KR20220021553A - Neurodegenerative diseases model animal and method for producing the same - Google Patents

Abstract

The present invention relates to an animal model of neurodegenerative diseases using PARIS overexpression. An animal model obtained by crossing a responder animal introduced with a vector capable of controlling the expression of PARIS according to the present invention with a Tet-OFF transgenic switch system and a driver animal containing a certain neuron-specific promoter may be induced to show dopamine cell death, motor disorder, brain tissue neuroinflammation, aggregate formation and mitochondrial abnormalities by overexpressing PARIS in specific neurons, and thus may be induced to show movement disorders (slow movement, dyskinesia) and non-motor disorders (anxiety, dysosmia) in a very short time to a degree similar to those of actual patients, and thus may be advantageously used as an animal model fully representing the lesions of degenerative brain diseases, especially mitochondrial dysfunction and aggregate lesions.

Description

Neurodegenerative disease animal model and manufacturing method thereof

The present invention relates to an animal model of a neurodegenerative disease using PARIS overexpression.

Neurodegenerative brain diseases have characteristic denatured protein aggregates as a major lesion. That is, in the case of Parkinson's disease and Lewy body dementia disease, the main lesion is a Lewy body composed of denatured α-synuclein.

Dementia is a representative chronic progressive degenerative brain disease, which is defined as a complex of symptoms that affects both mental, cognitive and social behavioral abilities, leading to disturbances in normal daily life. , is emerging as a serious social and economical health issue for the elderly. Dementia includes Alzheimer's disease, in which cognitive decline progresses without apparent cause, vascular dementia caused by cerebrovascular diseases such as recurrent cerebral infarction, Parkinson's dementia with Lewy body lesions, and Lewy bodies. It is classified as dementia, and dementia due to other diseases (Kumar V et al., Robbins and Cotran Pathologic Basis of Disease, Elsevier Saunders, 7th, 2004).

Lewy body diseases comprise a group of diseases characterized by the presence of proteinaceous neuronal inclusions called Lewy bodies (LBs). Clinically, two diseases can be distinguished: Parkinson's disease (PD) and dementia with Lewy bodies (DLB): Parkinson's disease is the most common progressive motor disease in the elderly , Lewy body dementia is the second most common cause of dementia after Alzheimer's disease (AD). The widespread distribution of Lewy bodies in almost all brain regions is a typical feature of Lewy body dementia, and the substantia nigra is most affected in Parkinson's disease. When first described, Lewy body dementia was thought to be a rare disease, but intensive investigations over the past few years have shown that it accounts for 10-15% of autopsied cases. The main symptoms of Lewy body dementia include variable cognitive impairment, recurrent visual hallucinations, and Parkinson's symptoms, nevertheless, the symptoms overlapping many Alzheimer's dementias frequently lead to a misdiagnosis of Lewy body dementia.

Parkinson's disease (PD) is the second most common age-related disease among neurodegenerative diseases after senile dementia. In this disease, motor function disorders such as hand tremor, rigidity syndrome, slow mobility, and postural instability appear, and emotional disorders such as emotional instability and fear are accompanied. In the case of Parkinson's patients, various nonmotor symptoms, such as olfactory dysfunction, depression, schizophrenia, and dementia, develop at each stage of the disease. The development of the disease is gradual, and pathologically, severe degeneration (about 70-80%) of the substantia nigra dopaminergic neurons of the brain is observed. In these cells, abnormal deposition lesions of a protein called Lewy body containing α-synuclein as a main component are confirmed. The elderly population in Korea is rapidly increasing, and the number of patients with Parkinson's disease is also continuously increasing. The exact cause of Parkinson's disease is still unknown. However, it has been reported that Parkinson's disease occurs due to the complex action of genetic factors and environmental factors. In particular, with the development of genetics in recent years, research on genetic factors of Parkinson's disease, which has not been known, is developing very rapidly.

On the other hand, using an animal model to discover a new treatment for cranial nerve disease is an essential element for discovering a new therapeutic target and conducting drug testing in the preclinical stage. The primary concern of neuroscientists is the selection of the most appropriate animal models useful to achieve their research goals. Investigators are often faced with the choice between models that are based on mechanisms that are not necessarily present in the patient, but have the basic pathological features of the disease, and animal models that are based on known etiological mechanisms, although not all clinical features are recapitulated. Therefore, researchers are challenging the development of animal models that conform to the etiology, symptoms, treatment, and physiological basis.

So far, a model animal expressing all the symptoms of human degenerative brain disease has not yet been established enough to be used as an effective disease model. is largely due to limitations of disease animal models. Accordingly, the need for a disease animal model using a species closer to humans has been raised due to the clear differences in dimenzone, breeding, lifespan and behavior from humans, and in the case of primates, due to the scarcity and cost and difficulty of breeding management Since there are restrictions that can be used for disease research only in very limited fields, there is an increasing demand for using animals that can perform more accurate intractable disease research at relatively low cost and facilities as model animals in the biomedical field. Conventional brain disease mouse model development has been mainly developed by drug administration, and in this case, results due to temporary brain damage can be observed, but it is difficult to proceed with specific and continuous brain disease-related research. In addition, although mice with brain disease caused by genetic modifications or deletions are being developed, there were many limitations such as homo-lethality, difficulties in animal management and proliferation, and deviation from clinical manifestations of lesions.

In particular, in the case of Parkinson's disease, which is caused by a deletion or dysfunction of the gene called parkin, which is 50% or more, a mouse model inducing Parkin's deletion was developed as an animal model for Parkinson's disease and used for basic research, but the lesion expressed in the animal model Compared to this clinical lesion, it is weak and takes a long time to induce, so there is a limit to its use as a preclinical animal model for treatment development research. In the case of a transgenic dominant Parkinson's gene model overexpressing α-synuclein, a representative Parkinson's dominant gene known to cause other Parkinson's disease, the death of dopaminergic neurons is sufficient compared to actual clinical lesions, despite the development of models using various promoters. The expression of Lewy body lesions was also inconsistent, and strong α-synuclein-A53T expression using the Prion promoter caused paralysis of the mouse due to the death of motor neurons in the spine. However, it has the disadvantage that it requires a long time of 7-8 months or more for the expression of α-synuclein aggregates and related lesions. In addition, it has limitations in the absence of Lewy body lesions and neuronal cell death for dopaminergic neurons in the midbrain, so it is not suitable as a model for Parkinson's disease. In the case of the recently developed stereotaxic injection model of PFF (α-synuclein preformed fibril) into the striatum brain region, expression/propagation of Lewy bodies, apoptosis of dopaminergic cells (about 50%), and motor abnormalities over 3 to 6 months. It has been reported, however, that lesion expression in this PFF model takes a long time over 6 months and 1 year, and the degree of dopaminergic cell death is not sufficient, which is different from the case of actual clinical Parkinson's patients. Therefore, it is difficult to induce consistent lesions according to quality control in PFF manufacturing, and if PFF purification is incomplete, artificial inflammatory lesions due to endotoxin contamination may be mixed.

As such, despite the extensive use of Lewy bodies animal models using various α-synuclein overexpression, the recent PFF injection model, and the extensive use of the Tau overexpression model, the existing mouse models have been used in human Parkinson's disease and neuronal cell death phenotype expression. They are distant and do not develop major disease lesions (Lewy body lesions, neurofibrillary tangles, motor abnormalities, cognitive impairment) or appear too late, making them unsuitable for in vivo validation of therapeutic lead substances. Therefore, in order to identify the causes of Parkinson's, Parkinson's dementia and Lewy body dementia, and to perform treatment methods through circuit research at the preclinical level, it is necessary to develop a mouse model that can faithfully represent the lesions of degenerative brain diseases.

It is an object of the present invention to provide a vector for preparing a neurodegenerative disease model.

It is also an object of the present invention to provide a responder animal and a driver animal for preparing a neurodegenerative disease model.

Another object of the present invention is to provide a neurodegenerative disease model mouse.

Another object of the present invention is to provide a method for preparing a neurodegenerative disease model.

In addition, it is an object of the present invention to provide a method for screening or verifying a therapeutic agent for a neurodegenerative disease.

In order to solve the above problems, the present invention provides a vector for preparing a neurodegenerative disease model, comprising a zinc finger protein 746 (PARIS) gene and a tetracycline regulatable transcription activator (tTa)-sensitive promoter (TetP).

In addition, the present invention provides a driver animal for preparing a neurodegenerative disease model, comprising a responder animal for preparing a neurodegenerative disease model injected with the vector, a tTA gene, and a neuron-specific promoter operably linked thereto.

In addition, the present invention provides an animal model of a neurodegenerative disease obtained by crossing the responder animal and the driver animal.

In addition, the present invention provides a method for preparing a cell/animal model of a neurodegenerative disease, comprising introducing the vector of the present invention into a neuron.

In addition, the present invention provides a method for screening or verifying a therapeutic agent for a neurodegenerative disease using a neurodegenerative disease model.

An animal model obtained by crossing a responder animal introduced with a vector capable of regulating the expression of PARIS according to the present invention with a Tet-OFF transgenic switch system and a driver animal containing a specific neuron-specific promoter is overexpressing PARIS in specific neurons. By inducing dopaminergic cell death, neuroinflammation of brain tissue, aggregate formation and mitochondrial abnormality in the field of play, movement disorders (dyskinesia, dyscoordination) and non-motor abnormalities (anxiety, olfactory dysfunction) to the same degree as in real patients in a very short period of time As it is induced, it is useful as an animal model that can faithfully represent lesions of degenerative brain diseases, particularly mitochondrial dysfunction, aggregate lesions, and neuronal cell death, which have resolved the problems of the absence of some conventional lesions and taking a long time to induce lesions. can be used

1 is a schematic diagram showing the detailed structure of a TetP-PARIS transgenic construct and a PARIS gene.
2 is a diagram showing the process of preparing a TetP-PARIS transgenic founder mouse by pronuclear injection of the TetP-PARIS construct:
A: TetP-PARIS construct validation;
B: Confirmation of expression of PARIS after transfection of P8 clone TetP-PARIS with pCMV-tTA; and
C: Selection through genotyping of TetP-PARIS founder line mice.
Figure 3 is a diagram showing the process of producing a DAT-PARIS transgenic mouse:
A: Schematic diagram of dopaminergic cell-specific PARIS-expressing Parkinson's disease mouse model construction cross-breeding strategy;
B: dopaminergic cell-specific PARIS expression mechanism; and
C: Confirmation of mouse genotype (PCR product of DAT-PF-tTA genotype WT: 184 bp, PCR product of mutant type: 232 bp; and PCR product of TetP-PARIS genotype Tet promoter: 173 bp, PCR product of PARIS: 343 bp ).
4 is a diagram confirming dopaminergic cell-specific PARIS expression in the PARIS Tg mouse model:
A: PARIS transgene expression protocol in PARIS Tg (AT-PF-tTA; TetP-PARIS) mice;
B: dopaminergic cell-specific transgene expression in PARIS Tg mice (3 months old); and
C: PARIS overexpression in the olfactory bulb of PARIS Tg mice.
5 is a diagram confirming movement and behavioral disorders in PARIS Tg mice:
A: motility experiments in the open field;
B: The ratio of dwell time in the central zone and the outer zone as an indicator of anxiety in the open field test;
C and D: Behavioral experiments with the pole test for measuring motion and the rotarod test for measuring motor coordination ability; and
E: Measuring olfactory function.
Figure 6 is a diagram confirming the dopaminergic neuron death phenotype of DAT-PARIS Tg:
A: Images of immunohistochemical staining of brain tissue sections of PARIS Tg mice and control mice with TH antibody;
B: Data counting the number of dopaminergic cells in the TH-stained SNpc region;
C: Images of striatal brain tissue sections from PARIS Tg mice and control mice immunohisto-stained with TH antibody; and
D: Graph quantifying TH intensity of striatal brain tissue sections immunohistochemically stained with TH antibody.
7 is a diagram confirming the expression of Parkinson's disease-related lesions in PARIS Tg mice:
A: The result of Western blot analysis confirming phosphorylated α-Syn (phospho S129-α-Syn) or α-Syn in the form of filament aggregates in the Triton X insoluble fraction of the midbrain brain tissue of PARIS Tg mice and control mice and the density of phosphorylated α-Syn (densitometry) quantitative graph; and
B: Immunofluorescence tissue imaging confirming neuroinflammation patterns in midbrain substantia nigra sections.
8 is a diagram confirming the mitochondrial abnormality of PARIS Tg mice:
A: Images confirming intracellular organelles including mitochondria in the midbrain substantia nigra of PARIS Tg mice and control mice;
B: Expression patterns of PARIS and mitochondrial marker proteins in midbrain tissues of PARIS Tg mice and control mice; and
C: Relative quantification graph of protein.
9 is a diagram showing the process of manufacturing a mouse model of Lewy body dementia:
A: Neuron-specific PARIS-expressing Lewy body dementia mouse model construction mating strategy schematic diagram;
B: Neuron-specific PARIS expression mechanism; and
C: Confirmation of the mouse genotype.
10 is a diagram confirming lesions caused by PARIS expression in the CamK-PARIS Tg Lewy body dementia model:
A: CamKII-PARIS Tg ( CamKII-tTA; TetP-PARIS ) PARIS transgene expression protocol in mice dietary schedule;
B: Analysis of survival curves of CamKII-PARIS Tg mice; and
C: Pathological image of CamKII-PARIS Tg mice.
11 is a diagram confirming the expression of PARIS in the CamKII-PARIS Tg Lewy body dementia mouse model:
A: PARIS expression patterns by brain tissue in CamKII-PARIS Tg mice;
B: Graph of quantitative analysis of the relative expression level of PARIS by brain tissue; and
C: Imaging confirming overexpression of PARIS in CTX, HIP and SN (substantia nigra) of CamK-PARIS Tg mice with FLAG antibody.
12 is a diagram confirming Lewy bodies and inflammatory lesions in CamKII-PARIS Tg mice:
A: Phosphorylated α-Syn immunohistochemical staining images from brain tissue sagital sections of CamKII-PARIS Tg mice and control mice; and
B: GFAP immunohistochemistry images in brain tissue, cerebral cortex (CTX) and hippocampus (Hippocampus) sections of CamKII-PARIS Tg mice and control mice.

