypes of the central nervous system and peripheral nervous system).

The present disclosure also encompasses a method for inducing the conversion of fibroblasts into iENP cells. The method comprises contacting the fibroblast with a kit of any aspect or embodiment of the present disclosure.

Optionally, the kit of the invention (i.e., 6TF, 7TF, 13TF, 15TF or 25TF) may further comprise an enhancer to increase the efficacy of the kit of the invention in the preparation of iENP cells. According to an embodiment of the present disclosure, the enhancer is selected from the group consisting of RepSox (a transforming growth factor-β (TGF-β) inhibitor), PP242 (an autophage activator), DZNep (a group of histone methyltransferase inhibitors), vitamin C (a DNA demethylating activator), and combinations thereof. According to an embodiment of the present disclosure, the enhancer is RepSox. According to another embodiment of the present disclosure, the enhancer is a combination of RepSox and PP242. According to still another embodiment of the present disclosure, the enhancer is a combination of RepSox, PP242, DZNep, and vitamin C.

When conceivable, the disclosure also encompasses polypeptides encoded by polynucleotides comprised by the kits 6TF, 7TF, 13TF, 15TF or 25TF. For example, a kit of the invention may comprise first to sixth polypeptides consisting of 6TF of CBX2 (SEQ ID NO: 1), HES1 (SEQ ID NO: 2), ID1 (SEQ ID NO: 3), TFAP2A ( Sequence numbers: 4), ZFP42 (SEQ ID NO: 5) and ZNF423 (SEQ ID NO: 6) are encoded by genes. Alternatively, the kit of the invention may comprise first to seventh polypeptides consisting of TFAP2A (SEQ ID NO: 4), ZFP42 (SEQ ID NO: 5), FOXG1 (SEQ ID NO: 8), NR2F2 (SEQ ID NO: 10), GATA3 (SEQ ID NO: 16), PAX6 (SEQ ID NO: 17) and SALL2 (SEQ ID NO: 18) are encoded by genes.

Another aspect of the present disclosure is directed to a method for screening for a drug candidate suitable for treating a neurological disorder, which is screened using a kit as described in any aspect or embodiment of the present disclosure. The method comprises: (a) transferring a polynucleotide of the kit of the invention into a fibroblast to induce the transformation of the fibroblast into an iENP cell; (b) cultivating the iENP cell of step (a) In the differentiation medium, the iENP cell is induced to transform into a stellate cell, an oligodendrocyte or a neuron; (c) the stellate cell, the oligodendrocyte or the neuron of step (b) Contacting one or more drugs to be tested; and (d) screening the candidate drug from the one or more drugs to be tested, wherein the drug candidate changes the phenotype of the stellate cell, the oligodendrocyte or the neuron Or genetic performance.

In step (a), the polynucleotide of the kit of the invention (i.e., 6TF, 7TF, 13TF, 15TF or 25TF) is transferred into the fibroblast. Exemplary methods for transferring polynucleotides into cells include, but are not limited to, calcium phosphate co-precipitation, electroporation, nucleofection, cell extrusion (cell) Squeezing, lightly pressing cell membranes, sonoporation (using high-intensity ultrasound to form pores in cell membranes), optical transfection (using a highly focused laser to create a tiny hole in the cell membrane), gene delivery (impalefection, DNA is attached to the surface of a nanofiber and injected into the cell), a gene gun (the DNA is attached to an inert solid nanoparticle and injected into the target nucleus), and magnetically transfected (magnetofection). To the target cells, viral transfection (viral transduction, using the virus as a vector, the DNA is delivered to the target cells) or by dendrimer, liposome and cationic polymer Transfection. In one embodiment, the polynucleotide is transferred to fibroblasts by viral transfection (e.g., lentivirus transfection). According to embodiments of the present disclosure, the expression of the polynucleotide induces the conversion of fibroblasts into iENP cells upon transfer. Alternatively, when the kit comprises a polypeptide encoded by the above 6TF, 7TF, 13TF, 15TF or 25TF polynucleotide, the polypeptide can be co-cultured with the fibroblast to achieve the same effect.

In step (b), the iENP cells of step (a) are cultured in a differentiation medium. The differentiation medium may contain specific differentiation factors such as ascorbic acid, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) depending on the needs of use. , fibroblast growth factor (FGF), sonic hedgehog (SHH) and N-[N-(3,5-difluorophenethyl)-L-alaninyl]-( S)-N-[N-(3,5-difluorophenacetyl)-L-alanyl-(S)-phenylglycine tert-butyl ester, DAPT), whereby iENP cells are differentiated into stars Cells, oligodendrocytes or neurons (may be central nervous system neurons or peripheral nervous system neurons).

Differentiated cells (i.e., differentiated stellate cells, oligodendrocytes, or neurons) can serve as a screening platform for those skilled in the relevant art to study novel mechanisms and/or screening candidates involved in neural message delivery pathways. drug. In investigating novel mechanisms, test cells can be administered to differentiated cells to discover potential molecules involved in the regulation of neural signaling pathways. Alternatively, the differentiated cells can be contacted with one or more test drugs to screen for a candidate drug that affects the phenotype or gene expression of the differentiated cells of steps (c) and (d).

According to some embodiments of the present disclosure, the fibroblast of step (a) is derived from a healthy individual.

According to some embodiments of the present disclosure, the fibroblast of step (a) is derived from an individual suffering from a neurological disease; for example, an individual suffering from a neurodevelopmental disease, a neurodegenerative disease, or a motor neuron disease . Exemplary neurodevelopmental diseases include, but are not limited to, autism spectrum disorder (ASD), fetal alcohol spectrum disorder, Down syndrome, attention deficit hyperactivity disorder (attention deficit hyperactivity disorder), Mendelsohn's syndrome, schizophrenia, and fragile-X syndrome. Exemplary neurodegenerative diseases include, but are not limited to, Alzheimer's disease (AD), Parkinson disease (PD), Huntington's disease (HD), amount Frontotemporal dementia (FTD), Friedreich's ataxia, age-related macular degeneration, and Creutzfeldt-Jakob disease. Motor neuron diseases include, but are not limited to, amyotrophic lateral sclerosis (ALS), primary lateral sclerosis (PLS), and spinal muscular atrophy (spinal muscular atrophy). SMA), progressive muscular atrophy (PMA), progressive bulbar palsy (PBP), pseudobulbar palsy, hereditary spastic paraplegia (HSP), library Kugelberg-Welander syndrome, Lou Gehrig's disease, Duchenne's paralysis, Werdnig-Hoffmann disease, and benign focal muscle atrophy ( Benign focal amyotrophy). According to some embodiments of the present disclosure, iENP derived from fibroblasts and cells differentiated from the iENP have pathological features similar to those of a neurological disease; accordingly, the iENP and differentiated cells provide a study for The treatment mode of potential drugs can be used to treat neurological diseases.

Another aspect of the present disclosure is directed to a method of treating an individual suffering from or suspected of suffering from a neurodegenerative disease. The methods of the invention comprise administering to the individual an effective amount of a drug candidate, wherein the drug candidate is screened according to any of the aspects and embodiments of the present disclosure.

The present disclosure further provides methods of treating an individual suffering from or suspected of having a neurodegenerative disease using a kit of the invention (ie, 6TF, 7TF, 13TF, 15TF, or 25TF). The method comprises: (a) isolating a fibroblast from the individual; (b) transferring the polynucleotide of the kit of the invention into the fibroblast to induce the transformation of the fibroblast into an iENP cell; Optionally, the iENP cell of step (b) is cultured in a differentiation culture medium to induce transformation of iENP cells into a stellate cell, an oligodendrocyte or a neuron; (d) the individual is cast An effective amount of iENP cells of step (b), or an effective amount of stellate cells, oligodendrocytes or neurons of step (c), is administered to slow or reduce the symptoms associated with the neurological condition.

In step (a), fibroblasts are isolated from an individual suffering from or suspected of having a neurological disorder. The individual is a mammal; for example, a human, a mouse, a rat, a monkey, a gorilla, a cat or a dog. Preferably, the individual is a human. The neurological disease treatable by the method of the invention is a neurodevelopmental disease, a neurodegenerative disease or a motor neuron disease.

Steps (b) and (c) of the method for treating a neurological disease are the same as steps (a) and (b) of the method for screening a candidate drug, respectively, and are not described herein for brevity.

In step (d), the subject is administered the iENP cells of step (b), or the stellate cells, oligodendrocytes or neurons induced in step (c). Depending on the particular therapeutic effect, the cells can be administered to the subject by any suitable route; for example, enteral, buccal, nasal, non-oral (eg intramuscular, intravenous, intraarterial, subcutaneous, intraperitoneal) , intracerebral, intraventricular or intraspinal injection), local or transmucosal and other routes of administration.

The disclosure also encompasses cells that are transformed by the kits of the invention (ie, 6TF, 7TF, 13TF, 15TF or 25TF), including iENP cells, stellate cells, oligodendrocytes, and neurons. According to some embodiments of the present disclosure, the iENP cells of the present invention can be differentiated into stellate cells, oligodendrocytes, and/or neurons, wherein the neurons can be a central nervous system neuron or a peripheral nervous system neuron. . The transformed cells can be used to treat neurological diseases. According to certain embodiments of the present disclosure, the transformed iENP cells can be administered to an individual suffering from or suspected of having a neurological disorder; in these embodiments, the transformed iENP cells are integrated into the individual. In the central nervous system or peripheral nervous system, and differentiate into stellate cells, oligodendrocytes, and neurons (which may be central nervous system neurons or peripheral nervous system neurons).

Generally, the neurological disease can be caused by physical injury, inflammation, aging or genetic mutation. Preferably, the neurological disease is a neurodevelopmental disease, a neurodegenerative disease or a motor neuron disease. Neurodevelopmental diseases that may be treated by the methods and/or cells of the invention include, but are not limited to, autism spectrum disorders, fetal alcohol syndrome, Down's syndrome, attention deficit hyperactivity disorder, Mendelssler syndrome, schizophrenia (schizophrenia) and fragile X chromosome syndrome. Neurodegenerative diseases include, but are not limited to, Alzheimer's disease, Parkinson's disease, Huntington's disease, frontotemporal dementia, Friedrich's movement disorder, age-related macular Degeneration and CJD. Exemplary motor neuron diseases include, but are not limited to, amyotrophic lateral sclerosis, primary lateral sclerosis, spinal muscular atrophy, progressive muscular atrophy, progressive bulbar palsy, pseudobulbar paralysis, Hereditary spastic paraplegia, KGB-Weland syndrome, Lujarig's disease, Duchenne, Wornick-Hoffman's disease and benign focal muscle atrophy.

In the following, a plurality of experimental examples are set forth to illustrate certain aspects of the present invention, and the present invention is not limited by the scope of the present invention. It is believed that the skilled artisan, after reading the description set forth herein, may fully utilize and practice the invention without undue interpretation. All publications cited herein are hereby incorporated by reference in their entirety. Example

Materials and methods

preparation iENP

By FOXG1 , GATA3 , MBD2 , MYCN (both from transOMIC technologies), SOX2 (FUW-teto-SOX2; Addgene), CBX2 , DACH1 , DEPDC1 , HES1 , ID1 , LHX2 , MYEF2 , NR2F2 , NR6A1 , OTX2a , PAX6a , SALL2 The coding sequences of SIX3 , SOX1 , SOX11 , TFAP2A , ZFP42 , ZIC2 , ZIC3 and ZNF423 (all taken from the cDNA of neural precursor cells derived from human ESC H9) construct a vector containing a candidate neural transcription factor. The coding sequence was transferred to a FUW or FUW-teto vector for subsequent experiments. The 1.3 kb PAX6 P1 promoter and the 1 kb SOX1 promoter were separately transferred to the FUW vector to construct PAX6: EGFP and SOX1: EGFP ; in this experiment, UbC: EGFP was used as a control group. iENP was prepared using lentiviral particles carrying the candidate transcription factors and 293FT cells according to standard preparation procedures. HD fibroblasts infected with CCD112SK foreskin fibroblasts with a lentivirus carrying a candidate transcription factor or reporter gene, isolated from a female patient and a male patient (both with 431 CAG repeat HTT), and isolated from AD The patient (AD1 with APOE4/E4 mutation, AD2 with PSEN1 E184D mutation, and AD3 with PSEN1 P264L mutation, all from the Coriell cell bank) AD fibroblasts, and then cultured the cells in fibroblast culture medium ( DMEM containing 10% fetal bovine serum). One day after infection, the cell culture medium was replaced with a neuro-inducible culture medium (containing N2 complement, 20 μg of bFGF per ml, 1% NEAA, 2 mM glutamic acid, 1 mM sodium pyruvate (Invitrogen), 2 μg per ml) Deoxytetracycline (Sigma), 10 ng of LIF (Invitrogen), 3 μM CHIR99021 (Sigma), and 2 μM SB431542 (R&D) DMEM/F12); the culture was replaced every 2 days. In the administration of small molecule treatment, 10 μM RepSox, 0.1 μM DZNep, 0.1 nM PP242 (Selleckchem) or 50 μg of vitamin C (Sigma) per ml was added to the nerve-inducing medium. One week after induction, cells expressing GFP were purified by BD FASCAria II classifier, planted in matrigel-coated cell culture plates, and iENP medium containing 2 μg of deoxytetracycline per ml (N2B27) was added. : 50% DMEM/F12, 50% Neurobasal, 0.5-fold N2 complement, 0.5-fold B27 complement, 10 μg bFGF per ml, 1% NEAA, 2 mM glutamic acid, 1 mM sodium pyruvate, 10 μL per ml LIF (Invitrogen), 3 μM CHIR99021 (Sigma) and 2 μM SB431542 (R&D)) (Fig. 1B). After 2 or 3 days, the cells spontaneously form a neurogenic globular structure. The neurosphere-like structure was collected and dispersed into single cells by trypsin, and then planted in a cell culture plate coated with ornithine-laminin, and added with 2 μg of deoxyhydroxyl per ml. Tetracycline iENP medium. The efficacy of iENP preparation was evaluated by two parameters: the percentage of cells that exhibited GFP driven by PAX6: EGFP or SOX1: EGFP after 6 days of lentiviral infection, and the percentage of neurosphere formation after 2 days of purification. After 2 to 3 passages of culture, deoxytetracycline was removed from the culture solution and subcultured every 7 days. After cultured in a culture medium containing no deoxytetracycline for 2 generations, RT-PCR analysis was performed to detect the expression of nerve genes, endogenous nerve genes and exogenous genes in iENP, and PCR analysis was used to detect the insertion of exogenous genes. And detecting the expression of neural genes by ICC analysis.

Differentiation and drug testing

Differentiation medium (Neurobasal, B27 complement, 1% NEAA, 2 mM glutamic acid, 1 mM sodium pyruvate, (Invitrogen), 300 μg of dbcAMP per ml, 50 μM ascorbic acid (Sigma), 20 Ng of BDNF per ml, 20 Ng of GDNF per ml, 50 Ng of NGF (Peprotech) per ml to detect general neural differentiation, and cortical neural differentiation medium, dopamine nerve differentiation medium and peripheral nervous system neural differentiation medium (cortical neural differentiation medium) : Neurobasal, N2 complement, B27 complement (Invitrogen), SHH per 100 ng of SHH, Noggin of 125 ng per ml, DKK of 250 ng per ml, BDNF of 10 ng per ml, bFGF (R&D) of 10 ng per ml, 2 μM XAV939, 100 nM LDN93189, 10 μM SB431542, 200 μM ascorbic acid, 200 μM dbcAMP (Sigma); Dopamine neural differentiation medium: DMEM/F12, N2 complement (Invitrogen), 20 Ng of BDNF per ml, 200 Ng per ml SHH, 100 ng of FGF8β (R&D) per ml, 200 μM ascorbic acid (Sigma); peripheral nervous system neural differentiation medium: DMEM/F12, N2 complement (Invitrogen), 3 μM CHIR99021, 10 μM S U5402, 10 μM DAPT, 200 μM dbcAMP) to detect differentiation of specific neurons. In the AD drug test, the iENP of AD and the control group were subjected to cortical differentiation. Seven days after the differentiation, the cells were administered with SB415286, 1-Azakenpaullone (Selleckchem) or DMSO (Sigma) for 2 days. In the HD experiment, after cell differentiation was induced, CGS21680 was administered as described above.

measuring Aβ

AD-iENP and CCD1112sk (CTL)-iENP were seeded in 24-well plates (8x10 ⁵ cells per well) and induced to differentiate into cortical neurons. The culture broth was collected after 20 days and stored at -80 °C for later analysis. Secreted Aβ42 and 40 were measured using Aβ42 and 40 human ELISA kits (KHB3544 and KHB3482, Thermo Fisher Scientific) and Benchmark plus microplate spectrophotometer (BIO-RAD). Each data is the result of three independent experiments.

Electrophysiological analysis

When the electrophysiological analysis results were recorded, neurons derived from iENP were co-cultured with mouse glial cells in neural maturation medium (B27: Neurobasal, B27 complement, 1% NEAA, 2 mM branic acid, 1 mM acetone). Sodium (Invitrogen), 20 ng of BDNF per ml, 20 ng of GDNF per ml, and 50 ng of NGF (Peprotech) per ml were cultured for 2 weeks. Mouse glial cells were isolated from the brain of P1 ICR mice and cultured for more than 3 generations to remove neurons, avoiding subsequent experiments; RT-PCR analysis and ICC analysis were used to detect the expression of Tuj1 mRNA and protein in mice. Quantity to confirm the result. Electrophysiological properties were determined by external cell patch clamp recording at room temperature with an external solution (115 mM NaCl, 2 mM KCl, 10 mM HEPES, 1.5 mM MgCl ₂ , 3 mM CaCl ₂ , 10 mOsm); The tubette was 5-10 MΩ and was filled with an internal solution (130 mM K-gluconate, 10 mM NaCl, 2 mM MgCl ₂ , 10 mM HEPES, 0.5 mM EGTA, 3 mM ATP). TTX (1 μM) in the external solution can be used to block the TTX-sensitive sodium channel. The sealing resistance of the entire cell module exceeds 1 GΩ. The cells were observed with 20 times Olympus BX51WI, water immersion lens and Sony CCD; the whole cell current clamp module was used to record the action potential; and the voltage was clamped with Signal software and Power 1401 (CED) controlled Multiclamp 700B (Molecular Devices). Module to record sodium current. Analyze the results with Microsoft Excel 2010.

Reverse transcription polymerase chain reaction (Reverse transcription-polymerase chain reaction, RT-PCR)

RNA was extracted using TRIzol reagent according to the standard extraction procedure (Molecular Research Center). The extracted RNA was reverse transcribed into cDNA using SuperScript III reverse transcriptase (Invitrogen). 25 ng of cDNA was used for each PCR. RT-PCR analysis was performed using GoTaq Green Master Mix (Promega). Quantitative PCR analysis was performed with SYBR ^® FAST 2x qRT-PCR Master Mix (KAPA) and 7900HT Fast Real-Time PCR System (Applied Biosystems).

Flow cytometry analysis

To assess the percentage of cells that would show GFP, cells were detached and resuspended in PBS. The results were recorded and analyzed using a BD FACSCalibur flow cytometer. The effect of removing a single factor was determined by the proportion of each group normalized in the control group.

Immunocytochemistry (immunocytochemical, ICC) Immunohistochemistry (immnohistochemical, IHC) analysis

ICC analysis was performed in the aforementioned manner. In the IHC analysis of the brain of the transplanted rat, the sample was dehydrated by using 20% sucrose in PBS, and then embedded in an OCT compound (Tissue-Tek). The sections were serially sectioned with a Leica CM3050S Sliding Microtome and each tissue section was 12 microns thick. Tissue sections were fixed with 4% paraformaldehyde for 30 minutes at room temperature and treated with ice methanol for 30 minutes. Analysis was performed using one of the primary and secondary antibody staining listed in Table 1. Cell performance messages were recorded using a Zeiss microscope and a Spot software. Table 1 Antibody primary antibody Secondary antibody

Animal experiment

Long-Evans rats (7-8 weeks old) underwent middle cerebral artery occlusion (MCAO) and common carotid artery occlusion (CCA occlusion) surgery, making them in an ischemic state 30 Minutes, then 50,000 undifferentiated iENP-6F and iENP-7F cells were injected into the rat brain (A/P: 0.3 cm, M/L: -2.0 cm, D/V: -2.8 cm, T/B:- 3.0 cm). After 12 weeks, the rats were sacrificed and fixed with 4% paraformaldehyde dissolved in 0.1 M PB buffer, after which the brain tissue was isolated. All animal experiments in this disclosure are approved by the Animal Care and Use Committee of the Academia Sinica and relevant experiments are performed in accordance with their operational standards.

Analysis of cell proliferation and death

The iENP-6F, iENP-7F, NP and GFP control cells were seeded in 24-well plates and cultured in iENP culture medium without deoxytetracycline. The number of cells was counted on days 1, 2, 3, 4 and 5. The results are expressed as relative amounts relative to the number of cells on day 1. Cell numbers were analyzed using BrdU (93-3943, Thermo Fisher Scientific) and TUNEL (G3250, Promega) assays according to standard protocols. The effects were observed with a Zeiss microscope and Spot software and analyzed with Metamorph software.

Microarray analysis

Total RNA of dermal fibroblasts, CCD112SK foreskin fibroblasts, neural precursor cells derived from human ESC H9, iENP-6F, iENP-7F, iENP-15F and iENP-13F were extracted using TRIzol reagent (Invitrogen). Each cell type was subjected to a second independent analysis. All gene expression results were analyzed by the Affymetrix Gene Expression Service Laboratory of the Academia Sinica. Wafers were scanned with Affymetrix GeneChip Scanner 7G and analyzed with GeneSpring X software (Agilent, Santa Clara, CA, USA). Standardized analysis was performed independently for each experimental data using Robust Multichip Average. Gene expression profiles were analyzed using Genespring software and Ingenuity Pathway Analysis software. The NCBI accession number for the microarray data of the present disclosure is GSE81554.

Example 1 Inducing the conversion of human fibroblasts into iENP

1.1 Screening transcription factor

To screen for potential transcription factors that can be used to prepare iENP, this study first used microarray analysis to compare gene expression profiles of various human ESC-ENP and fibroblast populations (Fig. 1A, panel a). Twenty-four transcription factors were selected, which exhibited higher levels of expression in human ESC-ENP than in fibroblasts (Fig. 1 panel b). NR2F2, which affects neural differentiation, is also selected. The hESC-ENP-based transcription factor is highly expressed in a heterogeneous ENP cell population derived from human ESCs, and the combination of 25 hESC-ENP transcription factors (25 TF) may induce fibroblast differentiation into different types of ENP. . Both PAX6 and SOX1 are known to be expressed in hESC-ENP. Two reporter systems ( PAX6: EGFP and SOX1: EGFP ) were established to detect neuronal transformation and evaluate the eliciting effect of ENP. The results of Figures 8A and 8B confirm that these reporters can behave in hESC-ENP.

To establish iENP, fibroblasts were subsequently co-infected with a lentivirus that encodes the above 25 transcription factors and a neural reporter (ie, PAX6: EGFP or SOX1: EGFP ) (Fig. 1B). After about 6 days of infection with lentivirus, PAX6:EGFP ⁺ cells with a rounded appearance were observed, while no change in appearance was observed in control fibroblasts infected with UbC:EGFP (Fig. 1C). Similar results were observed for fibroblasts infected with 25 transcription factors and SOX1:EGFP (Fig. 1C). The cell proportions of PAX6:EGFP ⁺ and SOX1:EGFP ⁺ were 5.31±0.38% and 6.31±0.45%, respectively (Fig. 2D and 4D). After purification and culture of PAX6:EGFP ⁺ or SOX1:EGFP ⁺ cells by FACS (Fig. 1B), the purified cells will spontaneously form neuron-like structures on the 2nd day after re-replantation (Fig. 1C) However, no neurogenic globular structure was observed in the control cells (Fig. 1C). Different analytical methods were then used to confirm the relevant properties of the putative iENP (iENP-25F) prepared with 25 transcription factors. Using ICC and RT-PCR analysis, it was found that PAX6:EGFP- and SOX1 : EGFP- iENP-25F exhibited general neuromarkers (eg, nestin, OTX2 and ZO1, Figure 1D), and neural genes (Fig. 1E). ).

To reduce the number of transcription factors required for the preparation of iENP (Figures 2A and 4A), each transcription factor was removed from each of the 25 transcription factors and reported with the remaining 24 transcription factors and PAX6:EGFP or SOX1:EGFP The daughter is co-infected with fibroblasts, and the transcription factor that induces iENP production is screened in two steps. Flow cytometry was then used to assess the effect of the removed transcription factors on the induction of PAX6:EGFP ⁺ or SOX1:EGFP ⁺ cells (Figures 2B and 4B). Based on this, 15 transcription factors ( CBX2 , DACH1 , FOXG1 , HES1 , ID1 , MYCN , NR2F2 , NR6A1 , SOX2 , SOX11 , TFAP2A , ZFP42 , ZIC2 , ZIC3 , ZNF423 ) and 13 transcription factors ( CBX2 , FOXG1 , GATA3) were identified . , HES1 , LHX2 , NR2F2 , NR6A1 , PAX6 , SALL2 , SOX11 , TFAP2A , ZFP42 , ZIC2 ); removal of these transcription factors significantly reduces PAX6:EGFP or compared to the number of cells induced by 25 transcription factors SOX1: production of EGFP ⁺ cells (Figs. 2B and 4B). To determine whether a smaller amount of a combination of transcription factors can be used to induce the conversion of fibroblasts to iENP, in a deoxytetracycline-inducible expression system, 15 or 13 transcription factors (15 TF or 13) that can be screened can be encoded. TF) lentivirus infects fibroblasts. After purification of PAX6:EGFP ⁺ or SOX1:EGFP ⁺ cells by FACS, it was confirmed that similar to iENP-25F, iENP-15F and iENP-13F could spontaneously form neuron-like structures; ICC and RT-PCR analysis showed These cells all express neuromarkers and genes (Fig. 9A-9B). From the results of PCR and RT-PCR analysis, it was revealed that the exogenous transgenic gene was integrated into the genomic DNA, and the expression of the endogenous ENP gene was activated after removal of deoxytetracycline (Fig. 9C-9D). . Further microarray results showed that the gene expression profiles of iENP-15F and iENP-13F were more similar to hESC-ENP than the native fibroblasts (Figs. 2E and 4E). More importantly, in vitro differentiation of iENP-15F and -13F confirmed that it spontaneously produced TUJ1 ⁺ neurons, GFAP ⁺ stellate cells, and GALC ⁺ oligodendrocytes (Fig. 9E). These results indicate that iENP-15F and iENP-13F have the general characteristics of NP and can form the main components of the human nervous system.

To determine the minimum number of transcription factors required to prepare iENP, another round of transcription factor screening (steps similar to those described above) was performed to select the most promising transcription factors for iENP production (2C and 4C). ). After the second round of transcription factor screening, the study found that CBX2 , HES1 , ID1 , TFAP2A , ZFP42 or ZNF423 (6F) and FOXG1 , GATA3 , NR2A2 , PAX6 , SALL2 , TFAP2A or ZFP42 were removed from the 15 TF and 13 TF combinations , respectively . (7F) significantly reduced the production of PAX6:EGFP ⁺ or SOX1:EGFP ⁺ cells (Fig. 2C and 4C). After infection with the above 6 TF or 7 TF, 10.54 ± 0.47% of PAX6: EGFP ⁺ cells and 11.22 ± 0.44% of SOX1: EGFP ⁺ cells (2D and 4D) were purified via FACS. Similar to the results of iENP-25F, -15F and -13F (Figures 1C and 9A), iENP-6F and iENP-7F isolated by FACS can also spontaneously form neurosphere-like structures (Fig. 2F and 4F). . It is worth noting that removal of either factor from the 6- or 7-TF combination significantly affects the production of PAX6:EGFP ⁺ or SOX1:EGFP ⁺ cells (Figures 11A-11B), as well as neuronal globular structures. Formation (Fig. 11C). Overall, these results indicate that in the 6- and 7-TF combinations, each transcription factor plays a key role in iENP production. In addition, PCR analysis confirmed the integration of exogenous transgenic genes in iENP-6F and iENP-7F gene DNA (Fig. 9C); RT-PCR analysis showed that removal of deoxytetracycline completely inhibited iENP-6F And the expression of the -7F exogenous transgenic gene, and activation of the corresponding endogenous gene performance (2G and 4G map). Gene expression analysis by GeneSpring software showed that the gene expression profiles of iENP-6F and -7F were more similar to those of hESC-ENP compared to their native fibroblasts (Figs. 2E and 4E), ICC and RT- PCR analysis also indicated that these cells exhibited the markers and genes of ENP (Figs. 2F, 2H, 4F and 4H). In addition, iENP-6F and -7F can maintain a normal karyotype (Fig. 9A) after subculture for more than 20 generations, and after being frozen and thawed, they can further proliferate without losing their NP characteristics. Overall, these results confirm that iENP-6F and -7F have similar cell morphology, biochemical and molecular properties to hESC-ENP.

1.2 Small molecule promotion iENP Generation

Based on several studies indicating that small molecules can increase the reorganization efficiency of different cell systems, this study will explore whether small molecule treatment can further improve the efficacy of iENP preparation. This study selected a series of candidate small molecules including TGF-β inhibitors, RepSox, autophagy activators, PP242, histone methyltransferase inhibitors, DZNep, DNA demethyl activators and vitamin C. These small molecules can promote pluripotent reforming or cell transformation. Accordingly, after infection of the fibroblasts with a virus encoding a 6- or 7-TF combination and a PAX6: EGFP or SOX1: EGFP reporter, the small molecules are administered to the cells either alone or in combination. FACS was used to analyze cells that would exhibit PAX6: EGFP or SOX1: EGFP , and then to assess the effect of single or multiple small molecules on iENP production (Figures 12A and 12B). Overall, the results indicate that administration of RepSox or RepSox plus PP242 significantly improved the efficacy of preparation of iENP-6F or iENP-7F (Figures 12A and 12B). Therefore, these results confirm that small molecules can increase the role of transcription factors and induce the conversion of fibroblasts into iENP.

Example 2 iENP In vitro pluripotency

Given that functional ENP can differentiate into stellate cells, oligodendrocytes, and neurons, this study then examined the ability of iENP to differentiate in vitro (Figures 3 and 5). 2-3 weeks after differentiation, GFAP ⁺ and GALC ⁺ cells can be observed in differentiated iENP-6F (Fig. 3A-3D) and -7F cells (Fig. 5A-5C) under conditions of neural differentiation. A large number of neuron-like cells (having medullary processes and exhibiting neuronal markers such as MAP2, NEUN, and TUJ1). It is worth noting that the synaptophysin (SYP) is also expressed together with the mature neuronal marker-NFH (Fig. 3E). In addition, ICC was used to quantitatively analyze the proportion of cells expressing TUJ1, GFAP and GALC in differentiated iENP. The results showed that the neuronal differentiation ability of iENP-6F was similar to that of hESC-ENP; whereas compared with hESC-ENP and iENP-6F, iENP- 15F has a weaker ability to produce neurons. This result demonstrates that removal of 9 transcription factors from the 15 TF combination further increases the neuronal propensity of iENP-6F (Fig. 3F). The differentiation ability of iENP-6F and -15F stellate cells and oligodendrocytes was significantly lower than that of human ESC-ENP stellate cells and oligodendrocytes (Fig. 3F), compared with iENP-6F. iENP-15F has a lower ability to produce oligodendrocytes (Fig. 3F). On the other hand, iENP-7F and -13F have similar neuronal differentiation capabilities, but their neural differentiation ability is lower than that of hESC-ENP (Fig. 5D). Both iENP-7F and -13F have lower ability to produce stellate cells and oligodendrocytes compared to hESC-ENP (Fig. 5D).

To determine whether iENP can differentiate into different neurons, different neuronal markers are then used to detect the iENP-6F and -7F neuronal cell populations (3G-3M and 5E-5J). ICC results showed that both iENP-6F and -7F produced different neuronal subtypes, including GABA ⁺ (3G and 5E maps), TBR1 ⁺ cortical neurons (3H and 5F maps), and TH ⁺ dopamine neurons ( 3I and 5K-(a)), HB9 ⁺ /ISL1 ⁺ motor neurons (3J, 3K, and 5H) and BRN3A ⁺ , PRPH ⁺ or NAV1.7 ⁺ peripheral neurons (3L, 3M, and 5I) -J map). Based on the ability of external stimulation to induce hESC-ENP to differentiate into specific neuronal subtypes, further testing of iENP in this study can also produce similar responses. Accordingly, iENP (Fig. 3N and 5K) was treated with differentiation conditions for preparing cortex, dopamine, and peripheral neurons. ICC analysis using anti-TBR1, TH or PRPH antibodies indicated that specific neuronal differentiation conditions significantly improved the ability of iENP to produce representative neuronal subtypes (cortex, dopamine or peripheral neurons) (Figs. 3N and 5K). These results confirm that iENP is pluripotent and produces a similar response to hESC-ENP for specific differentiation conditions.

Neurons derived from iENP are then tested for functional electrophysiological properties similar to neurons. Neurons derived from iENP were cultured in neurogenic medium for 2 weeks, and then subjected to whole-cell patch-clamp recording analysis. The results showed that the resting membrane potential of neurons derived from iENP-6F was -35.25±0.64 mV (3O- (a) Figure), while the resting membrane potential of neurons derived from iENP-7F is -64.3 ± 17.96 mV (Fig. 5L-(a)). In the current clamp module, the cell membrane is depolarized to induce action potentials (Fig. 3O-(b); and 5L-(b)), and spontaneous action potentials derived from iENP neurons are recorded (5L- (c) Figure). The inward current from the sodium channel media is blocked by TTX (an inhibitor of specificity for Na ⁺ ion channels) (Fig. 3O-(c) and 5L-(d)). These results indicate that neurons derived from iENP have functional electrophysiological properties similar to those of neurons.

Example 3 Transplanted iENP Integrates and differentiates into the rat brain

To analyze the differentiation ability of iENP in vivo, in this example, iENP was transplanted into the corpus callosum of the rat brain, and correlation analysis was performed after 12 weeks (Fig. 3P and 5M). First, it is tested whether iENP transplantation will cause tumor formation in the brain. H&E of brain sections and further RT-PCR and IHC analysis showed that after 12 weeks of transplantation, the expression of tumor-associated markers was not observed in the iENP-transplanted brain (Fig. 10). Interestingly, the study found that some of the transplanted cells metastasized to the ventricular zone (the neurogenic brain region, which expresses the radial nerve precursor cell marker - GFAP) (Fig. 3P-(a) and (b); and 5M- (a) and (b) map). Consistent with the results of in vitro differentiation, the transplanted iENP was partially localized into GFAP ⁺ stellate cells (3P-(c) and 5M-(c)), NG2 ⁺ oligodendrocytes (3P-(d) And (e) map; and 5M-(d) and (e) maps, and TUJ1 ⁺ or MAP2 ⁺ neurons (3P-(f) map and 3P-(i) map). Overall, these results indicate that iENP will integrate into rat brain tissue in vivo and differentiate into major neuronal cell types.

Example 4 iENP-6F and -7F Cell populations have different developmental tendencies

As mentioned above, iENP-6F and -7F have different propensity for neural differentiation. Based on this, the differences in the two cell population will be further explored. Microarray was first used to analyze the gene expression profile of the two iENP cell populations. The hot zone map results indicate that iENP-6F and -7F have similar gene expression profiles (Fig. 6A-(a)). By IPA analysis and detection of fold change and gene ontology, it was found that there were significant differences in the performance of 170 genes between iENP-6F and -7F (≥2 fold, Figure 6A-(b)). Among them, several genes associated with cell cycle and division have lower performance in iENP-7F than in iENP-6F (Fig. 6B-(a)); IPA analysis shows that compared to iENP-6F The message transmission pathway associated with cell death in iENP-7F is activated (Fig. 6B-(b)). Consistently, the growth curve of iENP-6F is similar to that of human ESC-ENP, while iENP-7F has a slower growth rate (Fig. 6B-(c)). Further analysis showed that iENP-6F had more BrdU ⁺ cell ratio and lower TUNEL ⁺ cell ratio than the iENP-7F (Fig. 6B-(d)).

To investigate the developmental propensities of iENP-6F and -7F, the expression of different regional markers in these cells was then examined (Fig. 6C-(a)). ICC was used to analyze the undifferentiated iENP cell population and its derived neurons (iENP-N). The results indicated that the iENP/iENP-N ratio of BF1 (forebrain marker) in iENP-6F/-N was significantly higher than that of iENP. -7F/-N; compared to iENP-7F/-N, iENP-6F/-N has lower performance of PITX3 (middle brain marker), HOXB4 (postbrain marker) and p75 or BRN3A (peripheral nervous system) The cell ratio of the marker) (Fig. 6C-(b) and Fig. 6D). Consistent with the ICC results, the comprehensive gene expression profiling and RT-qPCR analysis of iENP-6F and 7F indicated that the iENP-6F biased performance was related to the forebrain, midbrain and spinal cord compared to iENP-7F. Genes; compared to iENP-6F, iENP-7F is more biased to genes associated with the hindbrain and peripheral nervous system (Figures 6E and 6F). Overall, these results indicate that iENP-6F and -7F are different NP subpopulations with different neurogenic manifestations, growth rates, and developmental propensities.

Example 5 Ill state iENP Recurring pathological features

To explore the potential of iENP in establishing disease patterns, followed by an AD patient with ADOE4/E4 mutation (AD1), two AD patients with PSEN1 mutation (fAD, AD2 and AD3), and two HD patients (Male and female, having 41 CAG repeats in the HTT gene) were isolated from fibroblasts to prepare iENP. Similar to wild-type fibroblasts, after administration of a combination of 6-TF or 7-TF, AD- and HD-fibroblasts can be transformed into PAX6:EGFP- or SOX1:EGFP ⁺ cells (Fig. 13A), and These cell populations form neuron-like globular structures and exhibit NP markers/genes (Figs. 7A and 13B-13C). In addition, these putative AD- and HD-iENP can produce TUJ1 ⁺ neurons, GFAP ⁺ stellate cells, and GALC ⁺ oligodendrocytes (Fig. 7B).

Thereafter, it will be examined whether AD- and HD-iENP and the neurons they produce have pathological features of the disease in question. Neurons based on AD patients have higher levels of amyloid β (Aβ) and accumulate phosphorylated TAU protein (pTAU), first conditioned medium of neurons differentiated from AD- or control-iENP Among them, the extracellular expression of Aβ40 and Aβ42 was measured. The ELISA results showed that the two Aβ isoforms were produced by AD-iENP derived from two fAD-female cell populations with PSEN1 mutations (AD2 and AD3) compared to the control-iENP-derived neurons. There is a significantly higher amount of expression in neurons (Fig. 7C). In neurons derived from fAD-iENP (transformed from FAD-fibroblasts with PSEN1 E184D mutation (AD2)), Aβ42/Aβ40 also has a higher expression ratio; however, it is derived from fAD-iENP ( In neurons that were transformed from FAD-fibroblasts with the PSEN1 P264L mutation (AD3), a significantly increased Aβ42/Aβ40 ratio was not observed; previous studies have indicated that in cells that overexpress PSEN1 P264L, The Aβ42/Aβ40 ratio will increase slightly. To investigate the pathological properties of pTAU in neurons derived from AD-iENP, ICC analysis of neurons derived from AD1- and control-iENP was performed with antibodies recognizing pTAU (AT8); in some TUJ1 ⁺ neurons The expression of pTAU was observed in the cellular response, and the massive pTAU mainly accumulated in the cell body of AD-iENP-derived neurons, in the cortex of AD patients and neurons derived from AD-iPS. Similar results were observed (Fig. 7D-(a)). In addition, administration of GSK3β inhibitors to neurons derived from AD-iENP compared to DMSO-treated and control-iENP-derived neurons (SB415286 and 1-Aza significantly reduced aggregation of pTAU (Fig. 7D) ).