Hereinafter, the present invention will be described in detail by way of embodiments of the present invention with reference to the accompanying drawings. However, the following embodiments are presented as examples of the present invention, and when it is determined that detailed descriptions of well-known techniques or configurations known to those skilled in the art may unnecessarily obscure the gist of the present invention, the detailed description may be omitted, and , the present invention is not limited thereby. Various modifications and applications of the present invention are possible within the scope of equivalents interpreted therefrom and the description of the claims to be described later.

In addition, the terms used in this specification are terms used to properly express the preferred embodiment of the present invention, which may vary depending on the intention of a user or operator or customs in the field to which the present invention belongs. Accordingly, definitions of these terms should be made based on the content throughout this specification. Throughout the specification, when a part "includes" a certain component, it means that other components may be further included, rather than excluding other components, unless otherwise stated.

As used herein, the terms "about", "substantially" and the like are used in a sense at or close to the numerical value when the manufacturing and material tolerances inherent in the stated meaning are presented, and to aid understanding of the present application. It is used to prevent an unconscionable infringer from using the mentioned disclosure in an unreasonable way.

As used herein, the term "step for" or "step for" does not mean "step for".

In this specification, the term "combination of these" included in the expression of the Markush form means one or more mixtures or combinations selected from the group consisting of the components described in the expression of the Markush form, and the components It means to include one or more selected from the group consisting of.

In this specification, the description of "A and/or B" means "A, B, or A and B".

All technical terms used in the present invention, unless otherwise defined, have the meaning as commonly understood by one of ordinary skill in the art of the present invention. In addition, although preferred methods and samples are described herein, similar or equivalent ones are also included in the scope of the present invention. The contents of all publications herein incorporated by reference are incorporated herein by reference.

In one aspect, the present invention relates to a vector for preparing a neurodegenerative disease model, comprising a Parkin Interacting Substrate (PARIS) (or zinc finger protein 746, ZNF746) gene and a tetracycline regulatable transcription activator (tTa)-sensitive promoter.

In one embodiment, the tTA-sensitive promoter may be a Tet promoter (Tetracycline responsive promoter) operably linked to the PARIS gene.

In one embodiment, the PARIS gene may include the nucleotide sequence represented by SEQ ID NO: 1, the tTA-sensitive promoter may include the nucleotide sequence represented by SEQ ID NO: 2, and the vector of the present invention is SEQ ID NO: 3 It may include a nucleotide sequence represented by .

In one embodiment, the neurodegenerative brain disease may be a brain disease expressing denatured protein aggregates, and may include dementia, Alzheimer's disease, Lewy body dementia, Huntington's disease, Multiple system atrophy, amyolateral sclerosis, stroke, cerebral infarct, head trauma, cerebral arteriosclerosis, Parkinson's dementia and Parkinson's disease disease), and may be at least one selected from the group consisting of Parkinson's disease, Parkinson's dementia, and Lewy body dementia.

In one embodiment, the vector may be a vector for constructing a responder animal.

The PARIS (or ZNF746) of the present invention belongs to the KRAB-zinc finger family and is a domain that recruits factors involved in the formation of heterochromatin at the N-terminus of the KRAB (Kruppel associated box). ) has

As used herein, the term “operably linked” means that one nucleic acid fragment is associated with another nucleic acid fragment such that its function or expression is affected by the other nucleic acid fragment.

As used herein, the terms “gene”, “nucleic acid”, “polynucleotide” or “oligonucleotide” refer to a DNA molecule, RNA molecule, or analogs thereof. As used herein, the terms "nucleic acid", "polynucleotide" and "oligonucleotide" refer to, but are not limited to, DNA molecules such as cDNA, genomic DNA or synthetic DNA and RNA molecules such as guide RNA, messenger RNA or including synthetic RNA. Moreover, the terms "nucleic acid" and "polynucleotide" as used herein include single-stranded and double-stranded forms.

In addition, in the present invention, intracellular delivery of proteins, RNA, or DNA (including vectors) includes electroporation, liposomes, viral vectors, nanoparticles, PTD (Protein translocation domain) fusion protein methods, etc. Therefore, it can be carried out using various methods known in the art without limitation.

As used herein, the term "vector" refers to any nucleic acid comprising a competent nucleotide sequence that is inserted into a host cell and recombined with and inserted into the host cell genome, or that replicates spontaneously as an episome. Such vectors include linear nucleic acids, plasmids, phagemids, cosmids, RNA vectors, viral vectors, and the like.

As used herein, the term "host cell" refers to a eukaryotic or prokaryotic cell into which one or more DNA or vectors are introduced, and should be understood to refer not only to a particular subject cell, but also to its progeny or potential progeny. In fact, the progeny are not identical to the parent cell as certain modifications may occur in subsequent generations due to mutations or environmental influences, but are still included within the scope of the term as used herein.

The promoter used in the present invention can be of various nature and origin and has various properties. This is because the choice of the promoter used depends, inter alia, on the use and the gene in question when required. Thus, a promoter may be derived, for example, from a promoter that is strong or weak, ubiquitous or specific to a tissue/cell, or specific for a physiological or pathophysiological state (activity depends on the state of cell differentiation or a particular phase of the cell cycle). being). Promoters may be of eukaryotic, prokaryotic, viral, animal, plant, artificial, human, or the like origin. Specific examples of the promoter are promoters such as TK, GH, EF1-a, APO and CMV immediate early genes, or artificial promoters. When used in the present invention, such promoters are preferably "minimal", ie dependent on the presence of tTA. To this end, the native promoter can be cleaved enzymatically and preferably functionally coupled to at least one Op sequence before its activity can be tested. The skilled artisan can adjust the number of tetOp sequences and the distance to the selected promoter so that expression of the promoter is strictly tTA-dependent.

The tTA-sensitive promoter of the present invention is a promoter sensitive to the tTA transactivator, and its activity is increased in the presence of the transactivator. From a structural point of view, a promoter is a promoter comprising at least one site that binds a tTA factor in a sequence or at a functional distance from such a sequence (Gossen et al., 1992). Such a binding site or effector region (Op or tetOp) may have, for example, the nucleotide sequence of SEQ ID NO: 3 or a functional variant of this sequence. A variant may correspond to a sequence that has been genetically modified, for example, by mutation, deletion or addition of one or more base pairs and retains the ability to bind to a tTA transactivator. Preferably, the modification affects 10% or less of the nucleotide sequence of SEQ ID NO: 3. Additionally, the Op sequence may be present in more than one copy, for example from 1 to 10 copies. More preferably, in order to substantially control the expression of the nucleic acid, it is highly recommended that the promoter used in the present invention has a very low but not zero basal activity. As a result, it is observed that this promoter is active only in the presence of tTA and the regulation is very tight. Preferably, the promoter used in the present invention is a minimal promoter sensitive to the tTA transactivator. The minimum characteristic refers to a promoter with very low, if not zero, basic activity. Generally, such promoters contain minimal elements that are essential for transcriptional promoter function (eg, a TATA box) and lack the strength or other regions involved in the regulation of the native promoter. Such promoters can be made from various types of promoters by deletion and/or addition of elements (“silents”). The tTA of the present invention can act on the enhancer only in a state not bound to tetracycline or doxycycline to perform an enhancing function. However, when tTA forms a conjugate with tetracycline or doxycycline, the conjugate loses its ability to act on the enhancer and can no longer exert its enhancing function. Thus, the inclusion of tTA in a tetracycline/doxycycline-inducible system constitutes a so-called Tet-Off system that stops expression of a target gene in the presence of tetracycline or doxycycline.

In one aspect, the present invention relates to a responder animal for producing a degenerative nervous system brain disease model injected with the vector of the present invention, wherein the animal is a rat, mouse, guinea pig, monkey, dog, cat, rabbit, cow, It may be one or more non-human animals selected from the group consisting of sheep, pigs and goats, more preferably a mouse.

In one embodiment, the responder animal of the present invention can be crossed with an animal comprising a specific cell-specific promoter to overexpress PARIS in a specific cell.

In one aspect, the present invention relates to a vector for constructing a drive animal comprising a tTA gene and a neuron-specific promoter operably linked thereto.

In one embodiment, the neuron-specific promoter may be a dopamine neuron-specific promoter or a neuron-specific promoter.

In one embodiment, the dopaminergic neuron-specific promoter may be a promoter specifically expressed in dopaminergic neurons expressing one or more genes selected from the group consisting of TuJ1, MAP2, HuC/D, VMAT2, and DAT, dopaminergic It may be a receptor (dopamine transporter, DAT)-specific promoter, and may include the nucleotide sequence of SEQ ID NO: 4.

In one embodiment, the nerve cell-specific promoter may be CamKIIα (calmodulin-dependent protein kinase type II alpha chain), and may include the nucleotide sequence of SEQ ID NO: 5.

In one aspect, the present invention relates to a driver animal for preparing a neurodegenerative disease model, comprising a tTA gene and a neuron-specific promoter operably linked thereto.

In one embodiment, the driver animal may be manufactured by injecting the vector for manufacturing a drive animal of the present invention.

In one embodiment, the animal may further comprise a tTA gene and a tTA-sensitive promoter operably linked thereto.

In one embodiment, the animal may include a tTA gene and a dopaminergic neuron-specific promoter operably linked thereto, wherein the dopaminergic neuron is one selected from the group consisting of TuJ1, MAP2, HuC/D, VMAT2, and DAT. It may be a nerve cell expressing the above genes.

In one embodiment, the dopamine neuron-specific promoter may be a dopamine receptor (DAT)-specific promoter, and may include the nucleotide sequence of SEQ ID NO: 4.

In one embodiment, the nerve cell-specific promoter may be CamKIIα (calmodulin-dependent protein kinase type II alpha chain), and may include the nucleotide sequence of SEQ ID NO: 5.

In one embodiment, the driver animal may be used for making an animal model for Parkinson's disease or dementia.

In one aspect, the present invention relates to an animal model of a neurodegenerative disease obtained by crossing a responder animal and a driver animal of the present invention.

In one embodiment, the neurodegenerative disease animal model can regulate the expression of PARIS with a Tet-OFF system, and when doxycycline is treated, tTA can be inactivated to turn off the expression of PARIS.

In one embodiment, the neurodegenerative disease animal model is obtained by crossing a driver animal comprising a tTA gene and a dopaminergic neuron-specific promoter operably linked thereto with a responder animal of the present invention, PARIS gene, TetP-sensitive promoter , may be an animal model of Parkinson's disease in which both the tTA gene and a dopaminergic neuron-specific promoter exist, the animal model may overexpress PARIS specifically for dopaminergic neurons, and dopaminergic cell death and movement disorders within 1 to 5 months of age , olfactory dysfunction, mitochondrial structure abnormalities, or Lewy body lesions may occur.

In one embodiment of the present invention, PARIS overexpression in the whole brain of a model mouse induces Lewy body expression in the whole brain, thereby encroaching on Lewy bodies in the cerebral cortex and hippocampus in the late stage (late stage) of clinical Parkinson's disease and cognitive memory in Parkinson's patients ability to simulate disability.

In one embodiment, the neurodegenerative disease animal model is obtained by crossing a driver animal comprising a tTA gene and a neuron-specific promoter operably linked thereto with a responder animal of the present invention, PARIS gene, TetP-sensitive promoter, It may be an animal model of dementia disease in which both the tTA gene and a neuron-specific promoter exist, and dementia may be an animal model of dementia with Lewy bodies, and may express PARIS specifically in neurons.

In one aspect, the present invention relates to a composition for preparing an animal model of a neurodegenerative disease, comprising the vector of the present invention.

In one aspect, the present invention relates to a method for preparing a cell model of a neurodegenerative disease, comprising introducing a vector into a neuron.

In one embodiment, the neuron may be a neuron of a human, mouse, rat, rabbit, fruit fly, pig or monkey.

In one aspect, the present invention relates to a neurodegenerative disease cell model prepared by the above method.

In one aspect, the present invention prepares a responder animal by injecting the vector of the present invention into an animal other than a human; the responder animal is crossed with a driver animal comprising a neuron-specific promoter and a tTA gene; And it relates to a method for producing an animal model of a neurodegenerative disease, comprising selecting an animal having all of the PARIS gene, the TetP-sensitive promoter, the tTA gene, and the neuron-specific promoter.

In one embodiment, the animal may be one or more non-human animals selected from the group consisting of rat, mouse, guinea pig, monkey, dog, cat, rabbit, cow, sheep, pig and goat, more preferably a mouse.

In one aspect, the present invention relates to a method for screening a therapeutic agent for a neurodegenerative disease using the neurodegenerative disease model of the present invention.

In one embodiment, the neurodegenerative disease model may be a neurodegenerative disease cell model or a neurodegenerative disease animal model.

In one aspect, the present invention relates to a method of verifying the therapeutic effect of a neurodegenerative disease therapeutic agent of a neurodegenerative disease therapeutic agent using the neurodegenerative disease model of the present invention.

In one embodiment, the candidate substance for a therapeutic agent for a neurodegenerative disease may be a small molecule compound, an antibody, or a stem cell.

In one embodiment, the neurodegenerative disease model may be a neurodegenerative disease cell model or a neurodegenerative disease animal model.

The present invention will be described in more detail through the following examples. However, the following examples are only intended to embody the contents of the present invention, and the present invention is not limited thereto.

Example 1. TetP-PARIS responder mouse line development

In order to express a Parkinson-associated disease protein capable of mimicking mitochondrial dysfunction, the gene (SEQ ID NO: 1) of PARIS, which is a substrate protein of Parkin and is known to regulate mitochondrial production and antioxidant, was converted to PrP.tetP construct (TetP plasmid) By cloning into the XhoI site of (SEQ ID NO: 2), a TetP-PARIS construct (SEQ ID NO: 3) was prepared (FIG. 1A). The expression of PARIS in the TetP-PARIS construct can be regulated by tetP (Tetracycline regulatable promoter), which can be regulated by doxycycline, which is a tetracycline analog. In addition, a FLAG tag was attached to the C-terminus of the human PARIS gene to make detection easier ( FIG. 1B ). In order to verify several cloned TetP-PARIS constructs, a P8 clone in which the predicted insert size (PARIS = 2kb) was confirmed after excision with XhoI was obtained (Fig. 2A). After transfection of the P8 construct and pCMV-tTA into SH-SY5Y cells, the expression of PARIS with respect to the total protein was confirmed by western blot using a FLAG antibody and a PARIS antibody ( FIG. 2B ). As described above, the P8 plasmid whose PARIS sequence and expression was verified was cut with NotI restriction enzyme to remove the Blue Script region (pBS, 2950 bp), and linearized TetP-PARIS was injected into several mice by pronuclear injection to transgenic mice. was produced. Thereafter, genotyping PCR was performed on the genomic DNA extracted from each mouse tail using the TetP-specific primer set in Table 1 below, and the genotype of each mouse was confirmed by agarose gel electrophoresis. Through this, 29 founder lines were secured, and among them, the founder line #121 with the most TetP copy number was used for phenotype analysis (FIG. 2C).

primer sequence (5'->3') TetP F CGGGTCGAGTAGGCGTGTAC R TCTAGATGATCCCCGGGTACCGAG TetP-PARIS F GCGCTGCCCTTATTTTCATGTT R CCCGGTTCGACCTTCTAGGG DAT-PF-tTA-common F AGAAGAAGGAAACAGACTTCCTC DAT-PF-tTA-WT R TGATGAGGGTGGAGTTGGTC DAT-PF-tTA-mutant R GCTTGTTCTTCACGTGCCAGT Camk2a-tTA F CGC TGT GGG GCA TTT TAC TTT AG R CAT GTC CAG ATC GAA ATC GTC

Through this, a TetP-PARIS founder line capable of inducing expression of a PARIS protein that causes mitochondrial dysfunction as a Parkinson's disease-related protein was secured.

Example 2. Construction of dopamine cell-specific PARIS expression Parkinson's mouse model

2-1. Parkinson's mouse model PARIS Tg mouse crafting

In order to actually induce the expression of PARIS in the TetP-PARIS responder mouse obtained in the above example, it is necessary to cross with a driver line that simultaneously expresses tTA, so pDAT-PF-tTA capable of specifically expressing tTA in dopamine cells A mouse model of Parkinson's disease in which the mitochondrial toxicity-inducing protein PARIS is expressed in a dopaminergic cell-specific manner by crossing with a mouse model was constructed (FIG. 3A). pDAT-PF-tTA is a knock-in model of a transgenic cassette capable of amplifying tTA expression under the dopamine transporter (DAT) promoter. Inducing the expression of tTA, this tTA can be reattached to the lower TetP to induce the amplified expression of additional tTA (FIG. 3B). In other words, if tTA is specifically expressed in dopaminergic cells in which the DAT promoter is activated, and the TetP-PARIS responder construct is present at the same time, it is a system that can induce PARIS expression as well. Through this, dopaminergic cell-specific overexpression of PARIS is induced in cells in which pDAT-PF-tTA and TetP-PARIS are coexisted, and treatment with doxycycline inactivates tTA to turn off PARIS expression ( FIG. 3B ). After extracting genomic DNA from the tail to confirm the genotype of each of the crossed mice, a Parkinson model mouse ( PARIS Tg ) having pDAT-PF-tTA and TetP-PARIS simultaneously through genotyping PCR using the primer set in Table 1 was selected, and pDAT-PF-tTA heterozygous mice were used as controls ( Con ) ( FIG. 3C ).

2-2. PARIS Tg Confirmation of dopaminergic cell-specific PARIS expression in mice

Among PARIS Tg mice, the number of offspring expressing PARIS from the embryonic stage was smaller than expected, so it was determined that PARIS expression at the developmental stage caused problems in embryonic development, and the mated mice were maintained on doxycycline diet until they reached 1 month of age. (Fig. 4A). Thereafter, the expression of the PARIS gene was induced up to 2 and 3 months of age by replacing it with a regular diet at 1 month of age. Then, as a result of immunofluorescence staining analysis of the midbrain tissue of the PARIS Tg mouse with dopamine-specific TH (tyrosine hydroxylase) and FLAG, it was confirmed that PARIS was effectively induced in the dopaminergic neurons stained with TH in the midbrain of the PARIS Tg mouse. were (Fig. 4B). In addition, as confirmed by immunohistochemistry, it was confirmed that PARIS expression was induced even in the outer region of the olfactory bulb in which DAT-tTA is activated ( FIG. 4C ).

2-3. PARIS Tg Identification of movement disorders in mice

It was verified through various behavioral experiments whether the above-prepared PARIS Tg mouse could induce Parkinson's disease-related movement disorder due to PARIS expression in dopaminergic cells. Specifically, PARIS Tg ( DAT-PF-tTA; TetP- PARIS ) A mouse and a control group ( Con ) mouse were released in an open arena, and spontaneous exploration was measured through the SMART V 3.0 video tracking system, and the movement path was projected according to the movement of each mouse. (tracing) was confirmed for the control group and the Tg representative mouse group. In addition, as indicators for anxiety, the time taken to enter the central zone for the first time, the ratio of residence time in the central zone and the outer zone, and the total travel distance were confirmed ( n = Con 10 and Tg 12). As a result, it was clearly confirmed that the movement tendency in the corners of Tg mice gradually increased as the age of 2 and 3 months increased ( FIGS. 5A and 5B ). Through this, it can be inferred that anxiety symptoms appear in a progressive form in PARIS Tg. In addition, in order to confirm the slowing, i.e., movement, which is one of the main symptoms of Parkinson's disease, the mouse to be evaluated is acclimated on a pole (motor test pole) for at least 20 minutes, then the acclimated mouse is placed on top of the pole and the pole A pole test was performed, recording the total time taken to reach the lower end from the upper end of , it could be confirmed that the western sinus lesion appeared. In addition, to check the exercise coordination ability, set the rotarod device for 30 seconds to pre-run, then gradually increase the RPM (40 rpm/300sec) and record the time and rpm when the mouse goes down. As a result of performing the rotarod test, it was confirmed that 3-month-old PARIS Tg mice fell at a significantly lower rate and faster than the control group (FIG. 5D).

2-4. PARIS Tg Check the olfactory function of the mouse

Because PARIS Tg mice also overexpressed PARIS in the olfactory bulb, a hidden cookie finding test was performed to check the time it takes to find a food pellet hidden under a litter to check whether abnormal olfactory function is induced. As a result, in the case of PARIS Tg, it was found that the time it takes to find the food pellets hidden under the litter increases compared to the control group at the age of 3 months (FIG. 5E).

2-5. PARIS Tg Confirmation of dopaminergic neuronal cell death in mice

In order to confirm the death of dopaminergic neurons, a major lesion characteristic of Parkinson's disease, PARIS Tg mice and control mice ( Con ) were sleep anesthetized and fixed on a mouse surgical platform to perform reconstitution, 1XPBS and 4% paraformaldehyde ( wt/vol). After that, the mouse brain was extracted, post-fixed with 4% paraformaldehyde (wt/vol), and cryopreserved in 30% sucrose (wt/vol). (STR)) was sectioned with a 35 μm thickness and immunohistochemically stained with TH (tyrosine hydroxylase)-specific antibody, followed by sequentially biotinylated goat anti-rabbit IgG or anti-mouse IgG, and streptavidin-conjugated HRP. After incubation with (horseradish peroxidase), the degree of TH staining of each brain section was checked by exposure to 3,3-diaminobenzidine. Then, the number of dopaminergic cells in the SN pars compacta (SNpc) region was counted with an Optical Fractionator probe of Stereo Investigator, and the TH staining signal intensity in the STR region was quantified as an optical density using Image J, and then a bar graph indicated as Data were expressed as mean +/- standard error, and significance tests were calculated using an unpaired two-tailed student t -test (** P < 0.01 and *** P < 0.001). As a result, a gradual decrease in dopaminergic cells was confirmed from 2 months of age to 3 months of age, and at 3 months of age, more than 80% dopaminergic cell death was observed compared to the control group (Fig. 6A and B). In addition, consistent with this, it was confirmed that the axonal density of dopaminergic cells was significantly reduced in 3-month-old PARIS Tg striatum and NAc (Nucleus accumbens) ( FIGS. 6C and D ).

2-6. PARIS Tg Confirmation of the occurrence of aggregates in mice

In order to confirm the expression of Lewy bodies, a major lesion of Parkinson's disease, brain tissue sections (coronal brain sections, ventral midbrain (VM)) of the Tg mice and control mice were analyzed with antibodies (pS129) specific to Parkinson's disease-associated lesion features. -α-synuclein (pSYN) and filament conformation α-synuclein Lewy body lesions; and GFAP: inflammatory state) were confirmed by confocal microscopy. As a result, the structure of the Lewy body in the form of an aggregate was shown in the mesencephalic substantia nigra protein lysate of PARIS Tg (FIG. 7A). In addition, astrogliosis stained with GFAP was found in the midbrain substantia nigra of PARIS Tg mice as an inflammatory finding ( FIG. 7B ). In addition, PARIS, as a substrate protein of parkin, is a mitochondrial toxic protein that causes mitochondrial dysfunction and interference of antioxidant defense mechanisms through inhibition of the expression of PGC-1a. Mitochondrial structure abnormalities were confirmed using an electron microscope. As a result, unlike the clear cristate structure shown in the control group, the mitochondria with an abnormal structure were found in the midbrain substantia nigra of PARIS Tg ( FIG. 8A ). In addition, as a result of Western blot analysis of major mitochondrial marker proteins (SDHA: succinate dehydrogenase; HSP60: heat shock protein 60; VDAC: voltage dependent anion channel; and PHB1: prohibitin 1), pyruvate dehydrogenation in the midbrain substantia nigra of PARIS Tg It was confirmed that the expression of the enzyme (pyruvate dehydrogenase) and prohibitin 1 was significantly reduced compared to the control group (FIGS. 8B and C).

Through this, the mitochondrial dysfunction-simulating Parkinson's mouse model, developed through the hybridization of DAT-PF-tTA and TetP-PARIS in the present invention, is the PARIS Tg mouse model for major Parkinson's disease lesions (motor impairment, dopaminergic cell death, mitochondrial structural dysfunction). , Lewy body lesion and inflammatory expression) can be successfully induced in a short period of 3 months of age.

Example 3. Construction of a neuron-specific PARIS expression Parkinson's mouse model

3-1. Lewy body dementia mouse model CamKⅡα-PARIS produce

Brain diseases caused by abnormalities in the structure and function of mitochondria are not only Parkinson's disease, but also dementia (Lewy body dementia, Parkinson's dementia and Alzheimer's dementia) and prion disease. α-synuclein) is expressed in various brain regions, especially in the cerebral cortex and hippocampus, and is known to induce mitochondrial and neuronal dysfunction, mitochondrial dysfunction and Lewy body lesions in these cortex and hippocampus To simulate, TetP-PARIS responder mice were crossed with CamKIIα-tTA driver mice capable of inducing tTA expression in various neurons ( FIG. 9A ). The CamKIIα promoter is activated specifically for neurons in various brain regions, and by expressing tTA specifically for neurons, it induces the expression of PARIS in cells that also have the TetP-PARIS responder construct ( FIG. 9B ). Double transgenic mice ( CamKIIα-PARIS Tg ) with CamKIIα-tTA and TetP-PARIS simultaneously through genotyping PCR using the primer set in Table 1 after genomic DNA was extracted from the tail to confirm the genotype of each of the crossed mice. The presence was confirmed (FIG. 9C). Here, a mouse having CamKIIα-tTA alone was used as a control group ( Con ). Since CamKIIa -PARIS Tg mice were unable to obtain Tg mouse pups when they expressed PARIS from the embryonic stage, it was judged that PARIS expression in whole brain neurons at the developmental stage caused a problem in embryonic development, and was approximately 1 month old. Med mice were maintained on a doxycycline diet until maturity ( FIG. 10A ). After that, at 1 month of age, the normal diet was replaced with the normal diet to induce the expression of the PARIS gene, and as a result of analyzing the survival curve, Tg mice could not survive for more than 2 months (Fig. 10B) and showed pathological symptoms such as limb clasping (Fig. 10C). ).

3-2. CamKⅡα-PARIS Tg Confirmation of brain-specific PARIS expression in mice

Several brain regions of the CamK-PARIS Tg mouse (olfactory bulb, OB; cortex: cortex, CTX; hippocampus: hippocampus, HIP; cerebellum: cerebellum, CB; striatum: striatum, STR; dorsal midbrain: ventral midbrain, VM; brainstem: brainstem, BS) tissue was removed and shredded and then Western blot analysis was performed. As a result, it was confirmed that a fairly high level of PARIS was overexpressed in several brain regions, especially in tissues such as the olfactory bulb, cerebral cortex, and hippocampus. (FIG. 11A and B), and the expression of PARIS was verified once again by immunohistochemistry using FLAG antibody (FIG. 11C).

3-3. CamKⅡα-PARIS Tg Identification of dementia lesions in mice

In order to confirm whether Lewy body dementia-like lesions (Lewy body structure) showing degenerative protein aggregate lesions appear in a wide range of brain regions in CamKIIα-PARIS Tg mice of the present invention, brain tissue sections of CamKIIα-PARIS Tg mice by age were analyzed by pS129- The structure of Lewy bodies was verified by immunohistochemistry using an α-synuclein-specific antibody. As a result, it was shown that Lewy bodies-like structures labeled with pS129-α-synuclein were induced in the cerebral cortex and hippocampus of Tg mice expressing PARIS from 1.5 to 3 months of age ( FIG. 12A ). In addition, astrocytes stained with GFAP along with protein aggregate lesions in the cerebral cortex of Tg mice were confirmed as inflammatory findings ( FIG. 12B ).