Previous studies have indicated that DNA derived from HD-iPSC-derived neurons is susceptible to damage, and that administration of specific agonists to neurons derived from HD-iPSC to stimulate A _2A R reduces the extent/degree of DNA damage. To confirm whether HD-iENP and its neuron-derived cells can reproduce the pathological characteristics of HD, a specific A _2A R agonist, CGS21680, was administered to HD-iENP and control-iENP. Analysis of the expression of phosphorylated γH2AX (a marker of DNA damage) by ICC showed that HD-iENP and its neuron-derived cells had significantly higher amounts of γH2AX ⁺ nuclei compared to the control cell group (Fig. 7E). . In addition, CGS21680 stimulation significantly reduced the performance of gH2AX in HD-iENP and its neuron-derived cells, suggesting that activation of A _2A R can reduce DNA damage in these cells (Sections 7E-(b) and 7E-(d) )))). Overall, the above results indicate that pathological iENP and its neuron-derived cells can reproduce the pathological characteristics of AD and HD.

It is known that fibroblasts can be directly converted to iNP using different combinations of transcription factors. These iNPs have the general characteristics of neuronal pre-existing cells, such as neuronal markers/gene expression, proliferation and differentiation propensity. Unlike hESC-ENP, which can differentiate into the central nervous system and peripheral nervous system neuronal systems, iNP has a developmental potential for central nervous system subtypes. However, previous studies have focused less on whether these iNPs have the potential to develop into neuronal subtypes of the peripheral nervous system. This study confirmed that iENP-6F and iENP-7F can differentiate into central nervous system and peripheral nervous system neuron. Furthermore, similar to hESC-ENP, these cells respond to the same extracellular stimuli and produce specific neuronal subtypes. In these cells, the total genomic total transcript map also confirmed that the ENP induced by fibroblasts and their neurons derived from human ESC are highly similar. Accordingly, this study demonstrates that the iENP cell population reformed by the hESC-ENP transcription factor has a higher similarity to the embryonic NPC than the NPC derived from the adult brain.

Even though the two iENP cell populations of the present disclosure have similar NP characteristics, further analysis shows that the cell populations have different functional properties. First, the analysis confirmed that iENP-6F has a higher proliferative capacity and exhibits less apoptotic response than iENP-7F. Second, iENP-7F has a higher differentiation potential for neuronal lines than glial cell lines. Third, analysis of the neural differentiation potential of the iENP cell population revealed that iENP-7F has a regional preference for terminal neurons, while iENP-6F has a regional preference for frontal neurons. Transcription factors and neuronal reporters of iENP can be screened to account for the differences between iENP-6F and 7F. The present disclosure utilizes two neuronal reporters, PAX6 and SOX1, to detect and evaluate the neuronal transcriptional effects of hESC-ENP on neurons, thereby confirming that the 6- and 7-TF combinations can be used to induce iENP-, respectively. Production of 6F and -7F. On the other hand, PAX6 and SOX1 can also be used to screen iENP-6F and 7F cell populations. Therefore, screening through these neuronal reporters appears to determine the functional properties of the resulting iENP cell population. It is known that neural rosettes and neuroepithelial layers derived from human ESC are composed of different ENSC/ENP, which are involved in the neural development of the subsequent central and peripheral nervous systems. Therefore, 25 of the previously screened neuronal transcription factors that are highly expressed in hESC-ENP are likely to play a key role in the formation of heterogeneous NP cell populations. Accordingly, a specific neurological transcription factor combination was selected from the 25 transcription factors to induce fibroblast expression, and an iENP cell population having specific neuronal characteristics should be prepared. Collectively, the results indicate that the procedures set forth in this disclosure provide a related art method for efficiently preparing specific iENP cell populations by screening specific transcription factor combinations from 25 transcription factors, as well as using different neuronal reporters. A method for preparing a population of iENP cells. As to whether a specific hESC-ENP neurotransitor combination can be used to define the functional aspect of iENP, and the combination of transcription factors to promote the transformation of fibroblasts into iENP, further experiments are needed to clarify.

It is known that a combination of transcription factors (including all iPSC factors, only certain factors or only a single factor) can directly convert human or mouse fibroblasts into iNPs. Among the 25 transcription factors, the combination of the two transcription factors, -6 TF and 7 TF, can be further confirmed to induce the transformation of fibroblasts into iENP, respectively. Overall, the functional aspects of the transcription factors used to induce the production of iENP are associated with maintenance of neural development or nerve cells. In the 6-TF combination, these transcription factors are mainly involved in the differentiation of neurons and maintenance of NSC. In the 7-TF combination, most transcription factors are associated with early development of the central nervous system and peripheral nervous system, as well as early neural specificity. Unlike most transcription factor sets used to prepare iNPs, the function of the transcription factors of the present disclosure is independent of the production of human iPSCs, which shows that the process of inducing the conversion of fibroblasts into iENP does not require iPSC factors. Therefore, this result excludes the possibility of iENP production by the transient pluripotent state, thereby avoiding the canceration probability associated with the iPSC factor. It is worth noting that the two transcription factor combinations contain two transcription factors - TFAP2A and ZFP42/REX1 . It is known that TFAP2A is involved in the development of different tissues during embryonic development, especially neurodevelopment. ZFP42/REX1 will be expressed in ESC and NP, but it is not necessary for pluripotency in mice. However, infection of cells with lentiviruses encoding TFAP2A and ZFP42 did not produce iENP (results not shown), indicating that it is an indispensable factor in inducing iENP production, but alone is not sufficient to promote iENP production.

The iNP provides a cell-based platform for establishing neurodegenerative disease patterns and drug development. Accordingly, the present disclosure induces the preparation of iENP from fibroblasts of AD and HD patients, and demonstrates that the pathological iENP and its neuron-derived cells have pathological properties of HD and AD. For example, the examples of the present disclosure indicate that the proportion of Aβ variants and Aβ42/Aβ40 is rapidly increased in neurons derived from AD-iENP, and the expression of pTAU is also increased; administration of GSK3β inhibitors may be Decreasing the performance of pTAU, showing that neurons derived from AD-iENP reproduce at least part of the pathological features of AD. Pressure factors are known to cause DNA damage and increase neuronal cell death from HD patients. In addition, previous reports have also indicated that the A _2A R agonist can produce a beneficial effect in the HD transgenic animal model and the neuronal cell population derived from HD-iPSC. In view of this, the present disclosure demonstrates that DNA of HD-iENP and its neuron-derived cells is more susceptible to damage than iENP produced by healthy fibroblasts and its neuron-derived cells. In addition, administration of CGS21680 reduced DNA damage in HD-iENP and its neuron-derived cells. Collectively, these results indicate that to some extent, the iENP model of the present invention reproduces the pathological features associated with neurodegenerative diseases, thereby identifying relevant pathogenic mechanisms and screening novel therapeutic agents.

By transplanting iENP in vivo into the rat brain, it was confirmed that iENP can survive in the brain of mature individuals and differentiate into different neuronal subtypes. This result confirms that similar to hESC-ENP, iENP also has an in vivo differentiation tendency, and it is confirmed that the iENP established by the present invention can be used as an autologous cell source for treating neurodegenerative diseases such as AD and HD. However, even though the present disclosure has confirmed that no tumor formation was observed in the rat brain after 12 weeks of transplantation, the possibility of iENP canceration in the brain needs further confirmation.

Summarizing the above, the present disclosure demonstrates a method for directly converting human somatic cells into pluripotent iENP by overexpressing transcription factors enriched by human ESC-NP. The system of the present disclosure can be used to prepare a population of proliferative iNP cells (Fig. 6G) with a specific tendency to differentiate nerves, thereby exploring/developing novel mechanisms and therapeutic agents for neurogenic diseases.

Although the embodiments of the present invention are disclosed in the above embodiments, the present invention is not intended to limit the invention, and the present invention may be practiced without departing from the spirit and scope of the invention. Various changes and modifications may be made thereto, and the scope of the invention is defined by the scope of the appended claims.

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To make the above and other objects, features, advantages and embodiments can be more fully understood by reading the following description of the accompanying drawings: FIG 1A-1E using the first volume will show in hESC-ENP (hESC-ENP) 25 neuronal transcription factors to induce the conversion of human fibroblasts into iENP. Figure 1A: Confirmation of the neuronal transcription factors enriched by hESC-ENP by aligning the gene expression profiles of fibroblasts and hESC-ENP. (a) hESC-ENP (NP1: E-MEXP-2668, ArrayExpress database; NP2: ND collected by H9- SOX1: EGFP , day 18-NP) and fibroblasts (FB1, FB2 and FB3) The results of the hot zone map analysis of the gene expression profile. (b) Screening has a higher expression transcription factor in human ESC-ENP than fibroblasts. Figure 1B: Schematic diagram of an experimental strategy for directly converting fibroblasts into iENP. Figure 1C: Growth of cells collected by FACS after infection of fibroblasts with lentiviruses and neural reporters encoding the hESC-ENP transcription factor (25TF). UbC:EGFP- infected cells were used as a control group. Panel 1D: ICC staining with antibodies against specific antigens and analysis of iENP-25F cells similar to NP cell clustering/globular structure. Figure 1E: Analysis of the performance of specific genes in iENP-25F by RT-PCR. FB: fibroblast; NC: negative control (water). See also Figures 8A-8B. 2A-2H of FIG using PAX6: EGFP neural reported After 6 by transcription factors, transcription factors with which these induced fibroblasts (FB) into iENP. Figure 2A: Schematic diagram of an experimental strategy using the neural reporter PAX6:EGFP to reduce the number of transcription factors required to induce iENP (from 25 to 6). 2B and 2C: A single transcription factor was removed from the original 25-TF panel (Fig. 2B) and the 15-TF panel (Fig. 2C) to gradually screen for potential iENP factors for the preparation of iENP-6F. After removal of a single transcription factor from a combination of 25-TF or 15-TF, the results are expressed as the relative percentage of PAX6:EGFP ⁺ cells. Figure 2D: Comparison of the effect of 25-, 15- and 6-TF combinations on the induction of transformation of fibroblasts into PAX6:EGFP ⁺ cells. Figure 2E: Microarray analysis to determine the comprehensive gene expression thermogram of fibroblasts, hESC-ENP, iENP-6F and iENP-15F. Figure 2F: ICC staining analysis of iENPs-6F with antibodies against specific NP markers. Figure 2G: RT-PCR assay using mRNA extracted from iENP-6F to analyze endogenous and exogenous performance of 6 TF. Figure 2H: RT-PCR assays were performed using mRNA extracted from iENP-6F to analyze the performance of specific neural genes. FB: fibroblast; NC: negative control (water); plastid: the plastid of a specific gene. All quantitative data were obtained from three independent experiments and expressed as mean ± standard deviation. See also Figure 9-11. 3A-3P of FIG iENP-6F in vitro and in vivo pluripotency. Figure 3A-3E: Using anti-glial cell marker GFAP (Fig. 3A), oligodendrocyte marker GALC (Fig. 3B), specific neuronal markers (3C and 3D), and synaptic marker SYN (3E) The antibody of Figure) was subjected to ICC staining of differentiated iENP-6F. Figure 3F: Quantitative analysis and performance of TUJ1 ⁺ , GFAP ⁺ and GALC ⁺ cells in differentiated human ESC-NP (control group NPC), iENP-6F and iENP-15F. Figure 3G-3M: ICC staining of differentiated iENP-6F using antibodies against specific central nervous system and peripheral nervous system neuroantigens. Figure 3N: Specific stimuli can cause iENP-6F to produce a specific neuronal subtype. (a) Schematic diagram of the experimental procedure for inducing iENP-6F to produce a specific neuronal subtype. (b) ICC staining was performed using an antibody against a specific central nervous system and a neuropeptide of the peripheral nervous system to confirm the state of the differentiated iENP-6F under specific neuronal subtype differentiation conditions. (c) Quantitative analysis of specific neuronal subtypes induced by the conditions described in Figure 3N-(a). GF ^- : no inducer. GF ⁺ has an inducer; Figure 3O: Whole-cell patch-clamp recording of neurons derived from iENP-6F. (a) Current recording of neurons after 4 to 6 weeks of differentiation. (b) The action potential is induced by a current order of -50 to +120 pA. (c) Initiating internal Na ⁺ current and external Ca ²⁺ current at a voltage step of -40 to +50 mV. Tetrodotoxin (TTX) was used to block internal Na ⁺ currents. Figure 3P: In vivo transplantation of iENP-6F. (a) IHC staining using an antibody against human nuclear antigen (HuNu) to analyze a steroid containing the iENP-6F graft. (bi) IHC staining analysis of brain sections after 12 weeks of transplantation using antibodies against HuNu, Stem121 or specific neuroantigens. (j) Relative position of specific cells after transplantation. Scale bar = 10 microns (10 μm). All quantitative data were obtained from three independent experiments and expressed as mean ± standard deviation. See also Figure 10. FIGS. 4A-4H use of SOX1: EGFP neural reported After screening 7 kinds of transcription factors, transcription factors with which these induced into fibroblasts iENP. Figure 4A: Schematic diagram of an experimental strategy using the neural reporter SOX1:EGFP to reduce the number of transcription factors required to induce iENP (from 25 to 7). Figures 4B and 4C: A single transcription factor was removed from the original 25-TF panel (Fig. 4B) and the 13-TF panel (Fig. 4C), and the potential iENP factors used to prepare iENP-7F were screened step by step. After removing a single transcription factor from the TF combination, the results are expressed as the relative percentage of SOX1:EGFP ⁺ cells. Figure 4D: Comparison of the effect of 25-, 13- and 7-TF combination on the induction of transformation of fibroblasts into SOX1:EGFP ⁺ cells. Figure 4E: Microarray analysis to determine the comprehensive gene expression thermogram of fibroblasts, hESC-ENP, iENP-7F and iENP-13F. Figure 4F: ICC staining of iENPs-7F with antibodies against specific NP markers. Figure 4G: RT-PCR assays using mRNA extracted from iENP-7F to analyze the endogenous and exogenous manifestations of the seven transcription factors. Figure 4H: RT-PCR assays were performed on mRNA extracted from iENP-7F to analyze the performance of specific neural genes. FB: fibroblast; NC: negative control (water); plastid: the plastid of a specific gene. All quantitative data were obtained from three independent experiments and expressed as mean ± standard deviation. See also Figure 9-11. FIGS. 5A-5M of the differentiated iENP-7F pluripotency in vitro and in vivo. Figure 5A-5C: The differentiated iENP-7F was performed using antibodies against the glial cell marker GFAP (Fig. 5A), the oligodendrocyte marker GALC (Fig. 5B), and specific neuronal markers (Fig. 5C). ICC staining. Figure 5D: Quantitative analysis and performance of TUJ1 ⁺ , GFAP ⁺ and GALC ⁺ cells in differentiated human ESC-NP (control group NPC), iENP-7F and iENP-13F. 5E-5J: ICC staining of differentiated iENP-7F using antibodies against specific central nervous system and peripheral nervous system neuroantigens. Figure 5K: Specific stimuli can cause iENP-7F to produce a specific neuronal subtype. (a) ICC staining was performed using an antibody against a specific central nervous system and a neuropeptide of the peripheral nervous system to confirm the state of differentiated iENP-7F under specific neuronal subtype differentiation conditions. (b) Quantitative analysis of iENP-7F-derived neuronal subtypes induced by the conditions described in Figure 3N-(a). GF ^- : no inducer; GF ⁺ has an inducer. Figure 5L: Whole cell patch clamp recording of neurons derived from iENP-7F. (a) Current recording of neurons after 4 to 6 weeks of differentiation. (b) The action potential is induced by a current order of -80 to +60 pA. (c) Recording a spontaneously acting action potential with a subthreshold oscillatory potential of -40 mV. (d) Initiating internal Na ⁺ current and external Ca ²⁺ current at a voltage step of -40 to +50 mV. TTX is used to block internal Na ⁺ current. Figure 5M: In vivo transplantation of iENP-7F. (a) IHC staining of steroids containing iENP-7F grafts using anti-HuNu antibodies indicated that iENP would be transferred to the ventricular zone. (bi) IHC staining analysis of brain sections after 12 weeks of transplantation using antibodies against HuNu, Stem121 or specific neuroantigens. Scale bar = 10 microns (10 μm). All quantitative data were obtained from three independent experiments and expressed as mean ± standard deviation. See also Figure 10. FIGS. 6A-6G first iENP-6F and the difference of iENP-7F. Figure 6A-(a) and (b): Thermographic plot analysis of the comprehensive gene expression profiles of undifferentiated iENP-6F, iENP-7F and fibroblasts. Figure 6B: (a) Confirmation of dynamic changes in gene expression with specific GO. Red: increased performance; blue: reduced performance; (b) IPA analysis of activation pathways associated with cell death. (c) Growth curve analysis of specific cell populations. (d) ICC staining and quantitative analysis of iENP by BrdU and TUNEL tests. The nuclei were stained with DAPI (blue). Figure 6C: iENP preferences show signs of the brain area. (a) ICC staining of iENP with antibodies against specific brain region antigens. (b) Quantitative analysis of the percentage of cells in iENP that would be indicative of a particular brain region marker. Figure 6D: Quantitative analysis of the percentage of cells that express a particular brain region marker in neurons derived from iENP. Figure 6E: A pie chart illustrating the genes associated with brain region subtypes that increase and decrease between iENP-7F and -6F. Figure 6F: Measurement of relative expression of genes associated with brain regions in iENP-7F and -6F using RT-qPCR analysis. Figure 6G: Schematic diagram of a strategy for direct transformation of iENP with different differentiation propensities using different combinations of hESC-ENP transcription factors and neural reporters. FB: forebrain; MB: midbrain; HB: hindbrain; SC: spine. All quantitative data were obtained from three independent experiments and expressed as mean ± standard deviation. Figure 7A-7E Figure characterization of pathological iENP and its neuron-derived cells. Figure 7A: (a) AD-iENP and (b) HD-iENP appearance and ICC staining representative map, wherein the ICC is analyzed for the performance of nestin (Nestin). Figure 7B: Phase difference photographs of neurons derived from AD-iENP (a) and neurons derived from HD-iENP (b), and AD-iENP-derived cells using anti-GFAP, GALC and TUJ1 antibodies (a) And ICC staining results of HD-iENP-derived cells (b). Figure 7C-(a)-(c): Secreted Aβ42/40 ratio; Aβ42 and Aβ40 derived from AD-iENP neurons. AD2 and AD3: Patients with a PSEN1 mutation. Figure 7D: (a) The expression of pTAU in AD-iENP-derived neurons was analyzed by ICC staining using antibodies against TUJ1 and pTAU (AT8). (b) Quantitative analysis of the efficacy of 1-Aza and SB415286 in reducing pTAU expression in neurons derived from AD-iENP. AD1: A patient with an APOE4/E4 mutation. The control group was treated with DNSO. Figure 7E: ICC staining (a) and quantitative analysis (b) were used to detect the expression of γH2AX ⁺ cells in control and HD-iENP treated with control vehicle (DMSO)- and CGS 21680. (c) ICC staining and (d) quantitative analysis in the control vehicle (DMSO)- and CGS 21680-treated controls and HD-iENP-derived neurons, all quantitative data were obtained from γH2AX ⁺ cells. Three independent tests were performed and expressed as mean ± standard deviation. See also Figures 13A-13C. The first Figure 8A-8B construct and confirm PAX6: EGFP and SOX1: EGFP reporter is nervous. Figure 8A: Schematic representation of lentiviral construct - PAX6: EGFP and SOX1: EGFP . Figure 8B: ICC staining analysis of human ESC-derived neural rosettes (Day 20) transfected with PAX6: EGFP- or SOX1: EGFP- , respectively, with antibodies against GFP and PAX6 or SOX1. The nuclei were stained with DAPI (blue). 9A-9E of FIG confirmed iENP-15F and iENP-13F. Figure 9A: IC staining of iENP induced by a combination of 15 (selected by PAX6: EGFP ) and 13 (selected by SOX1: EGFP ) transcription factors with antibodies against specific NP markers. The nuclei were stained with DAPI (blue). Figure 9B: RT-PCR analysis of specific genes using mRNA extracted from undifferentiated iENP-15F and -13F. hESC-ENP and fibroblasts were used as positive control group and negative control group, respectively. Figure 9C: Integration of specific exogenous transgenic genes was analyzed by PCR assay using genomic DNA extracted from undifferentiated iENP-15F, iENP-6F, iENP-13F and iENP-7F. The plastid of a specific gene was used as a positive control group. Figure 9D: After removal of doxycycline, RT-PCR analysis of specific endogenous genes was performed using mRNA extracted from undifferentiated iENP-15F and iENP-13F. hESC-ENP was used as a positive control group. Figure 9E: ICC staining of differentiated iENP-15F and iENP-13F with antibodies against TUJ1, GFAP and GALC. Scale bar = 10 microns (μm). NC: Negative control group (water). The nuclei were stained with DAPI (blue). Figure 10 transplanted to neutral cerebral artery occlusion (middle cerebral artery occlusion, MCAO) causing damage to the brain of rats, iENP-6F and iENP-7 tumors do not occur. After 12 weeks of iENP-6F and iENP-7F transplantation, HE staining analysis was performed on the rat brain. Figure 11A-11C Diagram of the necessary transcription factor combinations for the preparation of iENP. Figure 11A-11B: Effect of removal of a single transcription factor from the 6 TF group on induction of PAX6: EGFP ⁺ cells (Fig. 11A), and removal of a single transcription factor from the 7 TF group on induction of SOX1: EGFP ⁺ cells ( Figure 11B). Figure 11C: Phase difference photographs of the formation of iENP-like cell clusters induced by a combination of transfected transcription factors. The formation of cell clustering was not observed after removal of a single transcription factor from the original 6 TF or 7 TF combination. 12A-12B of FIG small molecule treatment can be administered to improve the generated iENP. The effect of specific small molecules on the efficacy of preparation (Fig. 12A) iENP-6F and (Fig. 12B) iENP-7F. Error bars represent the mean ± standard deviation. Significance: *P<0.05. (VitC: vitamin C; PR: PP242 + RepSox; DV: DZNep + vitamin C; PRDV: PP242 + RepSox + DZNep + vitamin C). Preparation 13A-13C and FIGS confirmed HD-iENP and AD-iENP. Figure 13A: HD and AD fibroblasts form the induction rate of SOX1: EGFP ⁺ (assumed iENP-6F) and PAX6: EGFP ⁺ (assumed iENP-7F). Error bars represent the mean ± standard deviation. Figure 13B: ICC staining analysis of AD2- and AD3-iENP using a specific antibody to the neuronal marker NES. Figure 13C: RT-PCR analysis of specific genes using mRNA extracted from undifferentiated HD-iENP-6F, HD-iENP-7F, AD-iENP-6F and AD-iENP-7F. hESC-ENP and fibroblasts were used as positive control group and negative control group, respectively.