Through this, an aggregate-simulating Lewy body dementia mouse model through the crossing of CamKIIα-tTA and TetP-PARIS developed in the present invention CamKIIα-PARIS Tg mice exhibited 2-3 major Lewy body dementia lesions (Lewy body aggregate expression, inflammation expression). It can be seen that it can be successfully induced in a short period of months.

<110> Research and Business Foundation SUNGKYUNKWAN UNIVERSITY <120> NEURODEGENERATIVE DISEASES MODEL ANIMAL AND METHOD FOR PRODUCING THE SAME <130> R-2020-0364-KR-1 <160> 5 <170> KoPatentIn 3.0 <210> 1 <211> 1935 <212> DNA <213> Homo sapiens <400> 1 atggccgagg cggtcgcggc tccgatttct ccgtggacga tggcagccac gattcaggcc 60 atggagagga agattgaatc gcaggctgct cgcctgcttt ccctagaagg tcgaaccggg 120 atggccgaga agaagctggc tgattgcgag aagacagccg tggagttcgg gaaccagctg 180 gagggcaagt gggccgtgct ggggaccctg ctgcaggagt acgggctgct gcagaggcgg 240 ctggagaacg tggagaacct gctgcgcaac aggaacttct ggatcctgcg gctgcccccg 300 ggcagcaagg gggagtcccc taaggagtgg ggcaagctgg aggactggca gaaggagctc 360 tacaagcacg tgatgagggg caactacgag acgctggtct ccctggacta cgccatctcc 420 aagcccgagg tcctctccca gattgaacaa gggaaggagc cctgcaactg gcgccgccct 480 ggccccaaga ttccagatgt tcctgtggac cccagtccag gctcggggcc cccagttccc 540 gccccagacc tcttgatgca gatcaagcag gagggtgagc tccagctcca ggagcagcag 600 gccctgggcg tggaggcgtg ggcagccggg cagccagata ttgggg agga gccctggggc 660 ctcagccagc tggattccgg agcaggagac atctccacgg atgccacctc tggtgtccat 720 tccaactttt ccaccaccat cccgcccacc tcctggcaaa cggatctccc tccccaccat 780 ccctcttcag catgctcgga cgggaccctg aagctcaaca cagcagcctc cacggaagat 840 gtaaaaattg taataaaaac agaagtccag gaagaggagg tggtggccac acccgtacat 900 cctactgacc tagaggctca cgggaccctg tttggaccag gccaagccac acggttcttc 960 cctagtcctg cccaggaagg agcctgggaa agccagggca gctccttccc cagccaggac 1020 cctgtgctgg ggctgcgaga gcccgcccgg cctgagaggg acatgggtga gctcagtcct 1080 gctgtggccc aggaggagac ccctcctggg gactggctct tcggaggggt ccggtggggc 1140 tggaatttcc ggtgtaaacc gccagtgggc ctgaacccga ggacggggcc cgaggggctt 1200 ccttactcct ccccggacaa cggagaggcc atcttggacc ccagccaggc cccaaggcca 1260 ttcaacgaac cctgtaaata ccctggccgg accaaaggct ttggccacaa gccagggctg 1320 aagaagcacc ccgcggcgcc ccccgggggc cggcccttca cctgcgccac gtgtgggaag 1380 agcttccagc tgcaagtcag cctgagcgcg caccagcgca gctgtggggc gcccgacggg 1440 tcgggcccgg gcacaggcgg tggcggcagc ggcagtggcg gcggcggtgg cggcagc ggt 1500 gggggcagcg cacgggatgg cagcgccctt cggtgtgggg agtgcggccg ttgcttcacg 1560 cgccccgcgc acctcatccg ccatcgcatg ctgcacaccg gcgagcggcc cttcccctgc 1620 accgagtgtg agaagcgctt caccgaacgc tccaagctca tcgaccacta ccgaacgcac 1680 acgggcgtgc ggcccttcac ctgcaccgtc tgcggcaaaa gcttcatccg caaggaccac 1740 ctccgcaagc accagcgcaa ccatgcagcg ggcgccaaga ccccggcccg aggccagcca 1800 ctcccgacgc cgcccgcacc tcctgatccc ttcaagagcc ccgcctccaa aggacctttg 1860 gcctccacag accttgtgac cgactggact tgtggcctca gcgtcctggg acccaccgat 1920 ggcggggaca tgtga 1935 <210> 2 <211> 8584 <212> DNA <213> Artificial Sequence <220> <223> Tet promoter <400> 2 ctcgagcctt cctgcttgtt ccttcgcatt ctcgtggtct aggcttgctgggt ggt ggtct ggcttggtggt ggt ggtct ggct acctg aaatgtacag tagaccagtt gctctttgct 180 tcaggtccct ttgatggagt ctgtcatcag ccagtgctaa caccgggcca ataagaatat 240 aacaccaaat aactgctggc tagttggggc tttgtttttgg tttgttttt aga cctactggtgt agat gacagtga cacatgtaac ttagcatagg 360 caaagggttc tacaaccaaa gaagccactg tttggggatg gcgccctgga aaacagcctc 420 ccacctggga tagctagagc gtccacacgt ggaattcttt ctttactaac aaacgatagc 480 tgattgaagg caacaggaaa aaaaaaaatc aaattgtcct actgacgttg aaagcaaacc 540 tttgttcatt cccagggcac tagaatgatc tttagccttg cttggattga actaggagat 600 cttgactctg aggagagcca gccctgtaaa aagcttggtc ctcctgtgac gggagggatg 660 gttaaggtac aaaggctaga aacttgagtt tcttcatttc tgtctcacaa ttatcaaaag 720 ctagaattag cttctgccct atgtttctgt acttctattt gaactggata acagagagac 780 aatctaaaca ttctcttagg ctgcagataa gagaagtagg ctccattcca aagtgggaaa 840 gaaattctgc tagcattgtt taaatcaggc aaaatttgtt cctgaagttg ctttttaccc 900 cagcagacat aaactgcgat agcttcagct tgcactgtgg attttctgta tagaatatat 960 aaaacataac ttcaagctta tgtcttcttt ttaaaacatc tgaagtgtgg gacgccctgg 1020 ccgttccatc cagtactaaa tgcttaccgt gtgacccttg ggctttcagc gtgcactcag 1080 ttccgtagga ttccaaagca gacccctagc tggtctttga atctgcatgt acttcacgtt 1140 ttctatattt gtaactttgc atgtattttg ttttgtcata taaaaagt tt ataaatgttt 1200 gctatcagac tgacattaaa tagaagctat gatgaacacc tggcggggtt tgttctctct 1260 ccaatgatcc gagtccactg tttatcgcca gggtggcttg ggctcatttc acatccctgt 1320 ccctgagggg cctcgggtct tacctctggt cctgtcttgt ttccactggc tttgcatctt 1380 cccctaagtt atacttagcc ctgctgaaac acaaaagcac tcgtggggag gaggggtggg 1440 gagaggagac aagggggtga ggaggagggt tctcctgctc gggaagaggt ttgtttcctt 1500 ttcttttcaa tgtgtatcag tgtttttgcc tacatatagg actgttcacc acatacattg 1560 ccctcagagg tcaaaagaca gatcagatcc cctgggaact gaagttacaa atggttgtga 1620 gccactgtgg gtttggggac tcaaactagg tactctgaaa gaacagccac tgcttcaact 1680 ccagggggag tggtttttgt cctgaatcca gattcatttt ttcctctcct ctctctccct 1740 cccctccctt cttccccatt tgtttggaca tactgatcta cacctcaatc cataattatt 1800 ttgagatcat gttaattttt cttactgaac gtcaaaggct attctggaac tcaccaggta 1860 atccagactg gacttgagcc caaactgttt ctcctgcccc agtctttctt gtgctggcat 1920 tgtaaatgct aactcccatg cctggctcag acataactgt tttttggttt tttgtttttt 1980 tttttctttt gagacaggat atttttacat agtcctggtt gtcctggaac tca ctatgtc 2040 aaacaggctg gctttgaact tacagagatc tgcttgcccc ttgcctcctg agtgctggga 2100 ttacaggtct acacatctta gaaatagatt ttcttgatta aaatgaaact taagctccaa 2160 agtgcctgtt ttaaaccctc atagctcagg ttttgcaatt tcaggctcaa ccttttggcc 2220 caaaaggcca cacttgcaat tcactttgca tacctgtgta cattgtaagg gaaggcacgg 2280 gctcatggtg caggtgtcca ttgtgggaag gcacgggctc atggtgcagg tgtccattgt 2340 gggaaggcac cagtgcagct atatgatgtt cactactagg gattttcctg agcagccatc 2400 atgtacaaag gcatgaaact tgaagaagtg gtggaaacca caacgtgata gcgccacctg 2460 caggcccgtg aggatcaccc aaccctgctg gggaaaatcc cactcacaga attaagacag 2520 tcctgatatt cacaggtcat ataggagagc tcttggggag cgactccacc aagcagaaga 2580 gaagcctaaa gacaaagatc tgggcaagtg ctttttctta aagtcaggga ggagtacaca 2640 gaaggtagtg ggggatgggg gtatctgggg gtgtctttcg atgctactag atcagtcagc 2700 agtgtaaggt ggcagctttg accagaacag gtctgaccac actggttcac ctgctccgtt 2760 ggacagggta caactgtaga ggatgctagg cattgatgcc cacctcctgc aggactcagt 2820 cagtcagtcg attgcggccg ccaccgcggt ggagctccaa ttcgccctat agtgagtcg t 2880 attacaattc actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc 2940 aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc 3000 gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatgggac gcgccctgta 3060 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca 3120 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct 3180 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc 3240 acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat 3300 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc 3360 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc 3420 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta 3480 acaaaatatt aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc 3540 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg 3600 ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc 3660 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt 3720 gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct 3780 caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac 3840 ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact 3900 cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa 3960 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga 4020 taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt 4080 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga 4140 agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg 4200 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat 4260 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat 4320 tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc 4380 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga 4440 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc 4500 agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag 4560 gatct aggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc 4620 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt 4680 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt 4740 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat 4800 accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc 4860 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa 4920 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg 4980 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag 5040 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag 5100 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa 5160 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt 5220 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg 5280 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc 5340 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac 5400 cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct 5460 ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc 5520 gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact cattaggcac cccaggcttt 5580 acactttatg cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac 5640 aggaaacagc tatgaccatg attacgccaa gctcgaaatt aaccctcact aaagggaaca 5700 aaagctgggt accgggcccc ccctcgatcg aggtcgacgg tatcgataag cttgatatcg 5760 aattcctgca gcccggggga tccgcggccg ccgtcgagtt taccactccc tatcagtgat 5820 agagaaaagt gaaagtcgag tttaccactc cctatcagtg atagagaaaa gtgaaagtcg 5880 agtttaccac tccctatcag tgatagagaa aagtgaaagt cgagtttacc actccctatc 5940 agtgatagag aaaagtgaaa gtcgagttta ccactcccta tcagtgatag agaaaagtga 6000 aagtcgagtt taccactccc tatcagtgat agagaaaagt gaaagtcgag tttaccactc 6060 cctatcagtg atagagaaaa gtgaaagtcg agctcggtac ccgggtcgag taggcgtgta 6120 cggtgggagg cctatataag cagagctcgt ttagtgaacc gtcagatcgc ctggagacgc 6180 catccacgct gttttgacct ccatagaaga caccgggacc gatccagcct ccgcggcccc 6240 gaattcgagc tcggta cccg gggatcatct agagtggatc cgatcagcag accgattctg 6300 ggcgctgcgt cgcatcggtg gcaggtaagc gggctgctga agccaggcct tggcgagcac 6360 tcagccttcc gtcgtcaagc tcggctcact gcgcctctcg gggccttgag gccacgggga 6420 ctaggactgg gactgggact ggggctgagt ctggctggga ggtgactgta caccccctgt 6480 gcgcgactcc tggaggaacc gaatcccagg gcagccaggc cgggagccag cctttccttc 6540 ccgagccaga ttcacagctc agcatcgctg gggatggggg tggcatcttt tgactgtcct 6600 tggctgtttt cttctctctt tgtagtagct acagcgaaca taattttacc tcgttattcc 6660 accacagtca ttactccctt gcacagtttc attctcaacg tcgccgtgcg ccttcactgc 6720 cctgtctagg cgttttcatg attgtctatt ttcttgtact ttgaataccg tggtttaata 6780 gcagttgcgg ggtgcgcaga attctccatt tccttaagag aaactcctgg gagaatggga 6840 ctaaagacgt gcaaatttaa ttatatcgca aacaggaatc aaaattttgc attaaaatgc 6900 caaacatctt gaaaaattaa ctattcaatg aagaaaagga actactttac ctacacacac 6960 atccgagagc ttcgaggagg cgaaggaaat agaaagctaa gggatgattt gggttgtatt 7020 tgaatctgac acaagctttc catattattt atagcaggga ctaaacgatg agtcattttc 7080 tgaataagat gcaaattaaa g caagtttgt ttgttgtctt tacatctatt aaatagacag 7140 agacaatggc aacagcaacc ctaacctaga ggttgcctga aagtgtcagg tttgggaaca 7200 agtggccctg cttaagggct agaaagattg ctttacaacc aacaatcatg acttgacatt 7260 gcctggggtt ccttttgtct attccttttt taaaagacta gtgtttattt tatgtgcatg 7320 agtgttttgc atccacattc gcctgtatac acacctggtt ctgtggaggt caggagaggg 7380 tgctggatgc cctggcacta gagccttgga tggttatgtg agccctgcca caggggagct 7440 cagaaccaaa tccaggtcct ctggaagagc aaccagagct cttaaaactt ctaagtatcc 7500 ctccatcccc tttccatcat atttggaaag gagaaaactg ctacccatgc ctggcattta 7560 tttcagagat taactgtctg tgtaaaactt gacattgaaa gtgcactatt ctgtttccca 7620 ttcatactta gttgagacta ctgtaagtca gttagggctt tttttgtttg gttccttggt 7680 tagtttggag tgtgtttgtg agctcattaa caggctttca atatgtagct ggaatttgct 7740 gtgtagacca gacaggcctc aaatttgtgg caatcctccc tgcatcttcc cagaatgccc 7800 tggtacaggc ataaaccacc gtgcccagca gtaaaacaat ctggtgaggt attattagtc 7860 gtgtgctgtg acccagaaac cccactcctg gcaatttact gggaaggaac aaacaaaggg 7920 ctaggggagc catatggcct gcagtta gag aaaattagat ccaactgaaa aatcaaccta 7980 aaggtgtaaa agccaagcag ttaagaaact gacaagctca tgatggaagc cgaggccatc 8040 gtgaacactc ttcattttag gccccacgta tcactgggga caactgagag tcaaagtaca 8100 ggtaaggaga ccaaggcttt tcaggactca ggctgtctca gtgaaaagcc cagaagagca 8160 gtaattgaaa gagctcagac gatgtgtctg atctcctctg tttgtttgtt gctgtattat 8220 ttccactaac ttatttggga ggaaaaaaaa cagttcacag gcttcttttc ttgaaatact 8280 ggggattgct gggatcgaac ccagggatag gtttttagtt tctaaaataa catagatcat 8340 gccctgtttg ctttttggaa tatgtttgcg ctgcccttat tttcatgttc aaatactgct 8400 ccattttgcg tgactcttta gtattggttt gatgatttgc atattagatt agattgtatt 8460 tcagttctca gacttattta tcaattctag ttttctcttt ttgttgtttt aaaggactcc 8520 tgagtatatt tcagaactga accatttcaa ccgagctgaa gcattctgcc ttcctagtgg 8580 tacc 8584 <210> 3 <211> 10519 <212> DNA <213> Artificial Sequence <220> <223> TetP-PARIS construct <400> 3 ctcgagcctt cctgcttgtt ccttcgcatt ctcgtggtct aggctggggg aggggttatc 60 cacctgtagc tctttcaatt gaggtggttc tcattcttgc ttctctgtgt cccccatagg 120 ct aatacccc tggcactgat gggccctggg aaatgtacag tagaccagtt gctctttgct 180 tcaggtccct ttgatggagt ctgtcatcag ccagtgctaa caccgggcca ataagaatat 240 aacaccaaat aactgctggc tagttggggc tttgttttgg tctagtgaat aaatactggt 300 gtatcccctg acttgtaccc agagtacaag gtgacagtga cacatgtaac ttagcatagg 360 caaagggttc tacaaccaaa gaagccactg tttggggatg gcgccctgga aaacagcctc 420 ccacctggga tagctagagc gtccacacgt ggaattcttt ctttactaac aaacgatagc 480 tgattgaagg caacaggaaa aaaaaaaatc aaattgtcct actgacgttg aaagcaaacc 540 tttgttcatt cccagggcac tagaatgatc tttagccttg cttggattga actaggagat 600 cttgactctg aggagagcca gccctgtaaa aagcttggtc ctcctgtgac gggagggatg 660 gttaaggtac aaaggctaga aacttgagtt tcttcatttc tgtctcacaa ttatcaaaag 720 ctagaattag cttctgccct atgtttctgt acttctattt gaactggata acagagagac 780 aatctaaaca ttctcttagg ctgcagataa gagaagtagg ctccattcca aagtgggaaa 840 gaaattctgc tagcattgtt taaatcaggc aaaatttgtt cctgaagttg ctttttaccc 900 cagcagacat aaactgcgat agcttcagct tgcactgtgg attttctgta tagaatatat 960 aaaacataac ttcaagcttatgtcttcttt ttaaaacatc tgaagtgtgg gacgccctgg 1020 ccgttccatc cagtactaaa tgcttaccgt gtgacccttg ggctttcagc gtgcactcag 1080 ttccgtagga ttccaaagca gacccctagc tggtctttga atctgcatgt acttcacgtt 1140 ttctatattt gtaactttgc atgtattttg ttttgtcata taaaaagttt ataaatgttt 1200 gctatcagac tgacattaaa tagaagctat gatgaacacc tggcggggtt tgttctctct 1260 ccaatgatcc gagtccactg tttatcgcca gggtggcttg ggctcatttc acatccctgt 1320 ccctgagggg cctcgggtct tacctctggt cctgtcttgt ttccactggc tttgcatctt 1380 cccctaagtt atacttagcc ctgctgaaac acaaaagcac tcgtggggag gaggggtggg 1440 gagaggagac aagggggtga ggaggagggt tctcctgctc gggaagaggt ttgtttcctt 1500 ttcttttcaa tgtgtatcag tgtttttgcc tacatatagg actgttcacc acatacattg 1560 ccctcagagg tcaaaagaca gatcagatcc cctgggaact gaagttacaa atggttgtga 1620 gccactgtgg gtttggggac tcaaactagg tactctgaaa gaacagccac tgcttcaact 1680 ccagggggag tggtttttgt cctgaatcca gattcatttt ttcctctcct ctctctccct 1740 cccctccctt cttccccatt tgtttggaca tactgatcta cacctcaatc cataattatt 1800 ttgagatcat gttaattttt cttact gaac gtcaaaggct attctggaac tcaccaggta 1860 atccagactg gacttgagcc caaactgttt ctcctgcccc agtctttctt gtgctggcat 1920 tgtaaatgct aactcccatg cctggctcag acataactgt tttttggttt tttgtttttt 1980 tttttctttt gagacaggat atttttacat agtcctggtt gtcctggaac tcactatgtc 2040 aaacaggctg gctttgaact tacagagatc tgcttgcccc ttgcctcctg agtgctggga 2100 ttacaggtct acacatctta gaaatagatt ttcttgatta aaatgaaact taagctccaa 2160 agtgcctgtt ttaaaccctc atagctcagg ttttgcaatt tcaggctcaa ccttttggcc 2220 caaaaggcca cacttgcaat tcactttgca tacctgtgta cattgtaagg gaaggcacgg 2280 gctcatggtg caggtgtcca ttgtgggaag gcacgggctc atggtgcagg tgtccattgt 2340 gggaaggcac cagtgcagct atatgatgtt cactactagg gattttcctg agcagccatc 2400 atgtacaaag gcatgaaact tgaagaagtg gtggaaacca caacgtgata gcgccacctg 2460 caggcccgtg aggatcaccc aaccctgctg gggaaaatcc cactcacaga attaagacag 2520 tcctgatatt cacaggtcat ataggagagc tcttggggag cgactccacc aagcagaaga 2580 gaagcctaaa gacaaagatc tgggcaagtg ctttttctta aagtcaggga ggagtacaca 2640 gaaggtagtg ggggatgggg gtatctgggg g tgtctttcg atgctactag atcagtcagc 2700 agtgtaaggt ggcagctttg accagaacag gtctgaccac actggttcac ctgctccgtt 2760 ggacagggta caactgtaga ggatgctagg cattgatgcc cacctcctgtgc gagtcgtc cacctcctgtgc gagtcgtc attacaattc actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc 2940 aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc 3000 gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatgggac gcgccctgta 3060 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca 3120 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct 3180 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc 3240 acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat 3300 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc 3360 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc 3420 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta 3480 acaaaatatt aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc 3540 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg 3600 ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc 3660 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt 3720 gaaagt aaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct 3780 caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac 3840 ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact 3900 cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa 3960 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga 4020 taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt 4080 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga 4140 agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg 4200 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat 4260 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat 4320 tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc 4380 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga 4440 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc 4500 agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag 4560 gatctaggtg a agatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc 4620 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt 4680 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt 4740 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat 4800 accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc 4860 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa 4920 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg 4980 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag 5040 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag 5100 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa 5160 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt 5220 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg 5280 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc 5340 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac 5400 cgagcgcagc gagtcag tga gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct 5460 ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc 5520 gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact cattaggcac cccaggcttt 5580 acactttatg cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac 5640 aggaaacagc tatgaccatg attacgccaa gctcgaaatt aaccctcact aaagggaaca 5700 aaagctgggt accgggcccc ccctcgatcg aggtcgacgg tatcgataag cttgatatcg 5760 aattcctgca gcccggggga tccgcggccg ccgtcgagtt taccactccc tatcagtgat 5820 agagaaaagt gaaagtcgag tttaccactc cctatcagtg atagagaaaa gtgaaagtcg 5880 agtttaccac tccctatcag tgatagagaa aagtgaaagt cgagtttacc actccctatc 5940 agtgatagag aaaagtgaaa gtcgagttta ccactcccta tcagtgatag agaaaagtga 6000 aagtcgagtt taccactccc tatcagtgat agagaaaagt gaaagtcgag tttaccactc 6060 cctatcagtg atagagaaaa gtgaaagtcg agctcggtac ccgggtcgag taggcgtgta 6120 cggtgggagg cctatataag cagagctcgt ttagtgaacc gtcagatcgc ctggagacgc 6180 catccacgct gttttgacct ccatagaaga caccgggacc gatccagcct ccgcggcccc 6240 gaattcgagc tcggtacccg gg gatcatct agagtggatc cgatcagcag accgattctg 6300 ggcgctgcgt cgcatcggtg gcaggtaagc gggctgctga agccaggcct tggcgagcac 6360 tcagccttcc gtcgtcaagc tcggctcact gcgcctctcg gggccttgag gccacgggga 6420 ctaggactgg gactgggact ggggctgagt ctggctggga ggtgactgta caccccctgt 6480 gcgcgactcc tggaggaacc gaatcccagg gcagccaggc cgggagccag cctttccttc 6540 ccgagccaga ttcacagctc agcatcgctg gggatggggg tggcatcttt tgactgtcct 6600 tggctgtttt cttctctctt tgtagtagct acagcgaaca taattttacc tcgttattcc 6660 accacagtca ttactccctt gcacagtttc attctcaacg tcgccgtgcg ccttcactgc 6720 cctgtctagg cgttttcatg attgtctatt ttcttgtact ttgaataccg tggtttaata 6780 gcagttgcgg ggtgcgcaga attctccatt tccttaagag aaactcctgg gagaatggga 6840 ctaaagacgt gcaaatttaa ttatatcgca aacaggaatc aaaattttgc attaaaatgc 6900 caaacatctt gaaaaattaa ctattcaatg aagaaaagga actactttac ctacacacac 6960 atccgagagc ttcgaggagg cgaaggaaat agaaagctaa gggatgattt gggttgtatt 7020 tgaatctgac acaagctttc catattattt atagcaggga ctaaacgatg agtcattttc 7080 tgaataagat gcaaattaaa gcaagttt gt ttgttgtctt tacatctatt aaatagacag 7140 agacaatggc aacagcaacc ctaacctaga ggttgcctga aagtgtcagg tttgggaaca 7200 agtggccctg cttaagggct agaaagattg ctttacaacc aacaatcatg acttgacatt 7260 gcctggggtt ccttttgtct attccttttt taaaagacta gtgtttattt tatgtgcatg 7320 agtgttttgc atccacattc gcctgtatac acacctggtt ctgtggaggt caggagaggg 7380 tgctggatgc cctggcacta gagccttgga tggttatgtg agccctgcca caggggagct 7440 cagaaccaaa tccaggtcct ctggaagagc aaccagagct cttaaaactt ctaagtatcc 7500 ctccatcccc tttccatcat atttggaaag gagaaaactg ctacccatgc ctggcattta 7560 tttcagagat taactgtctg tgtaaaactt gacattgaaa gtgcactatt ctgtttccca 7620 ttcatactta gttgagacta ctgtaagtca gttagggctt tttttgtttg gttccttggt 7680 tagtttggag tgtgtttgtg agctcattaa caggctttca atatgtagct ggaatttgct 7740 gtgtagacca gacaggcctc aaatttgtgg caatcctccc tgcatcttcc cagaatgccc 7800 tggtacaggc ataaaccacc gtgcccagca gtaaaacaat ctggtgaggt attattagtc 7860 gtgtgctgtg acccagaaac cccactcctg gcaatttact gggaaggaac aaacaaaggg 7920 ctaggggagc catatggcct gcagttagag aaa attagat ccaactgaaa aatcaaccta 7980 aaggtgtaaa agccaagcag ttaagaaact gacaagctca tgatggaagc cgaggccatc 8040 gtgaacactc ttcattttag gccccacgta tcactgggga caactgagag tcaaagtaca 8100 ggtaaggaga ccaaggcttt tcaggactca ggctgtctca gtgaaaagcc cagaagagca 8160 gtaattgaaa gagctcagac gatgtgtctg atctcctctg tttgtttgtt gctgtattat 8220 ttccactaac ttatttggga ggaaaaaaaa cagttcacag gcttcttttc ttgaaatact 8280 ggggattgct gggatcgaac ccagggatag gtttttagtt tctaaaataa catagatcat 8340 gccctgtttg ctttttggaa tatgtttgcg ctgcccttat tttcatgttc aaatactgct 8400 ccattttgcg tgactcttta gtattggttt gatgatttgc atattagatt agattgtatt 8460 tcagttctca gacttattta tcaattctag ttttctcttt ttgttgtttt aaaggactcc 8520 tgagtatatt tcagaactga accatttcaa ccgagctgaa gcattctgcc ttcctagtgg 8580 taccatggcc gaggcggtcg cggctccgat ttctccgtgg acgatggcag ccacgattca 8640 ggccatggag aggaagattg aatcgcaggc tgctcgcctg ctttccctag aaggtcgaac 8700 cgggatggcc gagaagaagc tggctgattg cgagaagaca gccgtggagt tcgggaacca 8760 gctggagggc aagtgggccg tgctggggac cctgctgca g gagtacgggc tgctgcagag 8820 gcggctggag aacgtggaga acctgctgcg caacaggaac ttctggatcc tgcggctgcc 8880 cccgggcagc aagggggagt cccctaagga gtggggcaag ctggaggact ggcagaagga 8940 gctctacaag cacgtgatga ggggcaacta cgagacgctg gtctccctgg actacgccat 9000 ctccaagccc gaggtcctct cccagattga acaagggaag gagccctgca actggcgccg 9060 ccctggcccc aagattccag atgttcctgt ggaccccagt ccaggctcgg ggcccccagt 9120 tcccgcccca gacctcttga tgcagatcaa gcaggagggt gagctccagc tccaggagca 9180 gcaggccctg ggcgtggagg cgtgggcagc cgggcagcca gatattgggg aggagccctg 9240 gggcctcagc cagctggatt ccggagcagg agacatctcc acggatgcca cctctggtgt 9300 ccattccaac ttttccacca ccatcccgcc cacctcctgg caaacggatc tccctcccca 9360 ccatccctct tcagcatgct cggacgggac cctgaagctc aacacagcag cctccacgga 9420 agatgtaaaa attgtaataa aaacagaagt ccaggaagag gaggtggtgg ccacacccgt 9480 acatcctact gacctagagg ctcacgggac cctgtttgga ccaggccaag ccacacggtt 9540 cttccctagt cctgcccagg aaggagcctg ggaaagccag ggcagctcct tccccagcca 9600 ggaccctgtg ctggggctgc gagagcccgc ccggcctgag aggg acatgg gtgagctcag 9660 tcctgctgtg gcccaggagg agacccctcc tggggactgg ctcttcggag gggtccggtg 9720 gggctggaat ttccggtgta aaccgccagt gggcctgaac ccgaggacgg ggcccgaggg 9780 gcttccttac tcctccccgg acaacggaga ggccatcttg gaccccagcc aggccccaag 9840 gccattcaac gaaccctgta aataccctgg ccggaccaaa ggctttggcc acaagccagg 9900 gctgaagaag caccccgcgg cgccccccgg gggccggccc ttcacctgcg ccacgtgtgg 9960 gaagagcttc cagctgcaag tcagcctgag cgcgcaccag cgcagctgtg gggcgcccga 10020 cgggtcgggc ccgggcacag gcggtggcgg cagcggcagt ggcggcggcg gtggcggcag 10080 cggtgggggc agcgcacggg atggcagcgc ccttcggtgt ggggagtgcg gccgttgctt 10140 cacgcgcccc gcgcacctca tccgccatcg catgctgcac accggcgagc ggcccttccc 10200 ctgcaccgag tgtgagaagc gcttcaccga acgctccaag ctcatcgacc actaccgaac 10260 gcacacgggc gtgcggccct tcacctgcac cgtctgcggc aaaagcttca tccgcaagga 10320 ccacctccgc aagcaccagc gcaaccatgc agcgggcgcc aagaccccgg cccgaggcca 10380 gccactcccg acgccgcccg cacctcctga tcccttcaag