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<110> Academia Sinica <120> Sets for screening neurological drugs and their uses <130> P2998-TW <160> 27 <170> BiSSAP 1. 3   <210>   1   <211>   1599   <212>   DNA   <213>   Artificial sequence   <220>   <223>   CBX2 gene   <400>   1   Atggaggagc   Tgagcagcgt   Gggcgagcag   Gtcttcgccg   Ccgagtgcat   Cctgagcaag   60   Cggctccgca   Agggcaagct   Ggagtacctg   Gtcaagtggc   Gcggctggtc   Ctccaaacat   120   Aacagctggg   Agccggagga   Gaacatcctg   Gacccgaggc   Tgctcctggc   Cttccagaag   180   Aaggaacatg   Agaaggaggt   Gcagaaccgg   Aagagaggca   Agaggccgag   Aggccggcca   240   Aggaagctca   Ctgccatgtc   Ctcctgcagc   Cggcgctcca   Agctcaagga   Acccgatgct   300   Ccctccaaat   Ccaagtccag   Cagttcctcc   Tcttcctcca   Cgtcatcctc   Ctcttcctca   360   Gatgaagagg   Atgacagtga   Cttagatgct   Aagaggggtc   Cccggggccg   Cgagacccac   420   Ccagtgccgc   Agaagalgc   Ccagatcctg   Gtggccaaac   Ccgagctgaa   Gtagcccatc   480   Cggaagaagc   Ggggacgaaa   Gcccctgccc   Cgagagcaaa   Aggcaacccg   Aagacccgtg   540   Agcctggcca   Aggtgctgaa   Gaccgcccgg   Aaggatctgg   Gggccccggc   Cagcaagctg   600   Ccccctccac   Tcagcgcccc   Cgttgcaggc   Ctggcagctc   Tgaaggccca   Cgccaaggag   660   Gcctgtggcg   Gccccagtgc   Catggccacc   Cgagagaacc   Tggccagcct   Aatgaagggc   720   Atggccagta   Gccccggccg   Gggtggcatc   Agctggcaga   Gctccatcgt   Gcactacatg   780   Aaccggatga   Cccagagcca   Ggcccaggct   Gccagcaggt   Tggcgctgaa   Ggcccaggcc   840   Accaacaagt   Gcggcctcgg   Gctggacctg   Aaggtgagga   Cgcagaaagg   Ggagctggga   900   Atgagccctc   Caggaagcaa   Aatcccgaag   Gcccccagcg   Gtggggctgt   Ggagcagaaa   960   Gtggggaaca   Cagggggccc   Cccgcacacc   Catggtgcca   Gcagggtgcc   Tgctgggtgc   1020   Ccaggccccc   Agccagcacc   Cacccaggag   Ctgagcctcc   Aggtcttgga   Cttgcagagt   1080   Gtcaagaatg   Gcatgcccgg   Ggtgggtctc   Cttgcccgcc   Acgccaccgc   Caccaagggt   1140   Gtcccggcca   Ccaacccagc   Ccctgggaag   Ggcactggga   Gtggcctcat   Tggggccagc   1200   Ggggccacca   Tgcccaccga   Cacaagcaaa   Agtgagaagc   Tggcttccag   Agcagtggcg   1260   Ccaccccacc   Ctgccagcaa   Gagggactgt   Gtcaagggca   Gtgctacccc   Cagtgggcag   1320   Gagagccgca   Cagcccccgg   Agaagcccgc   Aaggcggcca   Cactgccaga   Gatgagcgca   1380   Ggtgaggaga   Gtagcagctc   Ggactccgac   Cccgactccg   Cctcgccgcc   Cagcactgga   1440   Cagaacccat   Cagtgtccgt   Tcagaccagc   Caggactgga   Agcccacccc   Cagcctcatc   1500   Gagcacgtat   Ttgtcaccga   Cgtcactgcc   Aacctcatca   Ccgtcacagt   Gaaggagtct   1560   Cccaccagcg   Tgggcttctt   Caacctgagg   Cattactga   1599   <210>   2   <211>   843   <212>   DNA   <213>   Artificial sequence   <220>   <223>   HES1 gene   <400>   2   Atgccagctg   Atataatgga   Gaaaaattcc   Tcgtccccgg   Tggctgctac   Cccagccagt   60   Gtcaacacga   Caccggataa   Accaaagaca   Gcatctgagc   Acagaaagtc   Atcaaagcct   120   Atttaggaga   Aaagacgaag   Agcaagaata   Aatgaaagtc   Tgagccagct   Gaaaacactg   180   Attttggatg   Ctctgaagaa   Atagagctcg   Cggcattcca   Agctggagaa   Ggcggacatt   240   Ctggaaatga   Cagtgaagca   Cctccggaac   Ctgcagcggg   Cgcagatgac   Ggctgcgctg   300   Agcacagacc   Caagtgtgct   Ggggaagtac   Cgagccggct   Tcagcgagtg   Catgaacgag   360   Gtgacccgct   Tcctgtccac   Gtgcgagggc   Gttaataccg   Aggtgcgcac   Tcggctgctc   420   Ggccacctgg   Ccaactgcat   Gacccagatc   Aatgccatga   Cccacccccg   Gcagccgcac   480   Cccgccttgc   Aggcgccgcc   Accgccccca   Ccgggacccg   Gcggccccca   Gcacgcgccg   540   Ttcgcgccgc   Cgccgccact   Cgtgcccatc   Cccgggggcg   Cggcgccccc   Tcccggcggc   600   Gccccctgca   Agctgggcag   Ccaggctgga   Gaggcggcta   Aggtgtttgg   Aggcttccag   660   Gtggtaccgg   Ctcccgatgg   Ccagtttgct   Ttcctcattc   Ccaacggggc   Cttcgcgcac   720   Agcggccctg   Tcatccccgt   Ctacaccagc   Aacagcggca   Cctccgtggg   Ccccaacgca   780   Gtgtcacctt   Ccaggggccc   Ctcgcttacg   Gcggactcca   Tgtggaggcc   Gtggcggaac   840   Tga   843   <210>   3   <211>   468   <212>   DNA   <213>   Artificial sequence   <220>   <223>   ID1 gene   <400>   3   Atgaaagtcg   Ccagtggcag   Caccgccacc   Gccgccgcgg   Gccccagctg   Cgcgctgaag   60   Gccggcaaga   Cagcgagcgg   Tgcgggcgag   Gtggtgcgct   Gtctgtctga   Gcagagcgtg   120   Gccatctcgc   Gctgcgccgg   Gggcgccggg   Gcgcgcctgc   Ctgccctgct   Ggacgagcag   180   Caggtaaacg   Tgctgctcta   Cgacatgaac   Ggctgttact   Cacgcctcaa   Ggagctggtg   240   Cccaccctgc   Cccagaaccg   Caaggtgagc   Aaggtggaga   Ttctccagca   Cgtcatcgac   300   Tacatcaggg   Accttcagtt   Ggagctgaac   Tcggaatccg   Aagttggaac   Ccccgggggc   360   Cgagggctgc   Cggtccgggc   Tccgctcagc   Accctcaacg   Gcgagatcag   Cgccctgacg   420   Gccgaggcgg   Catgcgttcc   Tgcggacgat   Cgcatcttgt   Gtcgctga   468   <210>   4   <211>   1296   <212>   DNA   <213>   Artificial sequence   <220>   <223>   TFAP2A gene   <400>   4   Atgttagttc   Acaggttttc   Agccatggac   Cgtcacgacg   Gcaccagcaa   Cgggacggca   60   Cggttgcccc   Agctgggcac   Tgtaggtcaa   Tctccctaca   Cgagcgcccc   Gccgctgtcc   120   Cacaccccca   Atgccgactt   Ccagccccca   Tacttccccc   Caccctacca   Gcctatctac   180   Ccccagtcgc   Aagatcctta   Ctcccacgtc   Aacgacccct   Acagcctgaa   Ccccctgcac   240   Gcccagccgc   Agccgcagca   Cccaggctgg   Cccggccaga   Ggcagagcca   Ggagtctggg   300   Ctcctgcaca   Cgcaccgggg   Gctgcctcac   Cagctgtcgg   Gcctggatcc   Tcgcagggac   360   Tacaggcggc   Acgaggacct   Cctgcacggc   Ccacaccgcc   Tcagctcagg   Actcggagac   420   Ctctcgatcc   Actccttacc   Tcacgccatc   Gaggaggtcc   Cgcatgtaga   Agacccgggt   480   Attacatcc   Cagatcaaac   Tgtaattaag   Aaaggccccg   Tgtccctgtc   Caagtccaac   540   Agcaatgccg   Tctccgccat   Ccctattaac   Aaggacaacc   Tcttcggcgg   Cgtggtgaac   600   Cccaacgaag   Tcttctgttc   Agttccgggt   Cgcctctcgc   Tcctcagctc   Cacctcgaag   660   Tacaaggtca   Cggtggcgga   Agtgcagcgg   Cggctctcac   Cacccgagtg   Tctcaacgcg   720   Tcgctgctgg   Gcggagtgct   Ccggagggcg   Aagtctaaaa   Atggaggaag   Atctttaaga   780   Gaaaaactgg   Acaaaatagg   Attaatctg   Cctgcaggga   Gacgtaaagc   Tgccaacgtt   840   Accctgctca   Catcactagt   Agagggagaa   Gctgtccacc   Tagccaggga   Ctttgggtac   900   Gtgtgcgaaa   Ccgaatttcc   Tgccaaagca   Gtagctgaat   Ttctcaaccg   Acaacattcc   960   Gatcccaatg   Agcaagtgac   Aagaaaaaac   Atgctcctgg   Ctacaaaaca   Gatatgcaaa   1020   Gagttcaccg   Acctgctggc   Tcaggaccga   Tctcccctgg   Ggaactcacg   Gcccaacccc   1080   Atcctggagc   Ccggcatcca   Gagctgcttg   Acccacttca   Acctcatctc   Ccacggcttc   1140   Ggcagccccg   Cggtgtgtgc   Cgcggtcacg   Gccctgcaga   Actatctcac   Cgaggccctc   1200   Aaggccatgg   Acaaaatgta   Cctcagcaac   Aaccccaata   Gccacacgga   Caacaacgcc   1260   Aaaagcagtg   Acaaagagga   Gaagcacaga   Aagtga   1296   <210>   5   <211>   933   <212>   DNA   <213>   Artificial sequence   <220>   <223>   ZFP42 gene   <400>   5   Atgagccagc   Aactgaagaa   Acgggcaaag   Acaagacacc   Agaaaggcct   Gggtggaaga   60   Gcccccagtg   Gggctaagcc   Caggcaaggc   Aagtcaagcc   Aagacctgca   Ggcggaaata   120   Gaacctgtca   Gcgcggtgtg   Ggccttatgt   Gatggctatg   Tgtgctatga   Gcctggccct   180   Caggctctcg   Gaggggatga   Tttctcagac   Tgttacatag   Aatgcgtcat   Aaggggtgag   240   Ttttctcaac   Ccatcctgga   Agaggactca   Ctttttgagt   Cctgtgaata   Cctaaagaaa   300   Ggatcagaac   Aacagctttc   Tcaaaaggtt   Ttcgaagcaa   Gctcccttga   Atgttctttg   360   Gaatacatga   Aaaaaggggt   Aaagaaagag   Cttccacaaa   Agatagtgtg   Agagaattcg   420   Cttgagtatt   Ctgagtacat   Gacaggcaag   Aagcttccgc   Ctggaggaat   Acctggcatt   480   Gacctatcag   Atcctaaaca   Gctcgcagaa   Tttgctagaa   Agaagccccc   Cataataataa   540   Gaatatgaca   Gtctgagcgc   Aatcgcttgt   Cctcagagtg   Gatgcactag   Gaagttgagg   600   Aatagagctg   Ccctgagaaa   Gcatctcctc   Attcatggtc   Cccgagacca   Cgtctgtgcg   660   Gaatgtggga   Aagcgttcgt   Tgagagctca   Aaactaaaga   Gacatttcct   Ggttcatact   720   Ggagagaagc   Cgtttcggtg   Cacttttgaa   Gggtgcggaa   Agcgcttctc   Tctggacttt   780   Aatttgcgta   Cgcacgtgcg   Catccacacg   Ggggagaaac   Gtttcgtgtg   Tccctttcaa   840   Ggctgcaaca   Ggaggtttat   Tcagtcaaat   Aacctgaaag   Cccacatcct   Aacgcatgca   900   Aatacgaaca   Agaatgaaca   Agagggaaag   Tag   933   <210>   6   <211>   3855   <212>   DNA   <213>   Artificial sequence   <220>   <223>   ZNF423 gene   <400>   6   Atgcataaga   Agagggttga   Agagggggag   Gcctcagact   Tctcgctggc   Ctgggattcc   60   Tccgtgacag   Cagcaggagg   Cctagaagga   Gagccagagt   Gcgatcagaa   Aaccagccgt   120   Gcgctggaag   Acaggaacag   Cgtgacaagt   Caagaggaga   Gaaatgagga   Tgatgaagac   180   Atggaggatg   Aatcaattta   Cacctgcgat   Cactgtcagc   Aggacttcga   Gtctctggca   240   Gacctgacgg   Accaccgggc   Ccaccgctgt   Cctggagatg   Gtgatgacga   Cccacaactc   300   Tcctgggtgg   Cctcgtctcc   Ctccagcaag   Gatgttgcgt   Cacccacgca   Gatgatcgga   360   Gatggttgtg   Acctcggcct   Cggcgaggag   Gaagggggca   Cgggcctgcc   Atacccttgc   420   Cagttctgcg   Acaagtcctt   Catccgcttg   Agctacttga   Agaggcacga   Gcagatccac   480   Agcgacaagc   Tgccgttcaa   Gtgcacctac   Tgcagccgcc   Tcttcaagca   Caagaggagc   540   Cgtgaccggc   Acatcaagct   Gcatacgggc   Gacaagaagt   Atcactgcca   Cgagtgcgag   600   Gcagccttct   Cccgcagcga   Ccacctcaag   Atccacctga   Agaccacag   Ctccagcaag   660   Cccttcaagt   Gcactgtgtg   Caagcgcggc   Ttctcctcca   Ccagctcgct   Gcagagccac   720   Atgcaggccc   Acaaaaagaa   Caaggagcat   Ctggccaagt   Cggagaagga   Agccaagaag   780   Gacgacttca   Tgtgcgacta   Ctgcgaggac   Accttcagcc   Agacggagga   Gctggagaag   840   Cacgtgctca   Cccgccaccc   Gcagctgtcc   Gagaaggcgg   Acctgcagtg   Cattcactgc   900   Cctgaggtct   Tcgtcgacga   Gaacacactg   Ctcgcccata   Tccaccaagc   Ccacgccaac   960   Cagaaacaca   Agtgccccat   Gtgccctgag   Cagttctcct   Cagtggaagg   Tgtctactgc   1020   Cacctggaca   Gccaccggca   Gcccgactcc   Agcaaccaca   Gtgtcagtcc   Cgaccctgta   1080   Ctgggcagcg   Tggcctccat   Gagcagcgcc   Acacccgact   Ccaggccctc   Tgtggagcgt   1140   Ggctccaccc   Cggactccac   Cttgaagccg   Ctgcgggggc   Agaagaagat   Gcgggatgac   1200   Gggcagggct   Ggaccaaggt   Ggtctatagc   Tgcccctatt   Gttccaagcg   Ggactttaac   1260   Agcctggccg   Tgctggagat   Ccacctgaag   Accatccacg   Cggacaagcc   Ccaggagagc   1320   Cacacatgtc   Agatctgcct   Ggactccatg   Cccaccctct   Acaacctcaa   Cgagcacgtt   1380   Cgcaagctgc   Acaagaacca   Tgcctaccct   Gtgatgcagt   Ttggcaacat   Ctctgccttc   1440   Cactgcaact   Actgccccga   Gatgttcgcc   Gacatcaata   Gcctgcagga   Gcacatccgc   1500   Gtctcccact   Gcggccccaa   Cgccaacccc   Tctgacggta   Ataatgcttt   Cttctgcaac   1560   Cagtgctcca   Tgggtttcct   Tactgagtcc   Tccctcaccg   Agcacatcca   Gcaggcccac   1620   Tgcagtgtgg   Gcagtgccaa   Actagagtct   Ccggtggtgc   Agcccacgca   Gtccttcatg   1680   Gaggtctatt   Cctgccccta   Ctgcaccaac   Tcccccatct   Ttggctccat   Cctgaaactc   1740   Accaagcaca   Tcaaggagaa   Ccacaagaac   Attccactgg   Cccacagcaa   Gaagtccaag   1800   Gccgagcaga   Gcccagtctc   Gtccgatgtg   Gaggtgtctt   Ccccgaagcg   Gcagcggctc   1860   Tcagcaagcg   Ccaactccat   Ctccaatggg   Gagtatcctt   Gcaatcaatg   Cgacctcaag   1920   Ttctccaact   Ttgagagctt   Ccagacccac   Ctgaagctgc   Acctggagct   Gctgctgcgg   1980   Aagcaagcgt   Gcccccagtg   Caaagaggac   Tttgactccc   Aggagtccct   Cctgcagcac   2040   Ctgacagtgc   Attacatgac   Cacgtcgacc   Cactatgtgt   Gcgagagctg   Cgacaagcaa   2100   Ttttcctcgg   Tggatgacct   Gcagaagcac   Ctgctggaca   Tgcacacctt   Tgtgttgtac   2160   Cactgcaccc   Tgtgtcagga   Ggtcttcgac   Tccaaggtgt   Ccatccaggt   Gcacctggcg   2220   Gtgaagcaca   Gcaatgagaa   Gaagatgtac   Cgctgcacgg   Cctgcaactg   Ggacttccgc   2280   Aaggaggctg   Acctgcaggt   Gcacgtcaaa   Cacagccacc   Tgggcaaccc   Ggccaaggct   2340   Cacaagtgca   Tcttctgtgg   Ggagaccttc   Agcaccgagg   Tggagctgca   Gtgccacatc   2400   Accacacaca   Gcaagaagta   Taactgtaag   Ttctgcagca   Aggccttcca   Cgccatcatc   2460   Ctgctggaga   Agcacctgcg   Ggagaagcac   Tgtgtgtttg   Atgctgcgac   Cgagaacggc   2520   Acggccaatg   Gggtaccccc   Aatggccacc   Aagaaagctg   Agcctgctga   Cctgcagggc   2580   Atgctgctta   Agaaccctga   Ggcacctaac   Agccatgagg   Ccagggagga   Tgacgtggac   2640   Gcgtcggagc   Ccatgtacgg   Ctgtgacatc   Tgtggggcgg   Cctacaccat   Ggaggtgctg   2700   Ctgcagaatc   Accggctgcg   Ggaccacaat   Atccggccgg   Gcgaggatga   Tggctcacgc   2760   Aagaaggctg   Agttattaca   Gggcagtcac   Aagtgcaacg   Tttgttcacg   Gactttcttc   2820   Tcggagaacg   Ggctacggga   Gcacctgcag   Acgcaccggg   Gccctgccaa   Gcactacatg   2880   Tgtcccatct   Gtggtgagcg   Cttcccttcg   Ctgctgacgc   Tcaccgaaca   Caaggtgacc   2940   Cacagcaaga   Gcctggacac   Gggcacctgt   Cgcatctgca   Agatgcccct   Gcagagcgag   3000   Gaggagttta   Ttgagcactg   Ccagatgcac   Cctgacctgc   Gcaactcact   Cacgggcttc   3060   Cgctgtgtgg   Tctgcatgca   Gacagtcact   Tccacgcttg   Agctcaagat   Ccatggcacc   3120   Ttccacatgc   Agaagctggc   Gggcagctca   Gcggcgtcct   Ccccacatagg   Ccaggggctg   3180   Cagaagctct   Acaagtgcgc   Cctgtgcctc   Aaggagttcc   Gcagcaagca   Ggacctggtg   3240   Aagcttgacg   Tcaatgggct   Gccctacggc   Ctctgcgccg   Gctgcatggc   Ccgcagcgcc   3300   Aacggacagg   Tgggtggcct   Ggccccgccc   Gagcccgccg   Accggccctg   Tgccggcctc   3360   Cgttgccccg   Agtgcagtgt   Caagtttgag   Agtgccgaag   Acctggagag   Ccacatgcag   3420   Gtggaccacc   Gtgacctcac   Gccggagacc   Agtgggcccc   Ggaaaggcac   Ccagacatcg   3480   Ccagtgcccc   Ggaaaaagac   Ataccagtgc   Atcaagtgcc   Agatgacctt   Cgagaacgag   3540   Agagagatcc   Aaatccacgt   Tgccaaccac   Atgattgagg   Aaggcatcaa   Ccacgagtgt   3600   Aagctgtgca   Accagatgtt   Cgactccccg   Gccaagctcc   Tctgtcacct   Cattgagcac   3660   Agcttcgagg   Gcatgggcgg   Caccttcaaa   Tgccccgtgt   Gtttcacagt   Cttcgtccag   3720   Gccaacaagt   Tgcagcagca   Catctttgcc   Gtgcacgggc   Aggaggacaca   Gatctacgac   3780   Tgctcacagt   Gccctcagaa   Gttcttcttc   Cagaccgagc   Tgcagaacca   Cacgatgagc   3840   Cagcacgcac   Agtga   3855   <210>   7   <211>   2127   <212>   DNA   <213>   Artificial sequence   <220>   <223>   DACH1 gene   <400>   7   Atggcagtgc   Cggcggcttt   Gatccctccg   Acccagctgg   Tcccccctca   Acccccaatc   60   Tccacgtctg   Cttcctcctc   Tggcaccacc   Acctccacct   Cttcggcgac   Ttcgtctccg   120   Gctccttcca   Tcggaccccc   Ggcgtcctct   Gggccaactc   Tgttccgccc   Ggagcccatc   180   Gcttcggcgg   Cggcggcggc   Ggccacagtc   Acctctaccg   Gcggcggcgg   Cggcggcggc   240   Ggcggcggca   Gcggaggcgg   Cggcggcagc   Agcggcaacg   Gaggcggcgg   Tggcggcggc   300   Ggcggtggca   Gcaactgcaa   Ccccaacctg   Gcggccgcga   Gcaacggcag   Cggcggcggc   360   Ggcggcggca   Tcagcgctgg   Cggcggcgtc   Gcttccagca   Cccccatcaa   Cgccagcacc   420   Ggcagcagca   Gcagcagcag   Tagcagcagc   Agcagcagca   Gcagtagtag   Cagcagcagc   480   Agtagcagca   Gcagctgcgg   Ccccctcccc   Gggaaacccg   Tgtactcaac   Cccgtcccca   540   Gtggaaaaca   Cccctcagaa   Taatgagtgc   Aaaatggtgg   Atctgagggg   Ggccaaagtg   600   Gcttccttca   Cggtggaggg   Ctgcgagctg   Atctgcctgc   Cccaggcttt   Cgacctgttc   660   Ctgaagcact   Tggtgggggg   Cttgcatacg   Gtctacacca   Agctgaagcg   Gctggagatc   720   Acgccggtgg   Tgtgcaatgt   Ggaacaagtt   Cgcatcctga   Ggggactggg   Cgccatccag   780   Ccaggagtga   Accgctgcaa   Actcatctcc   Aggaaggact   Tcgagaccct   Ctacaatgac   840   Tgcaccaacg   Caagttctag   Acctggaagg   Cctcctaaga   Ggactcaaag   Tgtcacctcc   900   Ccagagaact   Ctcacatcat   Gccgcattct   Gtccctggtc   Tcatgtctcc   Tgggataatt   960   Ccacacaacag   Gtctgacagc   Agccgctgca   Gcagctgctg   Ctgctaccaa   Tgcagctatt   1020   