agccccgcct ccaaaggacc 10440 tttggcctcc acagaccttg tgaccgactg gacttgtggc ct cagcgtcc tgggacccac 10500 cgatggcggg gacatgtga 10519 <210> 4 <211> 10000 <212> DNA <213> Artificial Sequence <220> <223> dopamine transporter (DAT) promoter <400> 4 ggatgtgcct aatctgacag aaacttgatg gagg aaacttgatg gggggaggagtc gagg at gggactcagt gagggaggga 120 ccaggaaggg gagcaccatt tgggatgtaa acaattaatt aattaattaa ttaattaatt 180 aattaaaaat aaaaataaat gtcatatcaa aaaaatgaaa taaagaaaag aaaagcaaaa 240 caaaaacaac aaacaatgaa caaaacagga tcatcctatc tgtgagaaat tcacaccgct 300 gaacttacat gccagcaagt tcccagagaa gaagggagca aaaaagttgt ccagggagtc 360 attattctag tatgatgcac actctaaact gtctaatgag aagacagtaa gaacataaag 420 gtagctccct actggctaca gcaatgctca caattaacac caaagggaca ataacagaaa 480 attcagccag aagaaggcac ccttccaagg tgcaaacaag agctagaaat caagagacat 540 ctgcaaagca tggaatggag agaaactgct ggttggtcaa gctctgccct gcaaaaccag 600 cttacaacac agagaaatag gcttccagac ccaggaagct aagcagacaa ctcatccccg 660 cagtctctccg 660 cagtctcccaagat acctggtgt 20 ttgggttcat aatgatgaaa aataagaagt acttatggag atagtagaaa accttgtctg 780 caccatctaa atgtctttta aagagtggtc atctcttgac aagttgggca atgtattctt 840 cagtacagca aaatatctgg tgcggatggc acaaaaaccc aacagggtgc accctggtca 900 agtgcttcct tcacaagtgc ttccttcaca agaggctaaa gcacggggtc ttggacagca 960 gacaccatga tctggagcgt gtgcgtgctt accctaagca gcagacagaa cagcctaaca 1020 aggagacagt gggaaaaggc cgggtgaaac aaaacccact cagtttgaaa aaggaccaag 1080 acgacacaga agaaaaatag aaagtcaaga gcaagctcgt agacattaag tgaaaggagc 1140 tctccctcct tttaatggta cctttttaat gaacgtctta gccaatccca gacccaccag 1200 cacatagaaa ctccactaag gggcaaatgt tgcttatctt gaagaacagg ctcaatcaat 1260 gtaggcatta ctctcttaat cagagcagag accctcagca agattgggtt tgttgatgat 1320 cagaaaaaaa aaagtgaggg ggtgtcatta accatcaccc aaggtaacag ccactccttc 1380 ctgtgccctg cagccccagc tctatggaat tctgggccag tatatagaga ggatgctatg 1440 cagctcttgg gaggctgcta cccacattgg attttctgag agtgcaacta ctcagggtga 1500 cccacacgtt tctaggtaac tcctcaccca agcatctgtt aagacactgt gctgattggt 1560 tcatcaa act ggactttggt ggaatcattt cttgttctgt ggtcagggcc ctatttggga 1620 taaacaagtg tttatttcca cttctctgag aagagtcaca caacaccaag gagtcctaac 1680 aggaaggagt ggcagaagaa acctgcttac ctcctggcag ccaggaaatg aagaaaatgc 1740 ccccactggg gtgggctctg ttcctttatc ctagcttata agattctgcc atgaagactg 1800 aggtagcttt ctctcctttt agcaatccct gtataaattc ctgcccagta ctcaagatgg 1860 caatccatta ttgtcttagt cactgttcta ttgctgtgaa gagacaccat gaccaaggca 1920 actgtatttt ttaattcttt ctttcatata ttacatcctg accaacattt cccttcctcc 1980 cagtctctct ccccacccac tcctcttgtt tcccttcaga aaagggcagg cctcccaagg 2040 atatcaacca aacatgacat atatgaagtt gtaataagac taggtacctc ccctcatatt 2100 gaggctggac aaggcaaccc agtaggagag caaagatctc aaaagcaggc aaagttggaa 2160 atagccccca ctctctctgt taggggtact acaaaaccaa gaaggtatac aacacggtat 2220 acatgtgtaa cgtgtgtgca gaggggctag gtcaagccca tgcaggatcc ctgttggtca 2280 gtctaagtga gcccacatga gtccaggttt gttgattctg tgggttttct tatggtgtcc 2340 ttgatccttc tggcccctac aatccttcct ccctttcttc cacaggactc accgagttct 2400 gcctaatgtt ta actgtggg tttttacatc tgtttctatc agttactgga tggagcctct 2460 ctgatggtga ctgagctagg ttcctgtcta taagtatcac agagtaacat ccgtatttgg 2520 ttctatccta tacctctggg ccatctgtcc tctggtttct ggtcctccag tcagtgtcag 2580 gggtgggctc cttcccatag catgggtctc aagctggacc agtcattagt tggccactcg 2640 cacaatttct gtgccatctt tacccgagca tatcatgctg gcaagacaaa ttgtacgtca 2700 aagatttagt ggctgggctg gtatccccat cccgccactg aaagtcttgt ctggttatag 2760 aagacagctg gttcaggttc catatcctcc attactagga gtcttggcta ggatcactgt 2820 attagattcc tgggaactgt cattgtacta gctttctacc tccctctcaa acttaacctc 2880 agttccagtt gcctctccca gtactctttc cctccatctt cccctaacct gatccatcct 2940 gttcccaatc cctacttgcc cccccccacg ccacttcccc atctacttgc catatctatt 3000 ctattttccc ttcccaggga gctccttgca tccctcatgc cctcctggct tagcctctct 3060 gggtatgtgg attgtagcaa gattattcca agacaattct tataaaagaa aacatttaat 3120 ttgtggcttg tttacaattt cagaggttac tctattatca ccatggcact ggaacattag 3180 cagtgaccta ctgatccaca gggaaaggga gagggagagg gagagggaga gggagaggga 3240 gagggagagg gagaggga ga gggagaggga gagggagagg aagagagcat ctggcataga 3300 cttttgatga ctaatatatg agcctatata tcctgcctta gttagggttt ctatcaatgt 3360 gaagagacac catgagattc caactcttac aaaggaaaac attaattggg gttggcttac 3420 aggtccagag gttcagttcc ttatcatcat ggtaggaagc atggcaagca tataggcagg 3480 catggtgttg gagaaggagc caagagttct acatatggac cagcaggcag caggaagaga 3540 cagacactgg gtctggctta gcatttaaaa tcccaaagcc aacccacaat gacacacctc 3600 ctccaaaaga ccacacccac cccaagaaga ccacactccc aaattactac tctctatgag 3660 cctatgtgag ccatttttct ttctttcttt tatttttaaa gatttattta tttattttat 3720 gtatatgagt acactgtagc tgtcttcaga cacaccagaa gaggacatca gataacattg 3780 cagatggttg tgagtcaccg tgtggttgct gggaattgaa ctctagacct ttggaagagc 3840 agtcagtgtt cttaaacact gagccatccc tccagcccca agccattttt cttcaaacca 3900 ccacataagc accacctgtt ttctcttttc ctttttttcc ttccttcctt ccttccttcc 3960 ctttcccaat gttgttccat tagagaaagt tgattgttcc tcccccatga atataaatga 4020 cagttatgtt gttaacattt acccaactgg ctgggtttac attcattcat tcattcattt 4080 tttaaaaaaa taaaaaataa aac aaaatat caaatcaaac tcagacaaaa cacacaaaca 4140 gaaagaaaag agcccaagaa agggcacaaa acccactcat ttgtatgctc agtaatccca 4200 taaaaacact aaactggaag ctataatact tatgctgaga acctggtaca accctgtgta 4260 tgctgcttca gtctctgtga gttcatgaga gttttgctca tgttgattta gaggtccttg 4320 ttcccttggt gtctttccac ctcctattct gtggggttcc ctgagctctg aaagaaagga 4380 tttatcttag ggtttccatt gctacaatga aacaccatga gcaaaagcaa gttgagggtg 4440 aaacttacac ttccacattg ctgatcacca ctgaaggaag tcaggacagg aactcaaaca 4500 tgacaagaac ctggaggcag gggctgatgc agaggccatt gagggatgct acttataggc 4560 ttgctcccat ggctttgttc agcttgcttt cttatggaac ccaggagcac cagcccagag 4620 ataatacctc ccacaatgga ctgggcactc ccacattggt aactaaaata tattacagct 4680 ggaattcacg gcattttctt gactgaggcc ccgctctgat gactctagag tgtgtcaaat 4740 tgacacaaaa ccaactagta caactgaccc cttgtcagct tgacaaacac cttcctttct 4800 tacgcatctc caagatctca ccttaaaaac ataaataacg taaaagtccc accgtcttaa 4860 caaattccaa acacgttaaa atgtcagtcc ctttaaaata gccaatctct ttcaaaaatc 4920 caaagtcgtt taaaattcaa aatctctca a ctgtgggtgc cagtaagata ctttcttact 4980 ttaagatgga aaaatcatgg catagtcaca atcaaataaa atcaaaacca aatttgaact 5040 gtccaatgac tgggatccac tagttatctt ctgaacccct ccaaagggct tgggtctctt 5100 ctccagctct gccttcttct gtgtcacaca cagcctgtct tctagactcc aaccggctcc 5160 actccatagc tgcttctgtt cataccatag tactggtgtc tccaaatctg atcccttgct 5220 gcaagtgggt tgcattttca ctaacagcct ctcatagctc tcttcctggt gctaagcctc 5280 agctgctctc catgactcct tcaagatttc aaaaccagta ccacctgggt aacttacaca 5340 ttaccaagtc cagctgccag catgaggtat acagccttgg ctatctctgg atcacagctg 5400 ctctgtgctc tcataaaaca cttcccagaa gatttcacct cagtggaggt ggtctcatct 5460 taatcatcac taagttctta gctccagcta accagcatca atttgtcgtc tcagtaatgc 5520 aaaggtttca cttcagtggt gttggtctct tgttaactgt gattggttct tcagccccag 5580 ctgacaaaaa taatagaatc ttaattcaaa atggcaaatg gccttgatag agtctaaact 5640 ttcctttgaa acttcacaag ccaagcctcc atcttctgct ctgctttcaa cattcttatc 5700 ttccaaactc aaaaaaaaaa aaagccaaac aaacaacaac aaaatagtcc aaggaagtct 5760 caaaactcaa tggcttttct agccaaaagt tcca aagtcc ttccacaacc cccccccaaa 5820 aaaaacatgg tcaaatctat ctgtcacagt actccactcc tggttccaac ttgtcttgga 5880 aacacccttg ttagggtttc tattgctgctgaa aacaaacaaaa caaggaacca tatc ta caaggat tattagtag caacacacacagata aa ccacacagt actccactcc tggttccaa aagtcc ttccacaacc cccccccaaa 5820 aaaaacatgg acttccacat tgctgtcagt tcatcactga aggaagtcag gacaggaacc tggaggcagg 6060 agctgatgca gaggccatgg aggggtgctg cttactggct tgcttctcct ggattgtgca 6120 gcctgctttc tcatagatcc caggactacc agcctaagga cgaaaccacc cacaatggga 6180 tgagccctcc cccattaatc actaattaag aaaatgcctt acagttggag ttttttgaag 6240 gcatttttct cagttaaggc cccctccatt cagataaccc tagcatgtgt caagtagaca 6300 taagactagt cagcacagtt ggttattctt acaagcttgg tgccaccatt gccctcgtgt 6360 atcttgcaga tggtacacca ctgtagataa agggtttgtg gctggcctgg tgtttacgtt 6420 tctcttttga tcgcccacag agtaccttct tgttacaaag atcctggaac atatgtgggc 6480 accagcttgg tatctttatg ttcgatgagt tgtgtttgtg ttgtcttcaa actggggact 6540 tacttgctac cagtttgtgg aaagcaacag tcttagcaat agcctgggtt gtttttgaaa 6600 tccagtggaa cacctttggc caacagttta gtttgatgta accccaaact cagtgctgga 6660 agcttcattt gatgacaaga catcaccatt tgggtctgtc tccgattgtt tgtcaattac 6720 acacacacac acacacacac cccttttgtt ttaggaggtt tctactatat tatccaatac 6780 tctgagccga tagcctcagc aacaactata aggaactgag ctctacattt agcaaaagtt 6840 ctaaca tgca aagatgggct gttcaactag gtttgattaa ctcagagcca accacactgg 6900 atctacctgc acacgcccag aaagtgaggt agagatgctt gcactcttag ttctctgtca 6960 tttgtgatga gggctgaaga cgcagtcagt cccctcaact catttctctg agagcacaca 7020 aagtcctact gtcctagctg catggttagg aatcaggtgt ggcaggagta aaggtttcct 7080 ggatatggga cgttgaccaa tatatgcatc acatggtctc tccctgtcac tgagctttga 7140 cactactaag ctggtgtgtt ttgtctgtat gccacctcac tttgttactt tactcctcac 7200 accataggta gtctaaaggt cccattgtgc aatggtattt ctttccatac accctgggta 7260 gtgcagatca ttcttttgta tcctcggggc tcttcttgga ttttgtctct gttttatgag 7320 tggtatgaac ctaagatctg ccctgtacct ctgttcttaa atgatcctct agctatttcc 7380 tttcttactc agccatctgg tttacttgat gacatcagtt gttttcagat gacttcagga 7440 ggcctacatt gggctcctgc ccctttcttg acaacagaat cactactgaa caccatgttg 7500 gtaaaatgca ctaaagataa cctcaaagtc accaaaatgc tttggtgtca catttaggaa 7560 ttaaaaattc aactctcctc ccttgctaat gcttggctga ttagtgagcc caagtctgag 7620 tctttcactt ccctgagtct gtcctgtatc ttctgtctgt ccccaacaag agtgtcccca 7680 ctctgtatgt a ttgagcaga gcagaaagca tagtatgaga aatgagaaat tctctacaga 7740 aatcttcaac agagttttac atggtattaa gttattgatt ctaactgacc tagaaatact 7800 cagtactaac aagaaaactt tatcaagcta tatccacagg ttgattccaa tgattgtgga 7860 tatttgttgt ataaggttgc ttccaggcca tttgctaatt tcagcaaatg attaccacaa 7920 cacacacaca cacacacaca cacacacaca cacacacaca cacacaactc tggatcaggg 7980 ccatggaaga gtatataact taagattaaa gttatgtatt agtgtaggtt gtgagagggc 8040 acactacttg ggaaactgag gcaagtaaag ttgcttgtta cattgttctc ttaaaggcaa 8100 gttaaacttc aaaaggctgg ttacaaagta ggatggaaga ttatgtcttc gatgatccca 8160 gaatctatta actcagttgt gaggtccagg aaagaccagc ctctttaatg taagaggtat 8220 ttgcataaca ctgttttatg acagaataga ggcaaagaga tagcctttag tgtgttagca 8280 actggaggtg attaccatta tctcctcaag tccctattac cagctggaat accaaggaca 8340 ttattcccct accctcatct acatgtggaa accataccct ctcgtgtcag atgctattta 8400 gagtcaggtc ttgggtttca ttggctctcc tcaaaaaaaa attttttttt gtcttataaa 8460 atggtcctgg ggagctctgc acacctctgc cctagggaga ccctagagaa cttcccattt 8520 gccgtgggag cagctct ctc caaatcctcc ctggggatct gagacttcta agcctttaca 8580 ggaataaatg tttgttgact gactccaagg ttgtaatgtt tctccacagc agctccagcg 8640 gaccactgac agctgtagca gtgtgccctc cccattctaa tctcacttgg aaaaacagct 8700 ctaaaaacag ctcctgctag gcatcagcct cattttgggg ggctgaaggg aagcgataag 8760 gccacaggac gtgggcacag agaaggaacc aggaagataa gggcccagcc attttggggt 8820 cccaactagt aggagacact gctaggaact tggtttagtg gagcctgcag gtgagatact 8880 tcttctaggg tccctcactc tggtcaaacc tctcagctat tctcagggaa agcagaaatg 8940 gaaaggaagg gcatgagggg gtgctgggaa tggggaaggc atgcagagag gaaaagggct 9000 taccctagga cctgctcaca tctgggtgtc ctggacctcc ctgggtggcc gaatgtcacg 9060 cccatctggg caggacgatt ccagttcctg gtcctttagc ccttgaaaca ggaatattgg 9120 ccacacaggc tttataggtc cctccaggaa aatcggtttc tgaagcaggc tgactggaag 9180 gagccttcga gtttggatca gatggcccgg atctgagtaa aaaagtatgc tcagaagaga 9240 acagaggcac acattacaaa catgcacttg tgaccatggg acctgagaaa ggtggcagag 9300 aatcaggacc catcaaaggg gtattggaac tttagtgcct aaggtgctca cggagtgagg 9360 agggaagagt gcctgagcaa ac tggtcagg gcaaggacga gacttgctgt gacctggcta 9420 ggaacgcagg tgggccacct ccacagcacc gggaggtgcg tcccacttgc tttgtctccg 9480 agcggaactg gagttggaaa atcttgctcg caggttggtt gttcagggac aagggagcag 9540 ggtcggagag tcatacaaca tcgaagggag aactggggag acccgggtga gccttgggag 9600 ctccgcacag atccacatgc gctcgccact tcactctctt cccgggatct gtttctgaca 9660 accttgctga aaccagttgt tgggagactc atgcaggcgc tggccgagga gagcgaacag 9720 ggacggagac ttggactgct ccctcctgga aaccaactgt ccctccggtc tgcaacgcgt 9780 ccaacctgga gatacactga tcccacgtgg ccgagactct ataaccttgc aaagacacct 9840 gccaaactcc ttgcctgcct aggctcagtg gcctctggct ccccgaagtc atcctagtcc 9900 ccaagtggtg caggggcgct gcccagcccg tcctccgccc accctgggcg gaacagaggc 9960 ggagccagtg ccagtgggcg cccccgacgg cgggcggggc 10000 <210> 5 <211> 10961 <212> DNA <213> Artificial Sequence <220> <223> CamKII alpha-promoter <400> 5 gggtcgacta aggcccgggc ggcctcgagg gggggcccgg tacccaattc gccctatagt 60 gagtcgtatt acgcgcgctc actggccgtc gttttacaac gtcgtgactg ggaaaaccct 120 ggcgttaccc aacttaatcg cagca catccccctt tcgccagctg gcgtaatagc 180 gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatgggac 240 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct 300 acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg 360 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt 420 gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca 480 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga 540 ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa 600 gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac 660 gcgaatttta acaaaatatt aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc 720 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac 780 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt 840 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag 900 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg 960 aactggatct caacagcggt aagatccttg agagttttcg ccc cgaagaa cgttttccaa 1020 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc 1080 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag 1140 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa 1200 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc 1260 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg 1320 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa 1380 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa 1440 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg 1500 gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag 1560 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg 1620 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt 1680 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt 1740 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac 1800 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagg a tcttcttgag 1860 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg 1920 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca 1980 gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga 2040 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca 2100 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc 2160 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca 2220 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa 2280 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc 2340 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc 2400 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg 2460 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat 2520 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca 2580 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca 2640 aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggt ttcccg 2700 actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact cattaggcac 2760 cccaggcttt acactttatg cttccggctc gtatgttgtg tggaattgtg agcggataac 2820 aatttcacac aggaaacagc tatgaccatg attacgccaa gcgcgcaatt aaccctcact 2880 aaagggaaca aaagctggag ctctaaggcc cgggcggcct cgacggtatc gataagcttc 2940 gatctttttt ccgtaaactc aataccaggc tgatgtccca ccggatctga tggcttaggg 3000 tggcagggaa tctcagttcc cctcagacac tctccctttg ctggttctca gggaggaggc 3060 aaggtcaagt cttcatctgt aggcacgtgg agggagggca cagaagccct cagctgaata 3120 gggtgggact tggggaaggg cagcaaccag gctgggttgc ctgggtcaca atcctgcctc 3180 tttcctgatg agtttccttt ttgccctcag gttacctata gcagcattct gcctcaatct 3240 cacccctaag atgagctctg gtgactttag gactccagtg tacacatgtg tctggggcca 3300 tggcagggtt tcttgctgac cttgtcacct tccagacaac ttgagtccat gaccctcttt 3360 ccagctctct gtggtgctct tggatatcag ctggagtatg gccagctggc tgctgctctg 3420 ttgaacaact caatgagaga acggacaggg taggctctga gaaatcttta cgttcctgga 3480 gcctcatgac ttgggagcct agtggaattc ttctcttttg gtccccaaca tctgggggga 3540 gggggaactg gctgagcctg agccactgta tagtgagggt gggggaaaca gctgtgaaag 3600 gagcctttga tttggtcttg aacacagttc ttccccacag ggccttgatt tccctacttg 3660 caaaggagta gggaaatatg agccttggct ctgcctacct cacgttgctg gtgctgtaga 3720 aaactggcca ggctgatggt tgtggaggag cctgtgaact tgattaaagt gccattatcc 3780 agaggcaaga gatgctggtc tgtgtgtgtg tgagtgtgtg tgtgtgtgtg tgtgtgtgtg 3840 tgtgtgtgtc ttggagacct gtgtgagctc gatgctagca gaggggacag aaggtaggtg 3900 ggaaggaatg aaggaatgaa ggaaggaaag aaggaaggtt gaaaggaaga aaggaaggaa 3960 ggaaggaagg aaggaaggaa ggaaggaagg aaggaaggaa ggaaggaagg tcctgccaca 4020 ggcttaccat gtagctgcag gcaaacccct gaccctctct gggcctaagt gtttctctac 4080 acacaatgga tgattcaaga gtccttactt ttggtggtta caggcacccc tgtgcacatt 4140 tgcatctggg gtggggggga cacaggcttg gtagtgttga ggaggggggt ggttgtagag 4200 cctgctagct gcacactgcg ttctgcatat ctcccttcag gtcccagtcg gcgcagtgtg 4260 tgtaggcgaa ggccctgctg tgaattttga aaatagttat ttttgtcact ggcaaaggag 4320 gccctgttag gactcgtcag cttgtggatg agcgggatgg gtggagtggg gtgggtgcgg 4380 tgccgccgtg gggggttacc gctgcttgca gggctgcatc gcccaggcag tgactgaatc 4440 ctgcatgagg gcctggccta ggctgtgggg aggagatgac cactgcgtcc tagatctttc 4500 cttagccctg tgcttccttt cttccttttt tccttaagat ttatttctaa tgatgcgtag 4560 gttgtgtatt tgtgtgtggg tatgtgcact tgaataaagg acccacagag gctatagaca 4620 tcagatctcc tagagctggg gttacagagg gcgtgagttg tccaacatgg gcaccggaaa 4680 ataaacttat gtgctctaca cgaccgagct gtctctccaa caccagccct cttcttttga 4740 cttttctcat ctccctcact tgtatgtttt cccttcctcg ataatgctga tacccagatg 4800 gtaggcacgg cccaggatag gcaggggtct ctgcgctcca gtggctgtag tggttttcct 4860 gcctgctctg aagaagacac tggctaaggt ggtgtcttag cctactgtat cctagaggtg 4920 ggttattcat agtctgccca ctgcccaaca cactctaggc ctgctggggc ctattctaac 4980 tctgcttgct ggcttgccac cctggcacac agtgtaggct tccctgtaga gccaggcttt 5040 agagaactgt atgagtactt ctctgagaac tgctggaggg gccctgcctg ccaggacttt 5100 tcaacttcca gccctgtcat ctatatcact tctgaggacc ccgtgtgggg gtcacgagaa 5160 caagaccata tgtagtgctt tctgttctcc cttgggtccc aggctctgaa tcaatctggt 5220 cccaag atat aagggatgat tggtgctgag gctggtgtct gtttctgaag ttttgaagac 5280 aagggttggc tcaagcctcc ctgtgttcag tcctccactc aatgcagaac tcagtgaact 5340 cagaattctc agcccagatg ccagcatagc ccagcatagc ccagcatagc ccaggagcta 5400 ctggagcatc agtttgaaac caggtccgca ggaactagtg ggcaacagtg tgtgaggcca 5460 gtggtctttg gggtattgta ttgaattgag aggtcctgct tagcagtcag catgcccaca 5520 acctgttctc tacggtggtc cggattcccc tcagcaagca cacctgaatc tttactacat 5580 cccagttcct ggttggctcc tgacttcggg ttactatggc tgtgatgaaa cactacgact 5640 aaaagcaatg tggggaagaa agttaatttt ttcactcgac cttccataga cagggttcat 5700 cactaaaagc agagggcaga ggaaaagata gctcagcggt taagagtgct gtctactcaa 5760 caaagaggtc ttgagttcaa ttcccagcaa ccacatggtg gctcacaact gtctatcctg 5820 ggatctgatg cccttttctg gcatacaggt atacatacag atagaggact catatacata 5880 aaataaataa ataaatcttt aaaagcaaca agggcagaaa ttcaagcagg gcagggaccc 5940 agaggccaaa gctgacgcag aggccatgga ggggtgttgc ttactggctt gctcctcatg 6000 gcttgctcag cctgttttct tatagaaccc acaaccacca ggccagagat gggaccaccc 6060 acaaagggca g agcctttcc ctatcaatca ctaatgggaa aacatcctgc agtcggatct 6120 tatgaagaca ttttctcagc tgagcttccc tcctatcaga taactctagc ttgtgtcaag 6180 gtgacttaaa actagccagc acagcacctt atgctcacat cacctgggtc cctttggaga 6240 ggacatagtt aaagggagcc cagaggcagt ccctaggcca caggtcttca ttgcccctct 6300 ctgggacgga ttagacaggc tgcagacctg ttagctggaa gagttagatt caggcaagag 6360 cttgaatctt tacctgatcc tggctatgga gtcctggcct ctaatgatca gctccctaac 6420 aacccaatgg agccatatac ctgcctgggc cacggctgtg tctcctcttc tttcagacac 6480 tcctggcttg cctaggacac aggctagcat cctgtcaatg ccaggaaggg gcacagcagg 6540 gaaagagcaa tgctgttggc ctgactgcca tcaactggtg tacctgttag agggcaacct 6600 ctattctctg caccttggtt cctagctcta agggatatgt ggcccctaaa ggtcttcata 6660 gcttgatatg ggaggcaggg gggctaagaa cagcgcaaga gtggtgagct tgcacagacc 6720 cggatttgat ctctgggtga gtgaggagga aatgagatgg gggtggggga agccctattt 6780 ctagctgtct tagcatagga actgaacctc cttctgcagg gcctgtgtca ctgccccttt 6840 cccccaggga gggcccctgc acggggcacc tcagggcaca gccctttttc cctccctcct 6900 ctcttagacc tggaatt act caacatcctg ccctgactca gttgctctcc cctcagaccc 6960 tcacagtctt ccttctcttc tggcccactt ttggctgagc ctgcccccaa ctttttctgc 7020 ccttagtggg acaggcccca tggggaccat tcagatggca cttttttccc cccctggggt 7080 ggttttctgt ggtggtgccc tattcaggca actgcaagac cctgtggcat ttagcatatg 7140 catgagagca catgaagaag ctagctatcc ctgtgtgctg aggattgtaa tcctctctca 7200 tccttccctt gtctcctgga acccagtcca gcctcctgtc cctcccgttg acacgagcca 7260 atgctggctc agcaaactcc agggctccca cccctggcca tcagcccttg gcacacaggc 7320 ttgtgcttga gtactgcaca cgtgttgcag ctggggtaca cgtgctggac tgttatgcct 7380 actgtggccc cgggggtgtg tgggaagtct ggcagaacca atccctccat cccccgatgc 7440 aatcatcagc ttattctctc acggccactc gggcatgctt gactccttga tgcccgccgc 7500 cactaggcac agctgccagc tttgtgggca cagaggatgt ggcgaattag tggtcatgcc 7560 tcctcagtgg aatggcaatt gcactcagca tgcaggtgtc taccaaaggc agtccctaca 7620 tccccgatgt actctcgaga cccatctaag gactagatct agtctctaga aggtcccatg 7680 cagatgtaag acagccctcc acagggagat tcttccagct agttctctat tatcagatgg 7740 gtctaagatc ctaggacctg cc tatccctt agccctgcat tcagcgagag aaggggtaaa 7800 gatgtgagga tgccagggag gaaggaaaag ggcacaagga agaaagaaag ggaaggaagc 7860 tggaagcatg gaaggacaaa gatggtgacc acagtagaat taggatccca tggttcctgt 7920 cagtggcttc ctgtgccttc ctgtgcctcc ctgagcccct ggggcatctt ctaaatgctt 7980 tgctggcctc tgagccaagc actgcatacc atcccgtggg gagtgacagg ccagcactgg 8040 tcaacgagga tgatggctac ttttgttcac agggtaacat ctccatggtt acagcctttg 8100 cacattcctc ttagtacttt accaatctca aagcagttgc caagcccttg ggccctaata 8160 agtgagggtc ccagtgccct cttttttaaa ttccttgcca tttgttttgc agaatttact 8220 gcaaataaag ccaaccccag gcaatgtcta aaccatgagt taacccccca gcaaggtctc 8280 agagaactgt gccccagaga gctgccaagg ttcagggagg agtatgagga gacaggattt 8340 ctagttcctt aataattcct tctgtctcag ccactgtgtt catcttgttt cagccacaaa 8400 actaccttta ttggtaagga acattattta cccagtttca cacttgaaga ggtccagaga 8460 cgttaacaca tcgattcaaa agcacagcct gtaagtcaca tagccactgt tagctgatcg 8520 acactatttc ccctgggcaa tggctgggtg attccaggga tccccttggg aacaggctag 8580 agcactggct ctcaacctgt gcgggtcg tg acctccttga ggggtagggg tgagggcagt 8640 gtcaaacaac ccttttacag gagtcgttta agaccgttgg gaaaaaaacc agatatttgc 8700 attatttttc gtaacagaag caagattata gttatggagt agtgacaaaa attatgttac 8760 agttggaggt cagcacagca tgaggaactg tatttaaggg ttgcggcatt aggaaggttg 8820 agaatcactg gcctagcgga tctgaatcag gaacacggac gtacagctct gcgccactcc 8880 tgccttcctc tggtgcctct agccttgccc atggtgttct gggcctgcct gctacccacc 8940 agctgtgcgg ccctgtgagc acaggccttt ctgctccgct ctgaattgcc acgttggcgg 9000 cagaagcggg aagcgtattg tgcgcagaaa caaaacggag tggttttttt tttccttttt 9060 ctgaaggtgg taatggtgca attagtggcg aagccatcac cccctcctcc ccggctcgcc 9120 tccctccttc ctctccacct cccttctctt tctttcctga gaaaaaaagt ggctgagttg 9180 aaaagatctc ccgtcaatct ttctgtaacg gactcaggaa gagggataga gggcccccta 9240 atgtttccag ggtcctcgag cctcagttgg gtcaggcact tgttggtgct ggagaatatt 9300 caaaggtacc actatgttcc ccacaaggga gttgagcaat ggattctgag gagcaagttt 9360 gaaacagaga atttgcgttc ccaggtcttg tgatctgccc cttgttcact gggggacaaa 9420 tgctggcatg agaccctgag acctctgctc agc caccttt ctctctctct ctctctttt 9480 ctctctctct ctctctctct ctctctctct ctctctctct ctctgtcctt aatggaggtg 9540 tgtgtgtgtt caagaccaag ctgcagtgtt ggagtgctttg tgga acctcattttt tgga acctcatttt tgga acctcattttt tctccatggg gttcttcttc tgattttcta gaaaatgaga tgggggtgca gagagcttcc 9720 tcagtgacct gcccagggtc acatcagaaa tgtcagagct agaacttgaa ctcagattac 9780 taatcttaaa ttccatgcct tgggggcatg caagtacgat atacagaagg agtgaactca 9840 ttagggcaga tgaccaatga gtttaggaaa gaagagtcca gggcagggta catctacacc 9900 acccgcccag ccctgggtga gtccagccac gttcacctca ttatagttgc ctctctccag 9960 tcctaccttg acgggaagca caagcagaaa ctgggacagg agccccagga gaccaaatct 10020 tcatggtccc tctgggagga tgggtgggga gagctgtggc agaggcctca ggaggggccc 10080 tgctgctcag tggtgacaga taggggtgag aaagcagaca gagtcattcc gtcagcattc 10140 tgggtctgtt tggtacttct tctcacgcta aggtggcggt gtgatatgca caatggctaa 10200 aaagcaggga gagctggaaa gaaacaagga cagagacaga ggccaagtca accagaccaa 10260 ttcccagagg aagcaaagaa accattacag agactacaag ggggaaggga aggagagatg 10320 aattagcttc ccctgtaaac cttagaaccc agctgttgcc agggcaacgg ggcaatacct 10380 gtctcttcag aggagatgaa gttgccaggg taactacatc ctgtctttct caaggaccat 10440 cccagaatgt ggcacccact agccgttacc atagcaactg cctctttgcc ccacttaatc 105 00 ccatcccgtc tgttaaaagg gccctatagt tggaggtggg ggaggtagga agagcgatga 10560 tcacttgtgg actaagtttg ttcgcatccc cttctccaac cccctcagta catcaccctg 10620 ggggaacagg gtccacttgc tcctgggccc acacagtcct gcagtattgt gtatataagg 10680 ccagggcaaa gaggagcagg ttttaaagtg aaaggcaggc aggtgttggg gaggcagtta 10740 ccggggcaac gggaacaggg cgtttcggag gtggttgcca tggggacctg gatgctgacg 10800 aaggctcgcg aggctgtgag cagccacagt gccctgctca gaagccccaa gctcgtcagt 10860 caagccggtt ctccgtttgc actcaggagc acgggcaggc gagtggcccc tagttctggg 10920ggcagcgggg gatccactag ttctagagcg gccgccaccg c 10961