Gctgaagcaa   Tgaaggtgaa   Aaaaatcaaa   Ttagaagcca   Tgagcaacta   Tcatgccagt   1080   Aataaccaac   Atggagcaga   Ctctgaaaac   Ggggacatga   Attcaagtgt   Cggactggaa   1140   Cttcctttta   Tgatgatgcc   Ccaccctcta   Attcctgtca   Gcctacctcc   Agcatctgtc   1200   Accatggcaa   Tgagccagat   Gaaccaccctc   Agcaccattg   Caaatatggc   Agcagcagca   1260   Caagttcaga   Gtcccccatc   Cagagttgag   Acatcagtta   Ttaaggagcg   Tgttcctgat   1320   Agcccctcac   Ctgccccctc   Tctggaggag   Gggagaaggc   Ctggcagtca   Cccatcatca   1380   Catcgcagca   Gcagcgtgtc   Cagctcccct   Gctcggactg   Agagctcttc   Tgacagaatc   1440   Ccggtccatc   Agaatgggtt   Gtccatgaac   Cagatgctga   Tgggcttatc   Accaaatgta   1500   Cttcctgggc   Ccaaagaggg   Agatttggcc   Ggtcatgaca   Tgggacatga   Gtcaaaaagg   1560   Atgcatattg   Aaaaagatga   Gaccccgctt   Tctacaccaa   Ccgcaagaga   Cagccttgac   1620   Aaactctctc   Taactgggca   Tggacaacca   Ctgcctccag   Gttttccatc   Tccttttctg   1680   Tttcctgatg   Gactgtcttc   Catcgagact   Cttctgacta   Acatacaggg   Gctgttgaaa   1740   Gttgccatag   Ataatgccag   Agctcaagag   Aaacaggtcc   Aactggaaaa   Aactgagctg   1800   Aagatggatt   Ttttaaggga   Aagagaacta   Agggaaacac   Ttgagaagca   Gttggctatg   1860   Gaacaaaaga   Atagagccat   Agttcaaaag   Aggctaaaga   Aggagaagaa   Ggcaaagaga   1920   Aaattgcagg   Aagcacttga   Gtttgagacg   Aaacggcgtg   Aacaagcaga   Acagacgcta   1980   Aaacaggcag   Cttcaacaga   Ttagctcagg   Gtcttaaatg   Actctctgac   Cccagagata   2040   Gaggctgacc   Gcagtggcgg   Cagaacagat   Gctgaaagga   Caatacaaga   Tggaagactg   2100   Tatttgaaaa   Ctactgtcat   Gtactga   2127   <210>   8   <211>   1470   <212>   DNA   <213>   Artificial sequence   <220>   <223>   FOXG1 gene   <400>   8   Atgctggaca   Tgggagatag   Gaaagaggtg   Aaaatgatcc   Ccaagtcctc   Gttcagcatc   60   Aacagcctgg   Tgcccgaggc   Ggtccagaac   Gacaaccacc   Acgcgagcca   Cggccaccac   120   Aacagccacc   Acccccagca   Ccaccacccac   Caccaccacc   Atcaccacca   Cccgccgccg   180   Cccgccccgc   Aaccgccgcc   Gccgccgcag   Cagcagcagc   Cgccgccgcc   Gccgcccccg   240   Gcaccgcagc   Ccccccagac   Gcggggcgcc   Ccggccgccg   Acgacgacaa   Gggcccccag   300   Cagctgctgc   Tcccgccgcc   Gccaccgcca   Ccaccggccg   Ccgccctgga   Cggggctaaa   360   Gcggacgggc   Tgggcggcaa   Gggcgagccg   Ggcggcgggc   Cgggggagct   Ggcgcccgtc   420   Gggccggacg   Gaagaggagaa   Gggcgccggc   Gccggggggg   Aggagaagaa   Gggggcgggc   480   Gagggcggca   Aggaggggga   Ggggggcaag   Gagggcgaga   Agaagaacgg   Caagtacgag   540   Aagccgccgt   Tcagctacaa   Cgcgctcatc   Atgatggcca   Tccggcagag   Ccccgagaag   600   Cggctcacgc   Tcaacggcat   Ctacgagttc   Atcatgaaga   Acttccctta   Ctaccgcgag   660   Aacaagcagg   Gctggcagaa   Ctccatccgc   Cacaatctgt   Ccctcaacaa   Gtgcttcgtg   720   Aaggtgccgc   Gccactacga   Cgacccgggc   Aagggcaact   Actggatgct   Ggacccgtcg   780   Agcgacgacg   Tgttcatcgg   Cggcaccacg   Ggcaagctgc   Ggcgccgctc   Caccacctcg   840   Cgggccaagc   Tggccttcaa   Gcgcggtgcg   Cgcctcacct   Ccaccggcct   Caccttcatg   900   Gaccgcgccg   Gctccctcta   Ctggcccatg   Tcgcccttcc   Tgtccctgca   Ccaccccccg   960   Gccagcagca   Ctttgagtta   Caacggcacc   Acgtcggcct   Accccagcca   Ccccatgccc   1020   Tacagctccg   Tgttgactca   Gaactcgctg   Ggcaacaacc   Actccttctc   Caccgccaac   1080   Ggcctgagcg   Tggaccggct   Ggtcaacggg   Gagatcccgt   Acgccacgca   Ccacctcacg   1140   Gccgccgcgc   Tagccgcctc   Ggtgccctgc   Ggcctgtcgg   Tgccctgctc   Tgggacctac   1200   Tccctcaacc   Cctgctccgt   Caacctgctc   Gcgggccaga   Ccagttactt   Tttcccccac   1260   Gtcccgcacc   Cgtcaatgac   Ttcgcagagc   Agcacgtcca   Tgagcgccag   Ggccgcgtcc   1320   Tcctccacgt   Cgccgcaggc   Cccctcgacc   Ctgccctgtg   Agtctttaag   Accctctttg   1380   Ccaagtttta   Cgacgggact   Gtctggggga   Ctgtctgatt   Atttcacaca   Tcaaaatcag   1440   Gggtcttctt   Ccaacccttt   Aatacattaa   1470   <210>   9   <211>   1395   <212>   DNA   <213>   Artificial sequence   <220>   <223>   MYCN gene   <400>   9   Atgccgagct   Gctccacgtc   Caccatgccg   Ggcatgatct   Gcaagaaccc   Agacctcgag   60   Tttgactcgc   Tacagccctg   Cttctacccg   Gacgaagatg   Acttctactt   Cggcggcccc   120   Gactcgaccc   Cccgggggga   Ggacatctgg   Aagaagtttg   Agctgctgcc   Cacgcccccg   180   Ctgtcgccca   Gccgtggctt   Cgcggagcac   Agctccgagc   Ccccgagctg   Ggtcacggag   240   Atgctgcttg   Agaacgagct   Gtggggcagc   Ccggccgagg   Aggaggcgtt   Cggcctgggg   300   Ggactgggtg   Gcctcacccc   Caacccggtc   Atcctccagg   Actgcatgtg   Gaggggcttc   360   Tccgcccgcg   Agaagctgga   Gcgcgccgtg   Agcgagaagc   Tgcagcacgg   Ccgcgggccg   420   Ccaaccgccg   Gttccaccgc   Ccagtccccg   Ggagccggcg   Ccgccagccc   Tgcgggtcgc   480   Gggcacggcg   Gggctgcggg   Agccggccgc   Gccggggccg   Ccctgcccgc   Cgagctcgcc   540   Cacccggccg   Ccgagtgcgt   Ggatcccgcc   Gtggtcttcc   Cctttcccgt   Gaacaagcgc   600   Gagccagcgc   Ccgtgcccgc   Agccccggcc   Agtgccccgg   Cggcgggccc   Tgcggtcgcc   660   Tcgggggcgg   Gtattgccgc   Cccagccggg   Gccccggggg   Tcgcccctcc   Gcgcccaggc   720   Ggccgccaga   Ccagggggggg   Cgaccacaag   Gccctcagta   Cctccggaga   Ggacaccctg   780   Agcgattcag   Atgatgaaga   Tgatgaagag   Gaagatgaag   Aggaagaaat   Cgacgtggtc   840   Actgtggaga   Agcggcgttc   Ctcctccaac   Accaaggctg   Tcaccacatt   Caccatcact   900   Gtgcgtccca   Agaacgagc   Cctgggtccc   Gggagggctc   Agtccagcga   Gctgatcctc   960   Aaacgatgcc   Ttcccatcca   Ccagcagcac   Aactatgccg   Ccccctctcc   Ctacgtggag   1020   Agtgaggatg   Cacccccaca   Gaagaagata   Aagagcgagg   Cgtccccacg   Tccgctcaag   1080   Agtgtcatcc   Ccccaaaggc   Taagagcttg   Agcccccgaa   Actctgactc   Ggaggacagt   1140   Gagcgtcgca   Gaaaccacaa   Catcctggag   Cgccagcgcc   Gcaacgacct   Tcggtccagc   1200   Tttctcacgc   Tcagggacca   Cgtgccggag   Ttggtaaaga   Atgagaaggc   Cgccaaggtg   1260   Gtcattttga   Aaaaggccac   Tgagtatgtc   Cactccctcc   Aggccgagga   Gcaccagctt   1320   Ttgctggaaa   Aggaaaaatt   Gcaggcaaga   Cagcagcagt   Tgctaaagaa   Aattgaacac   1380   Gctcggactt   Gctag   1395   <210>   10   <211>   1245   <212>   DNA   <213>   Artificial sequence   <220>   <223>   NR2F2 gene   <400>   10   Atggcaatgg   Ttagcagcac   Gtggcgcgac   Ccccaggacg   Aggtgcccgg   Ctcacagggc   60   Agccaggcct   Cgcaggcgcc   Gcccgtgccc   Ggcccgccgc   Ccggcgcccc   Gcacacgcca   120   Cagacgcccg   Gccaaggggg   Cccagccagc   Acgccagccc   Agacggcggc   Cggtggccag   180   Ggcggccctg   Gcggcccggg   Tagcgacaag   Cagcagcagc   Agcaacacat   Cgagtgcgtg   240   Gtgtgcggag   Acaagtcgag   Cggcaagcac   Tacggccagt   Tcacgtgcga   Gggctgcaag   300   Agcttcttca   Agcgcagcgt   Gcggaggaac   Ctgagctaca   Cgtgccgcgc   Caaccggaac   360   Tgtcccatcg   Accagcacca   Tcgcaaccag   Tgccagtact   Gccgcctcaa   Aaagtgcctc   420   Aaagtgggca   Tgagacggga   Agcggtgcag   Aggggcagga   Tgccgccgac   Ccagccgacc   480   Cacgggcagt   Tcgcgctgac   Caacggggat   Cccctcaact   Gccactcgta   Cctgtccgga   540   Tatatttccc   Tgctgttgcg   Cgcggagccc   Tatcccacgt   Cgcgcttcgg   Cagccaatgc   600   Atgcagccca   Acaacatcat   Gggtatcgag   Aacatttgcg   Aactggccgc   Gaggatgctc   660   Ttcagcgccg   Tcgagtgggc   Ccggaacatc   Cccttcttcc   Ccgacctgca   Gatcacggac   720   Caggtggccc   Tgcttcgcct   Cacctggagc   Gagctgtttg   Tgttgaatgc   Ggcgcagtgc   780   Tccatgcccc   Tccacgtcgc   Cccgctcctg   Gccgccgccg   Gcctgcatgc   Ttcgcccatg   840   Tccgccgacc   Gggtggtcgc   Ctttatggac   Cacatacgga   Tcttccaaga   Gcaagtggag   900   Aagctcaagg   Cgctgcacgt   Tgactcagcc   Gagtacagct   Gcctcaaggc   Cataggtctg   960   Ttcacctcag   Atgcctgtgg   Tctctctgat   Gtagcccatg   Tggaaagctt   Gcaggaaaag   1020   Tctcagtgtg   Ctttggaaga   Atacgttagg   Agccagtacc   Ccaaccagcc   Gacgagattc   1080   Ggaaagcttt   Tgcttcgcct   Cccttccctc   Cgcaccgtct   Cctcctcagt   Catagagcaa   1140   Ttgtttttcg   Tccgtttggt   Aggtaaaacc   Cccatcgaaa   Ccctcatccg   Ggatatgtta   1200   Ctgtccggca   Gcagttttaa   Ctggccgtat   Atggcaattc   Aataa   1245   <210>   11   <211>   1443   <212>   DNA   <213>   Artificial sequence   <220>   <223>   NR6A1 gene   <400>   11   Atggagcggg   Acgaaccgcc   Gcctagcgga   Gggggaggcg   Gcgggggctc   Ggcggggttc   60   Ctggagccctc   Ccgccgcgct   Ccctccgccg   Ccgcgcaacg   Gtttctgtca   Ggatgaattg   120   Gcagagcttg   Acccaggcac   Tatttctgtt   Tcagatgatc   Gggctgaaca   Acgaacctgt   180   Ctcatttgtg   Gggaccgcgc   Tacaggcttg   Cactatggga   Tcatctcctg   Tgagggctgc   240   Aaagggtttt   Tcaagcggag   Catttgcaac   Aaacgggtat   Atcgatgcag   Tcgtgacaag   300   Aactgtgtca   Tgtctcggaa   Gcagaggaac   Aggtgccagt   Actgccgcct   Gctcaaatgc   360   Ctccagatgg   Ggatgaaccg   Gaaggctatc   Agagaagatg   Gcatgcctgg   Aggccggaat   420   Aagagcattg   Ggccagtcca   Gatatcggaa   Gaagaaatcg   Aaaggatcat   Gtctgggcag   480   Gagtttgagg   Aagaggccaa   Tcactggagc   Aaccatggtg   Atagtgacca   Cagttcccct   540   Gggaacaggg   Cttcggagag   Caaccagccc   Tcaccaggct   Ccacactgtc   Ttccagtagg   600   Tctgtggaac   Tgaatggatt   Catggccttc   Agggaacagt   Acatgggaat   Gtctgtgcct   660   Ccacattacc   Aatatatacc   Gcaccttttt   Agctattctg   Gccactcacc   Acttctgccc   720   Caacaagctc   Gcagcctgga   Tccccagtca   Tacagtctga   Ttcaccagct   Gttatcagcc   780   Gaggacctgg   Aaccattggg   Cacgcccatg   Ttgattgaag   Atggatacgc   Tgtgacacag   840   Gcagaactat   Ttgccctgct   Ttgccgcctg   Gccgacgagc   Tgctctttag   Gcagattgcc   900   Tggatcaaga   Aactgccttt   Cttctgcgag   Ctctcaatca   Aggattacac   Gtgcctcttg   960   Agctctacgt   Ggcaggagct   Aatcctgctg   Tcttccctca   Ccgtttacag   Caagcagatc   1020   Tttggggaac   Tggctgatgt   Cactgccaag   Tactcgccct   Cggatgaaga   Actacacaga   1080   Tttagtgatg   Aagggatgga   Ggtgatcgag   Cggctcatct   Acctctatca   Caagttccat   1140   Cagctaaagg   Tcagcaacga   Ggagtatgct   Tgcatgaaag   Caattaactt   Cctaaatcaa   1200   Gatatcaggg   Gtctgaccag   Tgcctcacag   Ctggaacaat   Tgaataaacg   Atactggtac   1260   Atttgccagg   Attttactga   Atataaatac   Acacatcagc   Cgaaccgctt   Tcctgatctc   1320   Atgatgtgct   Tacctgagat   Tcgatatatt   Gcaggaaaga   Tggtgaatgt   Gcccctggag   1380   Cagctgcccc   Tcctctttaa   Ggtggtgctg   Cattcctgca   Agaccagtgt   Gggcaaggaa   1440   Tga   1443   <210>   12   <211>   954   <212>   DNA   <213>   Artificial sequence   <220>   <223>   SOX2 gene   <400>   12   Atgtacaaca   Tgatggagac   Ggagctgaag   Ccgccgggcc   Cgcagcaaac   Ttcggggggc   60   Ggcggcggca   Actccaccgc   Ggcggcggcc   Ggcggcaacc   Agaaaaacag   Cccggaccgc   120   Gtcaagcggc   Ccatgaatgc   Cttcatggtg   Tggtcccgcg   Ggcagcggcg   Caagatggcc   180   Caggagaacc   Ccaagatgca   Caactcggag   Atcagcaagc   Gcctgggcgc   Cgagtggaaa   240   Cttttgtcgg   Agacggagaa   Gcggccgttc   Atcgacgagg   Ctaagcggct   Gcgagcgctg   300   Cacatgaagg   Agcacccgga   Ttataaatac   Cggccccggc   Ggaaaaccaa   Gacgctcatg   360   Aagaaggata   Agtacacgct   Gcccggcggg   Ctgctggccc   Ccggcggcaa   Tagcatggcg   420   Agcggggtcg   Gggtgggcgc   Cggcctgggc   Gcgggcgtga   Accagcgcat   Ggacagttac   480   Gcgcacatga   Acggctggag   Caacggcagc   Tacagcatga   Tgcaggacca   Gctgggctac   540   Ccgcagcacc   Cgggcctcaa   Tgcgcacggc   Gcagcgcaga   Tgcagcccat   Gcaccgctac   600   Gacgtgagcg   Ccctgcagta   Caactccatg   Accagctcgc   Agactacat   Gaacggctcg   660   Cccacctaca   Gcatgtccta   Ctcgcagcag   Ggcacccctg   Gcatggctct   Tggctccatg   720   Ggttcggtgg   Tcaagtccga   Ggccagctcc   Agccccccg   Tggttacctc   Ttcctcccac   780   Tccagggcgc   Cctgccaggc   Cggggacctc   Cgggacatga   Tcagcatgta   Tctccccggc   840   Gccgaggtgc   Cggaacccgc   Cgcccccagc   Agacttcaca   Tgtcccagca   Ctaccagagc   900   Ggcccggtgc   Ccggcacggc   Cattaacggc   Acactgcccc   Tctcacacat   Gtga   954   <210>   13   <211>   1326   <212>   DNA   <213>   Artificial sequence   <220>   <223>   SOX11 gene   <400>   13   Atggtgcagc   Aggcggagag   Cttggaagcg   Gagagcaacc   Tgccccggga   Ggcgctggac   60   Acggaggagg   Gcgaattcat   Ggcttgcagc   Ccggtggccc   Tggacgagag   Cgacccagac   120   Tggtgcaaga   Cggcgtcggg   Ccacatcaag   Cggccgatga   Acgcgttcat   Ggtatggtcc   180   Aagatcgaac   Gcaggaagat   Catggagcag   Tctccggaca   Tgcacaacgc   Cgagatctcc   240   Aagaggctgg   Gcaagcgctg   Gaaaatgctg   Aaggacagcg   Agaagatccc   Gttcatccgg   300   Gaggcggagc   Ggctgcggct   Caagcacatg   Gccgactacc   Ccgactacaa   Gtaccggccc   360   Cggaaaaagc   Ccaaaatgga   Cccctcggcc   Aagcccagcg   Ccagccagag   Cccagagaag   420   Agcgcggccg   Gcggcggcgg   Cgggagcgcg   Ggcggaggcg   Cgggcggtgc   Caagacctcc   480   Aagggctcca   Gcaagaaatg   Cggcaagctc   Aaggcccccg   Cggccgcggg   Cgccaaggcg   540   Ggcgcgggca   Aggcggccca   Gtccggggac   Tacgggggcg   Cgggcgacga   Ctacgtgctg   600   Ggcagcctgc   Gcgtgagcgg   Ctcgggcggc   Ggcggcgcgg   Gcaagacggt   Caagtgcgtg   660   Tttctggatg   Aggaggagga   Cgacgacgac   Gacgacgacg   Agctgcagct   Gcagatcaaa   720   Caggagccgg   Acgaggagga   Cgaggaacca   Ccgcaccagc   Agctcctgca   Gccgccgggg   780   Cagcagccgt   Cgcagctgct   Gagacgctac   Aacgtcgcca   Aagtgcccgc   Cagccctacg   840   Ctgagcagct   Cggcggagtc   Ccccgaggga   Gcgagcctct   Acgacgaggt   Gcgggccggc   900   Gcgacctcgg   Gcgccggggg   Cggcagccgc   Ctctactaca   Gcttcaagaa   Catcaccaag   960   Cagcacccgc   Cgccgctcgc   Gcagcccgcg   Ctgtcgcccg   Cgtcctcgcg   Ctcggtgtcc   1020   Acctcctcgt   Ccagcagcag   Cggcagcagc   Agcggcagca   Gcggcgagga   Cgccgacgac   1080   Ctgatgttcg   Acctgagctt   Gaatttctct   Caaagcgcgc   Acagcgccag   Cgagcagcag   1140   Ctggggggcg   Gcgcggcggc   Cgggaacctg   Tccctgtcgc   Tggtggataa   Ggatttggat   1200   Tcgttcagcg   Agggcagcct   Gggctcccac   Ttcgagttcc   Ccgactactg   Cacgccggag   1260   Ctgagcgaga   Tgatcgcggg   Ggactggctg   Gaggcgaact   Tctccgacct   Ggtgttcaca   1320   Tattga   1326   <210>   14   <211>   1599   <212>   DNA   <213>   Artificial sequence   <220>   <223>   ZIC2 gene   <400>   14   Atgctcctgg   Acgcgggtcc   Gcagttcccg   Gccatcgggg   Tgggcagctt   Cgcgcgccac   60   Catcaccact   Ccgccgcggc   Ggcggcggcg   Gctgccgccg   Agatgcagga   Ccgtgaactg   120   Agcctggcgg   Cggcgcagaa   Cggcttcgtt   Gactccgccg   Ccgcgcacat   Gggagccttc   180   Aagctcaacc   Cgggcgcgca   Cgagctgtcc   Ccgggccaga   Gctcggcgtt   Cacgtcgcag   240   Ggccccggcg   Cccacccccg   Ctccgctgcg   Gctgccgctg   Cggccgcagc   Gctcgggccc   300   Cacgccgcgc   Acgttggctc   Ctactctggg   Ccgcccttca   Actccacccg   Ggacttcctg   360   Ttccgcagcc   Gcggcttcgg   Ggactcggcg   Ccgggcggcg   Ggcagcacgg   Gctgttcggg   420   Ccgggcgcgg   Gcggcctgca   Ccacgcgcac   Tcggacgcgc   Agggccacct   Cctcttcccg   480   Ggcctgccag   Agcagcacgg   Gccgcacggc   Tcgcagaatg   Tgctcaacgg   Gcagatgcgc   540   Ctcgggctgc   Ccggcgaggt   Gttcgggcgc   Tcggagcaat   Accgccaggt   Ggccagcccg   600   Cggaccgacc   Cctactcggc   Ggcgcaactc   Cacaaccagt   Acggccccat   Gaatatgaac   660   Atgggtatga   Acatggcagc   Agccgcggcc   Caccaccacc   Accaccacca   Ccaccacccc   720   Ggtgcctttt   Tccgctatat   Gcggcagcag   Tgcatcaagc   Aggaggtaat   Ctgcaagtgg   780   Atcgaccccg   Agcaactgag   Caatcccaag   Aagagctgca   Acaaaacttt   Cagcaccatg   840   Cacgagctgg   Tgacacacgt   Ctcggtggag   Cacgtcggcg   Gcccggagca   Gagcaaccac   900   Gtctgcttct   Gggaggagtg   Tccgcgcgag   Ggcaagccct   Tcaaggccaa   Atacaaactg   960   Gtcaaccaca   Tccgcgtgca   Cacaggcgag   Aaacccttcc   Cctgcccctt   Cccgggctgt   1020   Ggcaaagtct   Tcgcgcgctc   Cgagaacctc   Aagatccaca   Aaaggaccca   Cacaggggag   1080   Aagccgttcc   Agtgtgagtt   Tgagggctgc   Gaccggcgct   Tcgccaacag   Cagcgacagg   1140   Aagaagcaca   Tgcacgtcca   Cacctccgat   Aagccctatc   Tctgcaagat   Gtgcgacaag   1200   