Claims (29)

A vector for manufacturing a neurodegenerative disease model, comprising a Parkin Interacting Substrate (PARIS) gene and a tetracycline regulatable transcription activator (tTa)-sensitive promoter. The vector for preparing a neurodegenerative disease model according to claim 1, wherein the PARIS gene comprises the nucleotide sequence represented by SEQ ID NO: 1. The vector for manufacturing a neurodegenerative disease model according to claim 1, wherein the tTA-sensitive promoter is a Tet promoter operably linked to the PARIS gene (Tetracycline responsive promoter, TetP). The vector for preparing a neurodegenerative disease model according to claim 1, wherein the tTA-sensitive promoter comprises the nucleotide sequence shown in SEQ ID NO: 2. The vector for preparing a neurodegenerative disease model according to claim 1, comprising the nucleotide sequence represented by SEQ ID NO: 3. The method of claim 1, wherein the degenerative neurological brain disease is dementia, Alzheimer's disease, Lewy body dementia, Huntington's disease, multiple system atrophy, at least one selected from the group consisting of amyolateral sclerosis, stroke, cerebral infarct, head trauma, cerebral arteriosclerosis, Parkinson's dementia and Parkinson's disease; Vector for making neurodegenerative disease models. A responder animal for producing a neurodegenerative disease model injected with the vector of claim 1 . 8. The responder animal of claim 7, wherein the animal is at least one non-human animal selected from the group consisting of rat, mouse, guinea pig, monkey, dog, cat, rabbit, cow, sheep, pig and goat. A driver animal for preparing a neurodegenerative disease model, comprising a tTA gene and a neuron-specific promoter operably linked thereto. The driver animal for preparing a neurodegenerative disease model according to claim 9, wherein the animal is at least one non-human animal selected from the group consisting of rat, mouse, guinea pig, monkey, dog, cat, rabbit, cow, sheep, pig and goat. The driver animal for preparing a neurodegenerative disease model according to claim 9, further comprising a tTA gene and a tTA-sensitive promoter operably linked thereto. The driver animal for preparing a neurodegenerative disease model according to claim 9, comprising a tTA gene and a dopaminergic neuron-specific promoter operably linked thereto. The driver animal for manufacturing a neurodegenerative disease model according to claim 12, wherein the dopaminergic neurons are neurons expressing one or more genes selected from the group consisting of TuJ1, MAP2, HuC/D, VMAT2, and DAT. According to claim 12, wherein the dopaminergic neuron-specific promoter is a dopamine receptor (dopamine transporter, DAT)-specific promoter, a neurodegenerative disease model manufacturing driver animal. The driver animal for manufacturing a neurodegenerative disease model according to claim 9, wherein the nerve cell-specific promoter is CamKIIα (calmodulin-dependent protein kinase type II alpha chain). A neurodegenerative disease animal model obtained by crossing the responder animal of claim 7 and the driver animal of claim 9. The neurodegenerative disease animal model according to claim 16, wherein the expression of PARIS can be regulated by the Tet-OFF system. An animal model of Parkinson's disease obtained by crossing the responder animal of claim 7 and the driver animal of claim 12. 19. The animal model of Parkinson's disease according to claim 18, wherein the dopaminergic neuron specifically expresses PARIS. The animal model for Parkinson's disease according to claim 18, wherein dopaminergic cell death, movement disorders, olfactory dysfunction, mitochondrial structural abnormalities or Lewy body lesions occur. An animal model of dementia disease obtained by crossing the responder animal of claim 7 with the driver animal of claim 15. 22. The animal model of dementia disease according to claim 21, wherein the dementia is Lewy body dementia. The animal model for dementia disease according to claim 21, wherein the neuron-specific PARIS is expressed. A method for preparing a cell model for neurodegenerative diseases, comprising introducing the vector of claim 1 into neurons. The method for producing a neurodegenerative disease cell model according to claim 24, wherein the neuron is a neuron of a human, mouse, rat, rabbit, fruit fly, pig or monkey. A neurodegenerative disease cell model prepared by the method of claim 24 . 1) preparing a responder animal by injecting the vector of paragraph 1 into an animal other than a human;
2) crossing the responder animal with a driver animal comprising a neuron-specific promoter and a tTA gene; and
3) A method for producing an animal model of a neurodegenerative disease, comprising selecting an animal having all of the PARIS gene, the TetP-sensitive promoter, the tTA gene, and the neuron-specific promoter. A method for screening a therapeutic agent for a neurodegenerative disease using the neurodegenerative disease animal model of claim 16 or the neurodegenerative disease cell model of claim 26 . A method of verifying the therapeutic effect of a neurodegenerative disease therapeutic agent of a neurodegenerative disease therapeutic agent using the neurodegenerative disease animal model of claim 16 or the neurodegenerative disease cell model of claim 26 .

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