Tcctacacgc   Accccagctc   Gctgcggaag   Cacatgaagg   Tccatgagtc   Ctccccgcag   1260   Ggctctgaat   Cctccccggc   Cgccagctcc   Ggctatgagt   Cgtccacgcc   Cccggggctg   1320   Gtgtccccca   Gcgccgagcc   Cgagagcagc   Tccaacctgt   Ccccagggggg   Ggcggcagcg   1380   Gcggcggcgg   Ctgcggcggc   Ggcggccgcg   Gtgtccgcgg   Tgcaccgggg   Cggaggctcg   1440   Ggcagtggcg   Gcgcgggagg   Cggctcaggc   Ggcggcagcg   Gcagtggcgg   Gggcggcggc   1500   Ggggcgggcg   Gcgggggcgg   Cggcagctct   Ggcgggggca   Gcgggacagc   Cgggggtcac   1560   Agcggcctct   Cctccaactt   Caatgaatgg   Tacgtgtga   1599   <210>   15   <211>   1404   <212>   DNA   <213>   Artificial sequence   <220>   <223>   ZIC3 gene   <400>   15   Atgacgatgc   Tcctggacgg   Aggcccgcag   Ttccctgggc   Tgggagtggg   Cagcttcggc   60   Gcgccgcgcc   Accacgagat   Gcccaaccgt   Gaggcggcag   Gcatggggct   Gaatcccttc   120   Ggggactcaa   Cccacgccgc   Cgccgccgcc   Gccgccgccg   Ctgccttcaa   Gctgagccct   180   Gccgcggcgc   Acgatctatc   Ttcaggccag   Agctcggctt   Tcacgccgca   Gggttcgggc   240   Tacgccaacg   Ccctgggcca   Ccatcaccac   Caccatcacc   Atcatcacca   Caccagccag   300   Gtgcccagct   Acggtggcgc   Tgcctctgcc   Gccttcaact   Caacgcgcga   Gtttctgttc   360   Cgccagcgca   Gctccgggct   Cagtgaggcg   Gcctcgggtg   Gcgggcagca   Cgggctcttc   420   Gccggctcgg   Cgagcagcct   Gcatgctcca   Gctggcatcc   Ccgagccccc   Tagctacttg   480   Ctgtttcccg   Ggctgcatga   Gcagggcgct   Gggcacccgt   Cgcccacagg   Gcacgtggac   540   Aacaaccagg   Tccacctggg   Gctgcgtggg   Gagctgttcg   Gccgtgctga   Cccataccgc   600   Ccagtggcca   Gcccgcgcac   Ggacccctac   Gcggccggcg   Ctcagtttcc   Taactacagc   660   Cccatgaaca   Tgaacatggg   Agtgaacgtg   Gcggcccacc   Acgggcccgg   Cgccttcttc   720   Cgttatatgc   Ggcagcctat   Caagcaggag   Ctgtcgtgca   Aggtgadcga   Cgaggctcag   780   Ctgagccggc   Ccaagaagag   Ctgcgaccgg   Accttcagca   Ccatgcatga   Gctggtgaca   840   Catgtcacca   Tggagcatgt   Ggggggcccg   Gagcagaaca   Accacgtctg   Ctactgggag   900   Gagtgccccc   Gggagggcaa   Gtctttcaag   Gcgaagtaca   Aactggtcaa   Ccacatccga   960   Gtgcacacgg   Gcgagaagcc   Cttcccatgc   Cccttcccgg   Gctgcgggaa   Gatctttgcc   1020   Cgttctgaga   Acctcaagat   Ccacaagagg   Acccacacag   Gtgagaaacc   Tttcaaatgt   1080   Gaatttgaag   Gctgtgacag   Acgctttgcc   Aacagcagcg   Accgtaagaa   Gcacatgcat   1140   Gtgcatacct   Cggacaagcc   Ctatatctgc   Aaagtgtgcg   Acaagtccta   Cacgcacccg   1200   Agctccctgc   Gcaaacacat   Gaaggttcat   Gaatctcaag   Ggtcagattc   Ctccccgct   1260   Gccagttcag   Gctatgaatc   Ttccactcca   Cccgctatag   Cttctgcaaa   Cagtaaagat   1320   Accactaaaa   Ccccttctgc   Agttcaaact   Agcaccagcc   Acaaccctgg   Acttcctcct   1380   Aattttaacg   Aatggtacgt   Ctga   1404   <210>   16   <211>   1335   <212>   DNA   <213>   Artificial sequence   <220>   <223>   GATA3 gene   <400>   16   Atggaggtga   Cggcggacca   Gccgcgctgg   Gtgagccacc   Accaccccgc   Cgtgctcaac   60   Gggcagcacc   Cggacacgca   Ccaccccggc   Ctcagccact   Cctacagga   Cgcggcgcag   120   Tacccgctgc   Cggaggaggt   Ggatgtgctt   Tttaacatcg   Acggtcaagg   Caaccacgtc   180   Ccgccctact   Acggaaactc   Ggtcagggcc   Acggtgcaga   Ggtaccctcc   Gacccaccac   240   Gggagccagg   Tgtgccgccc   Gcctctgctt   Catggatccc   Taccctggct   Ggacggcggc   300   Aaagccctgg   Gcagccacca   Caccgcctcc   Ccctggaatc   Tcagcccctt   Ctccaagacg   360   Tccatccacc   Acggctcccc   Ggggcccctc   Tccgtctacc   Cccggccctc   Gtcctcctcc   420   Ttgtcggggg   Gccacgccag   Cccgcacctc   Ttcaccttcc   Cgcccacccc   Gccgaaggac   480   Gtctccccgg   Acccatcgct   Gtccacccca   Ggctcggccg   Gctcggcccg   Gcaggacgag   540   Aaagagtgcc   Tcaagtacca   Ggtgcccctg   Cccgacagca   Tgaagctgga   Gtcgtcccac   600   Tcccgtggca   Gcatgaccgc   Cctgggtgga   Gcctcctcgt   Cgacccacca   Ccccatcacc   660   Acctacccgc   Cctacgtgcc   Cgagtacagc   Tccggactct   Tcccccccag   Cagcctgctg   720   Ggcggctccc   Ccaccggctt   Cggatgcaag   Tccaggccca   Aggcccggtc   Cagcacagaa   780   Ggcagggagt   Gtgtgaactg   Tggggcaacc   Tcgaccccac   Tgtggcggcg   Agatggcacg   840   Ggacactacc   Tgtgcaacgc   Ctgcgggctc   Tatcacaaaa   Tgaacggaca   Gaaccggccc   900   Ctcattaagc   Ccaagcgaag   Gctgtctgca   Gccaggagag   Cagggacgtc   Ctgtgcgaac   960   Tgtcagacca   Ccacaaccac   Actctggagg   Aggaatgcca   Atggggaccc   Tgtctgcaat   1020   Gcctgtgggc   Tctactacaa   Gcttcacaat   Attacagac   Ccctgactat   Gaagaaggaa   1080   Ggcatccaga   Ccagaaacg   Aaaaatgtct   Agcaaatcca   Aaaagtgcaa   Aaaagtgcat   1140   Gactcactgg   Aggacttccc   Caagaacagc   Tcgtttaacc   Cggccgccct   Ctccagacac   1200   Atgtcctccc   Tgagccacat   Ctcgcccttc   Agccactcca   Gccacatgct   Gaccacgccc   1260   Acgccgatgc   Acccgccatc   Cagcctgtcc   Tttggaccac   Accacccctc   Cagcatggtc   1320   Accgccatgg   Gttag   1335   <210>   17   <211>   1269   <212>   DNA   <213>   Artificial sequence   <220>   <223>   PAX6 gene   <400>   17   Atgcagaaca   Gtcacagcgg   Agtgaatcag   Ctcggtggtg   Tctttgtcaa   Cgggcggcca   60   Ctgccggact   Ccaccccgca   Gaagattgta   Gagctagctc   Acagcggggc   Ccggccgtgc   120   Gacatttccc   Gaattctgca   Ggtgtccaac   Ggatgtgtga   Gtaaaattct   Gggcaggtat   180   Tacgagactg   Gctccatcag   Acccagggca   Atcggtggta   Gtaaaccgag   Agtagcgact   240   Ccagaagttg   Taagcaaaat   Agcccagtat   Aagcgggagt   Gcccgtccat   Ctttgcttgg   300   Gaaatccgag   Acagattact   Gtccgagggg   Gtctgtacca   Acgataacat   Accaagcgtg   360   Tcatcaataa   Acagagttct   Tcgcaacctg   Gctagcgaaa   Agcaacagat   Gggcgcagac   420   Ggcatgtatg   Ataaactaag   Gatgttgaac   Gggcagaccg   Gaagctgggg   Cacccgccct   480   Ggttggtatc   Cggggacttc   Ggtgccaggg   Caacctacgc   Aagatggctg   Ccagcaacag   540   Gaaggagggg   Gagagaatac   Caactccatc   Agttccaacg   Gagaagattc   Agatgaggct   600   Caaatgcgac   Ttcagctgaa   Gcggaagctg   Caaagaaata   Gaacatcctt   Tacccaagag   660   Caaattgagg   Ccctggagaa   Agaggttgag   Agaacccatt   Atccagatgt   Gtttgcccga   720   Gaaagactag   Cagccaaaat   Agatctacct   Gaagcaagaa   Tacaggtatg   Gttttctaat   780   Cgaagggcca   Aatggagaag   Agaagaaaaa   Ctgaggaatc   Agagaagaca   Ggccagcaac   840   Acacctagtc   Atattcctat   Cagcagtagt   Ttcagcacca   Gtgtctacca   Accaattcca   900   Caacccacca   Caccggtttc   Ctccttcaca   Tctggctcca   Tgttgggccg   Aacagacaca   960   Gccctcacaa   Acacctacag   Cgctctgccg   Cctatgccca   Gcttcaccat   Ggcaaataac   1020   Ctgcctatgc   Aacccccagt   Ccccacccamg   Acctcctcat   Actcctgcat   Gctgcccacc   1080   Agcccttcgg   Tgaatgggcg   Gagttatgat   Acctacaccc   Ccccacatat   Gcagacacac   1140   Atgaacagtc   Agccaatggg   Cacctcgggc   Accacttcaa   Caggactcat   Ttccccggt   1200   Gtgtcagttc   Cagttcaagt   Tcccggaagt   Gaacctgata   Tgtctcaata   Ctggccaaga   1260   Ttacagtaa   1269   <210>   18   <211>   3024   <212>   DNA   <213>   Artificial sequence   <220>   <223>   SALL2 gene   <400>   18   Atgtctcggc   Gaaagcagcg   Gaaaccccaa   Cagttaatct   Cggactgcga   Aggtcccagc   60   Gcgtctgaga   Acggtgatgc   Tagcgaggag   Gatcaccccc   Aagtctgtgc   Caagtgctgc   120   Gcacaattca   Ctgacccaac   Tgaattcctc   Gcccaccaga   Acgcatgttc   Tactgaccct   180   Cctgtaatgg   Tgataattgg   Gggccaggag   Aaccccaaca   Actcttcggc   Ctcctctgaa   240   Cccggccctg   Agggtcacaa   Taatcctcag   Gtcatggaca   Cagagcatag   Caacccccca   300   Gattctgggt   Cctccgtgcc   Cacggatccc   Acctggggcc   Cagagaggag   Aggagaggag   360   Tctccagggc   Atttcctggt   Cgctgccaca   Ggtacagcgg   Ctgggggagg   Cgggggcctg   420   Atcttggcca   Gtcccaagct   Gggagcaacc   Ccattacctc   Cagaatcgac   Ccctgcaccc   480   Cctcctcctc   Caccaccccc   Tccgccccca   Ggggtaggca   Gtggccactt   Gaatatcccc   540   Ctgatcttgg   Aagagctacg   Ggtgctgcag   Cagcggcaga   Tccatcagat   Gcagatgact   600   Gagcaaatct   Gcaggcaggt   Gctgttgctt   Ggctccttag   Gccagacggt   Gggtgcccct   660   Gccagtccct   Cagagctacc   Tgggacaggg   Actgcctctt   Ccacacaagcc   Cctactaccc   720   Ctcttcagcc   Ccatcaagcc   Tgtccaaacc   Agcaagacac   Tggcatcttc   Ctcctcctcc   780   Tcctcttcct   Cttcaggggc   Agaaacgccc   Aagcaggcct   Tcttccacct   Ttaccaccca   840   Ctggggtcac   Agcatccttt   Ctctgctgga   Ggggttgggc   Gaagccacaca   Acccacccct   900   Gccccttccc   Cagccttgcc   Aggcagcaca   Gatcagctga   Ttgcctcgcc   Tcatctggca   960   Ttcccaagca   Ccaggggact   Actggcagca   Cagtgtcttg   Gggcagcccg   Aggccttgag   1020   Gccactgcct   Ccccagggct   Cctgaagcca   Aagaatggaa   Gtggtgagct   Gagctacgga   1080   Gaagtgatgg   Gtcccttgga   Gaagcctggt   Ggaaggcaca   Aatgccgctt   Ctgtgccaaa   1140   Gttattggca   Gtgacagtgc   Cctgcagatc   Caccttcgtt   Cccacacggg   Tgagaggccc   1200   Tataagtgca   Atgtctgtgg   Aaaccgtttt   Accacccgtg   Gcaacctcaa   Agtgcatttc   1260   Caccggcatc   Gtgagaagta   Cccacatgtg   Cagatgaacc   Cacacccagt   Accagagcac   1320   Ctagactatg   Tcattaccag   Cagtggcttg   Ccttatggta   Tgtccgtgcc   Accagagaag   1380   Gccgaggagg   Aggcagccac   Tccaggtgga   Ggggttgagc   Gcaagcctct   Ggtggcctcc   1440   Acaacagcac   Tcagtgccac   Agagagcctg   Actctgctct   Ccaccagtgc   Aggcacagcc   1500   Acggctccag   Gactccctgc   Tttcaataag   Tttgtgctca   Tgaaagcagt   Ggaacccaag   1560   Aataaagctg   Atgaaaacac   Ccccccaggg   Agtgagggct   Cagccatcag   Tggagtggca   1620   Gaaagtagca   Cggcaactcg   Catgcaacta   Agtaagttgg   Tgacttcact   Accaagctgg   1680   Gcactgctta   Ccaaccactt   Caagtccact   Ggcagcttcc   Ccttccccta   Tgtgctagag   1740   Cccttggggg   Cctcaccctc   Tgagacatca   Aagctgcagc   Aactggtaga   Aaagattgac   1800   Cggcaaggag   Ctgtggcggt   Gacctcagct   Gcctcaggag   Cccccaccac   Ctctgcccct   1860   Gcaccttcat   Cctcagcctc   Ttctggacct   Aaccagtgtg   Tcatctgtct   Ccgagtgctt   1920   Agctgtcctc   Gggccctacg   Ccttcattat   Ggccaacatg   Gaggtgagag   Gcccttcaaa   1980   Tgcaaagtgt   Gtggcagagc   Cttctccacc   Aggggtaatc   Tgcgtgcaca   Tttcgtgggc   2040   Cacaaggcca   Gtccagctgc   Ccgggcacag   Aattcctgcc   Ccatctgcca   Gaagaagttc   2100   Accaatgctg   Tcactctgca   Gcagcatgtc   Cggatgcacc   Tggggggcca   Gatccccaac   2160   Ggtggtactg   Cactccctga   Aggtggagga   Gctgctcagg   Agaatggctc   Cgagcaatct   2220   Acagtctccg   Gggcagggag   Tttcccccag   Cagcagtccc   Agcagccatc   Accggaagag   2280   Gagttgtctg   Aggaggagga   Agaggaggat   Gaggaagaag   Aggaagatgt   Gactgatgaa   2340   Gattccctgg   Cagggagagg   Ctcagagagt   Ggaggtgaga   Aggcaatatc   Agtgagaggt   2400   Gattcagaag   Aggcatctgg   Ggcagaggag   Gaggtgggga   Cagtggcggc   Agcagccaca   2460   Gctgggaagg   Agatggacag   Taatgagaaa   Actactcaac   Agtcttcttt   Gccaccacca   2520   Ccaccacctg   Acagcctgga   Tcagcctcag   Ccaatggagc   Agggaagcag   Tggtgtttta   2580   Ggaggcaagg   Aagagggggg   Caaaccggag   Agaagctcaa   Gtccggcatc   Agcactcacc   2640   Ccagaagggg   Aagccaccag   Cgtgaccttg   Gtagagagag   Tgagcctgca   Ggaggcaatg   2700   Agaaaggagc   Caggagagag   Cagcagcaga   Aaggcctgcg   Aagtgtgtgg   Ccaggccttt   2760   Ccctcccagg   Cagctctgga   Ggagcatcag   Aagacccacc   Ccaaggaggg   Gccgctcttc   2820   Acttgtgttt   Tctgcaggca   Gggctttctt   Gagcgggcta   Ccctcaagaa   Gcatatgctc   2880   Ctggcacacc   Accaggtaca   Gccctttgcc   Ccccatggcc   Ctcagaatat   Tgctgctctt   2940   Tctctagtcc   Ctggctgttc   Gccttccatc   Acctccacag   Ggctctcccc   Ctttccccga   3000   Aaagatgacc   Ccaccatccc   Atga   3024   <210>   19   <211>   1221   <212>   DNA   <213>   Artificial sequence   <220>   <223>   LHX2 gene   <400>   19   Atgctgttcc   Acagtctgtc   Gggccccgag   Gtgcacgggg   Tcatcgacga   Gatggaccgc   60   Agggccaaga   Gcgaggctcc   Cgccatcagc   Tccgccatcg   Accgcggcga   Caccgagacg   120   Accatgccgt   Ccatcagcag   Tgaccgcgcc   Gcgctgtgcg   Ccggctgcgg   Gggcaagatc   180   Tcggaccgct   Actacctgct   Ggcggtggac   Aagcagtggc   Acatgcgctg   Cctcaagtgc   240   Tgcgagtgca   Agctcaacct   Ggagtcggag   Ctcacctgtt   Tcagcaagga   Cggtagcatc   300   Tactgcaagg   Aagactacta   Caggcgcttc   Tctgtgcagc   Gctgcgcccg   Ctgccacctg   360   Ggcatctcgg   Cctcggagat   Ggtgatgcgc   Gctcgggact   Tggtttatca   Cctcaactgc   420   Ttcacgtgca   Ccacgtgtaa   Caagatgctg   Accacgggcg   Accacttcgg   Catgaaggac   480   Agcctggtct   Actgccgctt   Gcacttcgag   Gcgctgctgc   Agggcgagta   Ccccgcacac   540   Ttcaaccatg   Ccgacgtggc   Agcggcggcc   Gctgcagccg   Cggcggccaa   Gaggcggggg   600   Ctgggcgcag   Caggggccaa   Ccctctgggt   Cttccctact   Acaatggcgt   Gggcactgtg   660   Cagaaggggc   Ggccgaggaa   Acgtaagagc   Ccgggccccg   Gtgcggatct   Ggcggcctac   720   Aacgctgcgc   Taagctgcaa   Cgaaaacgac   Gcagagcacc   Tggaccgtga   Ccaggcatac   780   Ccgagcagcc   Agaagaccaa   Gcgcatgcgc   Acgtccttca   Agcaccacca   Gcttcggacc   840   Atgaagtctt   Actttgccat   Taaccacaac   Cccgacgcca   Aggacttgaa   Gcagctcgcg   900   Caaaagacgg   Gcctcaccaa   Gcgggtcctc   Caggtctggt   Tccagaacgc   Ccgagccaag   960   Ttcaggcgca   Acctcttacg   Gcaggaaaac   Acgggcgtgg   Acaagtcgac   Agacgcggcg   1020   Ctgcagacag   Ggacgccatc   Gggcccggcc   Tcggagctct   Ccaacgcctc   Gctcagcccc   1080   Tccagcacgc   Ccaccaccct   Gacagacttg   Actagcccca   Ccctgccaac   Tgtgacgtcc   1140   Gtcttaactt   Ctgtgcctgg   Caacctggag   Ggccatgagc   Ctcacagccc   Ctcacaaacg   1200   Actcttacca   Accttttcta   a   1221   <210>   20   <211>   1236   <212>   DNA   <213>   Artificial sequence   <220>   <223>   MBD2 gene   <400>   20   Atgcgcgcgc   Acccgggggg   Aggccgctgc   Tgcccggagc   Aggaggaggg   Ggagagtgcg   60   Gcgggcggca   Gcggcgctgg   Cggcgactcc   Gccatagagc   Aggggggcca   Gggcagcgcg   120   Ctcgccccgt   Ccccggtgag   Cggcgtgcgc   Agggaaggcg   Ctcggggcgg   Cggccgtggc   180   Cgggggcggt   Ggaagcaggc   Gggccggggc   Ggcggcgtct   Gtggccgtgg   Ccggggccgg   240   Ggccgtggcc   Ggggacgggg   Acggggccgg   Ggccggggcc   Gcggccgtcc   Cccgagtggc   300   Ggcagcggcc   Ttggcggcga   Cggcggcggc   Tgcggcggcg   Gcggcagcgg   Tggcggcggc   360   Gccccccggc   Gggagccggt   Ccctttcccg   Tcggggagcg   Cggggccggg   Gcccagggga   420   Ccccgggcca   Cggagagcgg   Gaagaggatg   Gattgcccgg   Ccctcccccc   Cggatggaag   480   Aaggaggaag   Tgatccgaaa   Atctgggcta   Agtgctggca   Agagcgatgt   Ctactacttc   540   Agtccaagtg   Gtaagaagtt   Cagaagcaag   Cctcagttgg   Caaggtacct   Gggaaatact   600   Gttgatctca   Gcagttttga   Cttcagaact   Ggaaagatga   Tgcctagtaa   Attacagaag   660   Aacaaacaga   Gactgcgaaa   Cgatcctctc   Aatcaaaata   Agggtaaacc   Agacttgaat   720   Acaacattgc   Caattagaca   Aacagcatca   Attttcaaac   Aaccggtaac   Caaagtcaca   780   Aatcatccta   Gtaataaagt   Gaaatcagac   Ccacaacgaa   Tgaatgaaca   Gccacgtcag   840   Cttttctggg   Agaagaggct   Acaaggactt   Agtgcatcag   Atgtaacaga   Acaaattata   900   Aaaaccatgg   Aactacccaa   Aggtcttcaa   Ggagttggtc   Caggtagcaa   Tgatgagacc   960   Cttttatctg   Ctgttgccag   Tgctttgcac   Acaagctctg   Cgccaatcac   Agggcaagtc   1020   Tccgctgctg   Tggaaaagaa   Ccctgctgtt   Tggcttaaca   Catctcaacc   Cctctgcaaa   1080   Gcttttattg   Tcacagatga   Agacatcagg   Aaacaggaag   Agcgagtaca   Gcaagtacgc   1140   Aagaaattgg   Aagaagcact   Gatggcagac   Atcttgtcgc   Gagctgctga   Tacagaagag   1200   Atggatattg   Aaatggacag   Tggagatgaa   Gcctaa   1236   <210>   twenty one   <211>   2436   <212>   DNA   <213>   Artificial sequence   <220>   <223>   DEPDC1 gene   <400>   twenty one   Atggagagtc   Agggtgtgcc   Tcccgggcct   Tatcgggcca   Ccaagctgtg   Gaatgaagtt   60   Accacatctt   Ttcgagcagg   Aatgcctcta   Agaaaacaca   Gacaacactt   Taaaaaatat   120   Ggcaattgtt   Tcacagcagg   Agaagcagtg   Gattggcttt   Atgacctatt   Aagaaataat   180   Agcaattttg   Gtcctgaagt   Tacaaggcaa   Cagactatcc   Aactgttgag   Gaaatttctt   240   Aagaatcatg   Taattgaaga   Tatcaaaggg   Aggtggggat   Cagaaaatgt   Tgatgataac   300   Aaccagctct   Tcagatttcc   Tgcaacttcg   Cacttataaa   Ctctaccacg   Aaggtatcca   360   Gaattgagaa   Aaaacaacat   Agagaacttt   Tccaaagata   Aagatagcat   Ttttaaatta   420   Cgaaacttat   Ctcgtagaac   Tcctaaaagg   Catggattac   Atttatctca   Ggaaaatggc   480   Gagaaaataa   Agcatgaaat   Aatcaatgaa   Gatcaagaaa   Atgcaattga   Taatagagaa   540   Ctaagccagg   Aagatgttga   Gaagagtttgg   Agatatgtta   Ttctgatcta   Cctgcaaacc   600   Attttaggtg   Tgccatccct   Agaagagtc   Ataaatccaa   Aacaagtaat   Tccccaatat   660   Ataatgtaca   Acatggccaa   Tacaagtaaa   Cgtggagtag   Tttaactaca   Aaacaaatca   720   Gatgacctcc   Ctcactgggt   Attatctgcc   Atgaagtgcc   Tagcaaattg   Gccaagaagc   780   Aatgatatga   Ataatccaac   Tttagttgga   Tttgaacgag   Atgtattcag   Aacaatcgca   840   Gattattttc   Tagatctccc   Tgaacctcta   Cttacttttg   Aatattacga   Attatttgta   900   Aacattttgg   Ttgtttgtgg   Ctacatcaca   Gtttcagata   Gatccagtgg   Gatacataaa   960   Attcaagatg   Atccacagtc   Ttcaaaattc   Cttcacttaa   Acaatttgaa   Ttccttcaaa   1020   Tcaactgagt   Gccttcttct   Cagtctgctt   Catagagaaa   Aaaacaaaga   Agaatcagat   1080   Tctactgaga   Gactacagat   Aagcaatcca   Ggatttcaag   Aaagatgtgc   Taagaaaatg   1140   Cagctagtta   Atttaagaaa   Cagaagagtg   Agtgctaatg   Acataatggg   Aggaagttgt   1200   Cataatttaa   Tagggttaag   Taatatgcat   Gatctatcct   Ctaacagcaa   Accaaggtgc   1260   Tgttctttgg   Aaggaattgt   Agatgtgcca   Gggaattcaa   Gtaaagaggc   Atccagtgtc   1320   Tttcatcaat   Cttttccgaa   Catagaagga   Caaaataata   Aactgttttt   Agagcttaag   1380   Cccaaacagg   Aattcctgtt   Gaatcttcat   Tcagaggaaa   Atattcaaaa   Gccattcagt   1440   Gctggtttta   Agagaacctc   Tactttgact   Gttcaagacc   Aagaggagtt   Gtgtaatggg   1500   Aaatgcaagt   Caaaacagct   Ttgtaggtct   Cagagtttgc   Ttttaagaag   Tagtacaaga   1560   Aggaatagtt   Atatcaatac   Accagtggct   Gaaattatca   Tgaaaccaaa   Tgttggacaa   1620   Ggcagcacaa   Gtgtgcaaac   Agctatggaa   Agtgaactcg   Gagagtctag   Tgccacaatc   1680   Aataaaagac   Tctgcaaaag   Tacaatagaa   Ctttcagaaa   Attctttact   Tccagcttct   1740   Tctatgttga   Ctggcacaca   Aagcttgctg   Caacctcatt   Tagagagggt   Tgccatcgat   1800   Gctctacagt   Tatgttgttt   Gttacttccc   Ccacacaacat   Gtagaaagct   Tcaactttta   1860   Atgcgtatga   Tttcccgaat   Gagtcaaaat   Gttgatatgc   Ccaaacttca   Tgatgcaatg   1920   Ggtacgaggt   Cactgatgat   Acataccttt   Tctcgatgtg   Tgttatgctg   Tgctgaagaa   1980   Gtggatcttg   Atgagcttct   Tgctggaaga   Ttagtttctt   Tcttaatgga   Tcatcatcag   2040   Gaaattcttc   Aagtaccctc   Ttacttacag   Actgcagtgg   Aaaaacatct   Tgactactta   2100   Aaaaagggac   Atattgaaaa   Tcctggagat   Ggactatttg   Ctcctttgcc   Aacttactca   2160   Tactgtaagc   Agattagtgc   Tcaggagttt   Gatgagcaaa   Aagtttctac   Ctctcaagct   2220   Gcaattgcag   Aacttttaga   Aaatattatt   Aaaaacagga   Gtttacctct   Aaaggagaaa   2280   Gaagaaaaac   Taaaacagtt   Tcagaaggaa   Tatcctttga   Tatatcagaa   Aagatttcca   2340   Accacggaga   Gtgaagcagc   Actttttggt   Gacaaaccta   Caatcaagca   Accaatgctg   2400   Attttaagaa   Aaccaaagtt   Ccgtagtcta   Agataa   2436   <210>   twenty two   <211>   1803   <212>   DNA   <213>   Artificial sequence   <220>   <223>   MYEF2 gene   <400>   twenty two   Atggcggacg   Ccaacaaggc   Cgaggtgccc   Ggggccactg   Gtggcgacag   Cccgcacctg   60   Cagcccgcag   Agccgccggg   Cgagccgcgg   Cgagagccgc   Accccgcgga   Ggcggagaag   120   Cagcagccgc   Agcacagcag   Cagctccaat   Ggcgttaaaa   Tggagaatga   Tgaatcagca   180   Aaagaagaga   Aatctgactt   Aaaggaaaaa   Tctacaggaa   Gtaagaaggc   Caatagattt   240   Catccttatt   Caaaagacaa   Gaattcgggc   Gctggagaaa   Gaagagggtcc   Aaatcgtaac   300   Agagttttca   Ttagcaacat   Cccatatgac   Atgaaatggc   Aagctattaa   Agatctaatg   360   Agagagaaag   Ttggtgaggt   Tacatacgtg   Gagctcttta   Agtaggcgga   Aggaaaatca   420   Aggggttgtg   Gtgtggttga   Attcaaagat   Gaagaatttg   Taaagaaagc   Cctagaaact   480   Atgaacaaat   Atgatcttag   Tggaagaccc   Cttaatatta   Aagaggatcc   Tgatggagaa   540   Aatgctcgta   Gggcattgca   Gcgaacagga   Ggatcatttc   Caggaggaca   Cgtccctgat   600   Atgggatcag   Ggttgatgaa   Tttaccacct   Tccatactca   Ataatccaaa   Cattcctcct   660   Gaagtcatca   Gtaatttgca   Ggccggtaga   Cttggttcca   Caatttttgt   Tgccaatctt   720   Gacttcaaag   Ttggttggaa   Gaagctaaag   Gaagtgttca   Gcatagctgg   Aactgtgaag   780   Cgggcagata   Ttaaagaaga   Caaagatggc   Aagagcagag   Gaatgggcac   Tgtcactttt   840   Gagcaagcaa   Ttgaagcagt   Tcaagcaatt   Tctatgttca   Atgggcagtt   Tttatttgat   900   Agactatgc   Atgtgaaaat   Ggatgacaag   Tctgttcctc   Atgaagagta   Ccgttcacat   960   Gatggtaaaa   Caccacaatt   Accacgtggt   Cttggaggca   Ttgggatggg   Acttggtccg   1020   Ggtggacagc   Ctattagtgc   Cagccagttg   Aacataggtg   Gagtaatggg   Aaatttaggt   1080   Ccaggtggta   Tgggaatgga   Tggtccaggt   Tttggaggaa   Tgaatagaat   Tggaggagga   1140   Atagggtttg   Gtggtctgga   Agcaatgaat   Agcatgggag   Gatttggagg   Agttggccga   1200   Atgggagagc   Tgtaccgtgg   Tgcgatgact   Agtagcatgg   Agcgagattt   Tggacgtggt   1260   Gatattggaa   Taaatcaagg   Ctttggagat   Tcctttggta   Gacttggcag   Tgcaatgatt   1320   Ggagggtttg   Caggaagaat   Agtagcttct   Aacatgggtc   Cagtaggatc   Tggaataagt   1380   Ggtggaatgg   Gtagcatgaa   Cagtgtgact   Ggaggaatgg   Gtaggggact   Ggaccggatg   1440   Agttccagct   Ttgatagaat   Gggaccaggt   Ataggagcta   Tactggaaag   Gagcatcgat   1500   Atggatcgag   Gatttttatc   Gggtccaatg   Ggaagcggaa   Tgagagagag   Aataggctcc   1560   Aaaggcaacc   Agatatttgt   Cagaaatcta   Cctttgact   Tgacttggca   Gaaactaaaa   1620   Gagaaattca   Gtcagtgtgg   Tcatgtaatg   Tttgcagaaa   Taaaaatgga   Gaatggaaag   1680   Tcaaaaggct   Gtggaacagt   Cagatttgac   Tccccagaat   Cagctgaaaa   Agcctgcaga   1740   Ataatgaatg   Gcataaaaat   Cagtggcaga   Gaaattgatg   Ttcgcttgga   Tcgtaatgca   1800   Taa   1803   <210>   twenty three   <211>   870   <212>   DNA   <213>   Artificial sequence   <220>   <223>   OTX2a gene   <400>   twenty three   Atgatgtctt   Atcttaagca   Accgccttac   Gcagtcaatg   Ggctgagtct   Gaccacttcg   60   Ggtatggact   Tgctgcaccc   Ctccgtgggc   Tacccggcca   Cccccggaga   Acagcgccgg   120   Gagaggacga   Cgttcactcg   Ggcgcagcta   Gatgtgctgg   Aagcactgtt   Tgccaagacc   180   Cggtacccag   Acatcttcat   Gcgagaggag   Gtggcactga   Aaatcaactt   Gcccgagtcg   240   Agggtgcagg   Tatggtttaa   Gaatcgaaga   Gctaagtgcc   Gccaacaaca   Gcaacaacag   300   Cagaatggag   Gtcaaaacaa   Agtgagacct   Gccaaaaaga   Agacatctcc   Agctcgggaa   360   Gtgagttcag   Agagggaac   Aagtggccaa   Ttcactcccc   Cctctagcac   Ctcagtcccg   420   Accattgcca   Gcagcagtgc   Tcctgtgtct   Atctggagcc   Cagcttccat   Ctccccactg   480   Tcagatccct   Tgtccacctc   Ctcttcctgc   Atgcagaggt   Cctatcccat   Gacctatact   540   Caggcttcag   Gttatagtca   Agtagatgct   Ggctcaactt   Cctactttgg   Gggcatggac   600   Tgtggatcat   Atttgacccc   Tatgcatcac   Cagcttcccg   Gaccaggggc   Cacactcagt   660   Cccatgggta   Ccaatgcagt   Caccagccat   Ctcaatcagt   Ccccagcttc   Tctttccacc   720   Cagggatatg   Gagcttcaag   Cttgggtttt   Aactcaacca   Ctgattgctt   Ggattataag   780   Gaccaaactg   Cctcctggaa   Gcttaacttc   Aatgctgact   Gcttggatta   Taaagatcag   840   Acatcctcgt   Ggaaattcca   Ggttttgtga   870   <210>   twenty four   <211>   999   <212>   DNA   <213>   Artificial sequence   <220>   <223>   SIX3 gene   <400>   twenty four   Atggtattcc   Gctcccccct   Agacctctat   Tcctcccact   Tcttgttgcc   Aaacttcgcc   60   Gattctcacc   Accgctccat   Acttctggcg   Agtagcggcg   Gcgggaacgg   Tgcgggaggc   120   Ggcggcggcg   Cgggaggcgg   Cagcggcggc   Gggaacggtg   Cgggaggcgg   Cggtgctggc   180   Ggagcaggcg   Gcggcggcgg   Cggcggctcc   Agggcccccc   Cggaagagtt   Gtccatgttc   240   Cagctgccca   Ccctcaactt   Ctcgccggag   Caggtggcca   Gcgtctgtga   Gacgctggag   300   Gagacgggcg   Acatcgagcg   Gctgggccgc   Ttcctctggt   Cgctgcccgt   Ggcccccggg   360   Gcgtgcgagg   Ccatcaacaa   Acacgagtcg   Atcctgcgcg   Cgcgcgccgt   Ggtcgccttc   420   Cacacgggca   Acttccgcga   Cctctaccac   Atccttgaga   Accacaagtt   Caccaaggag   480   Tctcacggca   Agctgcaggc   Catgtggctc   Gaggcgcact   Accaggaggc   Cgagaagctg   540   Cgcggccgcc   Cactcggccc   Ggtggacaag   Taccgcgtgc   Gcaagaagtt   Cccgctgcca   600   Cgcaccatct   Gggacggcga   Gcagaagacg   Cattgcttca   Aggaggggac   Tcggagcctg   660   Ttgcgggagt   Ggtacctaca   Ggacccctac   Cccaacccca   Gcaagaaacg   Cgaactggcg   720   Caggccaccg   Gcctcactcc   Cacacaagta   Ggcaactggt   Ttaagaaccg   Gcggcagcgc   780   Gaccgcgccg   Cggcggccaa   Gaacaggctc   Cagcaccagg   Ccattggacc   Gaggggcatg   840   Cgctcgctgg   Ccgagcccgg   Ctgccccacg   Cacggctcgg   Cagagtcgcc   Gtccacggcg   900   Gccagcccga   Ccaccagcgt   Gtccagcctg   Acggagcgcg   Cagacaccgg   Cacctccatc   960   Ctctcggtaa   Cctccagcga   Ctcggaatgt   Gatgtatga   999   <210>   25   <211>   1176   <212>   DNA   <213>   Artificial sequence   <220>   <223>   SOX1 gene   <400>   25   Atgtacagca   Tgatgatgga   Gaccgacctg   Cactcgcccg   Gcggcgccca   Ggcccccacg   60   Aacctctcgg   Gccccgccgg   Ggcgggcggc   Ggcgggggcg   Gaggcggggg   Cggcggcggc   120   Ggcgggggcg   Ccaaggccaa   Ccaggaccgg   Gtcaaacggc   Ccatgaacgc   Cttcatggtg   180   Tggtcccgcg   Ggcagcggcg   Caagatggcc   Caggagaacc   Ccaagatgca   Caactcggag   240   Atcagcaagc   Gcctgggggc   Cgagtggaag   Gtcatgtccg   Aggccgagaa   Gcggccgttc   300   Atcgacgagg   Ccaagcggct   Gcgcgcgctg   Cacatgaagg   Agcacccgga   Ttacaagtac   360   Cggccgcgcc   Gcaagaccaa   Gacgctgctc   Aagaaggaca   Agtactcgct   Ggccggcggg   420   Ctcctggcgg   Ccggcgcggg   Tggcggcggc   Gcggctgtgg   Ccatgggcgt   Gggcgtgggc   480   Gtgggcgcgg   Cggccgtggg   Ccaggccctg   Gagagcccag   Gcggcgcggc   Gggcggcggc   540   Tacgcgcacg   Tcaacggctg   Ggccaacggc   Gcctaccccg   Gctcggtggc   Ggcggcggcg   600   Gcggccgcgg   Ccatgatgca   Ggaggcgcag   Ctggcctacg   Ggcagcaccc   Gggcgcgggc   660   Ggcgcgcacc   Cgcacgcgca   Ccccgcgcac   Ccgcacccgc   Accacccgca   Cgcgcacccg   720   Cacaacccgc   Agcccatgca   Ccgctacgac   Atgggcgcgc   Tgcagtacag   Ccccatctcc   780   Aactcgcagg   Gctacatgag   Cgcgtcgccc   Tcgggctacg   Gcggcctccc   Ctacggcgcc   840   Gcggccgccg   Ccgccgccgc   Tgcgggcggc   Gcgcaccaga   Actcggccgt   Ggcggcggcg   900   Gcggcggcgg   Cggccgcgtc   Gtcgggcgcc   Ctgggcgcgc   Tgggctctct   Ggtgaagtcg   960   Gagcccagcg   Gcagcccgcc   Cgccccagcg   Cactcgcggg   Cgccgtgccc   Cggggacctg   1020   Cgcgagatga   Tcagcatgta   Cttgcccgcc   Ggcgaggggg   Gcgacccggc   Ggcggcagca   1080   Gcggccgcgg   Cgcagagccg   Gctgcactcg   Ctgccgcagc   Actaccaggg   Cgcgggcgcg   1140   Ggcgtgaacg   Gcacggtgcc   Cctgacgcac   Atctag   1176   <210>   26   <211>   2004   <212>   DNA   <213>   Artificial sequence   <220>   <223>   PAX6: EGFP gene <400> 26 tgaggtgtgt cctaatcgtg cggcattcaa caaatggact tctggtgtgt ggtcagaaga 60 gaaaagccat ttacttactt tcctccccgg ttttctggca acagctgaag gggagctgcc 120 tccgtggact gagcagaccc aggagaggga gtcgtggtgc ggagacacac gcaccacaca 180 cagatgaccg gtggcacaca cgacacacgc tgacataccg acatcgccag tgggacacac 240 acacacacac acacacacac acacacacac acagagagag agagaatccc tcccagcatt 300 ggtcatccgc ccccccaccc aggcttccac tccccctccc ctcttatctc ccctggcttc 360 ccctcctctc gggcgctgcg aaaagcagcc gcacttagtc aacaaatggc acgtgggaga 420 agttggtgag tgtttggtga ggactcttca gggcttttca caagaaccct ctgtacacaa 480 agtaagtggc gtgtttactc gggcctctcc agccagagct gtgcctctgc tccgctgcgc 540 accgcggctt ccgaaaggag aaaggagaga agaaagggcg gggagagcgg ggtggaggat 600 ttggacaggc cctggaggct tgggctgggg aggcctctgg cctcgtttag ttctcggccc 660 ggcaacctcc tctcggccta ggcttcgccg cggcctccgc agctggaatg gagctgccag 720 gacccagtga cgctcccgcc cctttcctct tcttccaagg ggccaggtgg gctggggtgc 780 ggccgccgct gtgctctgtg tcttggggcc Ccggctggga tggggtgggg gcgggcgggg 8 40 gcggggcggc aggccacgct gtcctggagt tggcaagaaa ggacagcaca gaaacttgca 900 ccctccgagg actgggagtc ccgagtccag cttaggggga gtgggggcgc gacccccaac 960 ccagaaacct tcacttgacc gctcaagttc gcggcagcag ggcgggccgc gccgaatctc 1020 ggcgtgcgcg gagcggggag atgcaggcga gcgccagagc ccgggctcgg gggccctgcg 1080 ccggggagag gagccgggac ccaccggcgg agccgaaaac aagtgtattc atattcaaac 1140 aaacggacca attgcaccag gcggggagag ggagcatcca atcggctggc gcgaggcccc 1200 ggcgctgctt tgcataaagc aatattttgt gtgagagcga gcggtgcatt tgaagcttag 1260 atctggatcc cctctagagt cgagatggtg agcaagggcg aggagctgtt caccggggtg 1320 gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc 1380 gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc 1440 aagctgcccg tgccctggcc caccctcgtg accaccctga cctacggcgt gcagtgcttc 1500 agccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc 1560 tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag 1620 gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag 1680 gagga cggca acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat 1740 atcatggccg acaagcagaa gaacggcatc aaggtgaact tcaagatccg ccacaacatc 1800 gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc 1860 cccgtgctgc tgcccgacaa ccactacctg agcacccagt ccgccctgag caaagacccc 1920 aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc 1980 ggcatggacg agctgtacaa gtaa 2004 <210> 27 <211> 1810 <212> DNA <213> Artificial Sequence <220> <223> SOX1: EGFP gene <400> 27 caacccaatc gttaatcatt cggaacgcgc gggcggggag cggcgaggag ggcgagctcg 60 gggttcgccg ccgccgccgc cgccgcgcgc gcgcgctcag gaagcggtgt ggctgtcacc 120 ccctcccggg cctcctcccc cctccttcct gctttgctcc ccctccttcc tcccctcctc 180 cccgctccgc cgcccgcgcc cagtgtatct actccctccc cacgtcactc gccagcgcgc 240 catgcaaatc accgccgccg ccggctccca ttggccgcgg cgcgctcatt taatggcagc 300 ccgggcccgg cgtatggctg ctgggccccg cgcgccgccg gccccgcgtg cgcctccgct 360 ccgagcgcac ggccccgggc aggcagcggg cagcccatcc cgggctcggc ggccccggct 420 ctccggccct ctccgcgagc ccgcgctcct cccgctgtcc ccgggcccct ccctggctgc 480 accgtaatcg ccccctgcag gcccccctgc gcctcccccc ccccgccact ggcgcctggc 540 ttcccccggg cacctgggac cagcacatgc ccagcgcacg cggcgcgccg ccctgctaga 600 agttgcagcc tccgagttgg aggccgctga ggaccgagcg caggaggaag gagacagcgc 660 gcagcggcgg ccggcgagga gacagcacac cccgggccgg gcccagcgca ccgctcccgg 720 ccccaaaagc ggagctgcaa cttggccacg actgcacctg tttgcaccgc tccgccgagg 780 gcgcctgggc tgcggtggcg gcgaagacgg Cgaccccgac cgtcggcctc tttggcaagt 8 40 ggtttgtgca tcaggagaaa ctttccacct gcgagccgaa ccggcgccga gtgcgtgtgt 900 ttctgccttt ttttgttgtc gttgcctcca cccctcccca ttcttctctc cgctaggacc 960 cccccgcccc cgtctcactc cgtctgaatt cctctccgtc tccctcccac cccggccgtc 1020 tatgctccag gccctctcct cgcggtgccg gtgaacccgc cagccgcccc gggatccacc 1080 ggtcgccacc atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt 1140 cgagctggac ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga 1200 tgccacctac ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc 1260 ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga 1320 ccacatgaag cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg 1380 caccatcttc ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg 1440 cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat 1500 cctggggcac aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa 1560 gcagaagaac ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt 1620 gcagctcgcc gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc 1680 cgaca Accac tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga 1740 tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct 1800 gtacaagtaa 1810

Claims (15)

A kit for screening a drug candidate for treating a neurological disease, comprising a first to a sixth polynucleotide comprising CBX2 (SEQ ID NO: 1) and HES1 (SEQ ID NO: 2), respectively. , ID1 (SEQ ID NO: 3), TFAP2A (SEQ ID NO: 4), ZFP42 (SEQ ID NO: 5), and ZNF423 (SEQ ID NO: 6). The kit according to claim 1, further comprising a seventh to a fifteenth polynucleotide comprising DACH1 (SEQ ID NO: 7), FOXG1 (SEQ ID NO: 8), and MYCN (SEQ ID NO: 9), NR2F2 (sequence number: 10), NR6A1 (sequence number: 11), SOX2 (sequence number: 12), SOX11 (sequence number: 13), ZIC2 (sequence number: 14), and ZIC3 (sequence number: 15) Gene. The kit of claim 2, further comprising a sixteenth to a twenty-fifth polynucleotide comprising GATA3 (SEQ ID NO: 16), PAX6 (SEQ ID NO: 17), SALL2 (sequence) No.: 18), LHX2 (sequence number: 19), MBD2 (sequence number: 20), DEPDC1 (sequence number: 21), MYEF2 (sequence number: 22), OTX2a (sequence number: 23), SIX3 (sequence number: 24) and the gene of SOX1 (sequence number: 25). The kit of claim 1 further comprising a reporter polynucleotide comprising the sequence of SEQ ID NO: 26. The kit of claim 1 further comprising an enhancer selected from the group consisting of RepSox, PP242, DZNep, vitamin C, and combinations thereof. A method for screening a drug candidate for treating a neurological disease, comprising: (a) transferring the first to sixth polynucleotides described in claim 1 into a fibroblast, thereby inducing the The fibroblast is transformed into an induced embryonic neural precursor cell; (b) the induced embryonic neural precursor cell of step (a) is cultured in a differentiation culture medium, thereby inducing transformation of the induced embryonic neural precursor cell into a star a cell, an oligodendrocyte or a neuron; (c) contacting the stellate cell of step (b), the oligodendrocyte or the neuron with one or more drugs to be tested; and (d) The candidate drug is screened in the one or more test drugs, wherein the candidate drug changes the phenotype, gene expression of the stellate cell, the oligodendrocyte or the neuron. The method of claim 6, wherein the fibroblast is derived from an individual suffering from a neurological disease. The method of claim 7, wherein the neurological disease is a neurodegenerative disease. The method of claim 6, wherein in the step (a), the first to sixth polynucleotides are co-transferred into the fibroblast with a reporter polynucleotide, wherein the polynucleoside is reported The acid comprises the sequence of SEQ ID NO: 26. A use of the first to sixth polynucleotides according to claim 1, which is for preparing a medicament for treating an individual suffering from or suspected of suffering from a neurological disease. The use of claim 10, wherein the neurological disease is a neurodegenerative disease. An induced embryonic neural precursor cell prepared by using the kit of claim 1, which can differentiate into a stellate cell, an oligodendrocyte or a neuron. A stellate cell prepared using the kit of claim 1. An oligodendrocyte cell prepared using the kit of claim 1. A neuron prepared using the kit of claim 1.