KR20190056164A - Transgenic animal inducing reactive astrocyte and preparation method thereof - Google Patents

Abstract

The present invention, according to one aspect, provides a transgenic animal that induces reactive astrocytes, a method for producing the same, and a method for screening a substance for treating brain diseases using a transgenic animal. Accordingly, the reactivity of astrocytes can be controlled, and an animal model for focal infection can be produced. In addition, the present invention can be used to screen a substance for treating various diseases in which reactive astrocytes are involved, such as brain inflammation, degenerative brain diseases, dementia, and stroke.

Description

[0001] The present invention relates to a transgenic animal inducing reactive astrocyte and preparation method thereof,

To a method for producing the same, and to a method for screening a substance for treating brain diseases using a transgenic animal.

Degenerative brain disease is a brain disease that occurs as a result of age-related degenerative diseases. It is known that degenerative brain diseases cause progressive dysfunctions such as cognitive disorder and movement disorder due to the gradual deformation or loss of function of certain brain cells of the brain and spinal cord. Dementia is a disease with general impairment of systemic functions such as memory impairment and loss of judgment. Alzheimer's dementia (dementia of Alzheimer type), 20-30% of dementia of vascular type, Alcoholic dementia and Parkinson's disease dementia, and about 15 ~ 20% are known to develop both Alzheimer type and vascular dementia.

Astrocyte, also known as astroglia, is a starlike cell in the brain and spinal cord. The astrocytes function in biochemical support of the endothelial cells forming the blood-brain barrier, in providing nutrition to the nerve tissue, maintaining the extracellular ion balance, and repairing the brain and spinal cord after traumatic injury. However, reactive astrocyte cells are known to be transformed into astrocytes that are toxic to nerve cells and can release signals that can kill neurons (Liddelow et al., Nature 541 (7638) : 481-487).

Therefore, it is necessary to develop an animal model for inducing reactive astrocytes and develop a method for screening substances for treating brain diseases using the animal model.

To provide a transgenic animal that induces reactive astrocytic cells.

Thereby producing a transgenic animal capable of inducing reactive astrocytic cells.

A method for screening a substance for treating brain diseases using a transgenic animal that induces reactive astrocytic cells.

One aspect is a transgenic animal that induces reactive astrocytic cells,

A first gene cassette operatively linked to a nucleotide sequence encoding a glial fibrillary acidic protein (GFAP) promoter sequence and a Cre recombinase-estrogen receptor T2 (Cre-ERT2) polypeptide; And

A transcription termination nucleotide sequence is located between two LoxP nucleotide sequences and a second gene cassette comprising a nucleotide sequence encoding a Diphtheria Toxin Receptor (DTR) downstream of any one of the LoxP nucleotide sequences is transformed,

Wherein the CreERT2-LoxP system is activated by administration of an estrogen antagonist to induce expression of DTR in a reactive astrocytic cell.

The term " astrocyte ", also referred to as astroglia, is a star-like brain cell present in the central nervous system of vertebrate animals. The astrocytes function as nutrients to nerve cells, support endothelial cells in vascular-brain barrier, absorb and transmit neurotransmitters, regulate ion concentrations in extracellular space, regulate synaptic transmission, restore the nervous system, or a combination of these Can be performed. When injured in the brain, astrocytes can become proliferative (astrocytosis), swelling, and astrogliosis.

The term " reactive astrocyte " refers to astrocytes that are abnormally proliferated by the transformation of astrocytes. Reactive astrocytes may abnormally proliferate due to central nervous system trauma, infection, ischemia, stroke, autoimmune reactions, neurodegenerative diseases, and the like. Reactive astrocytes may inhibit the regeneration of neurons or secrete toxic substances to induce the death of other astrocytes or neurons.

The term " transgenic animal " refers to an animal capable of artificially regulating the expression of a target gene by transforming an exogenous target gene into an animal. The animal may be a mammal other than a human, for example, a mouse, a rat, a guinea pig, a dog, a pig, a cow, a goat, a horse, a sheep, a cat, and an ape.

The transgenic animal is further characterized in that a nucleotide sequence encoding a glial fibrillary acidic protein (GFAP) promoter sequence and a Cre recombinase-estrogen receptor T2 (Cre-ERT2) polypeptide is operably linked The first gene cassette was transformed.

The glial fibrillary acidic protein (GFAP) is an intermediate filament protein expressed in various cells of the central nervous system including astrocytes and ependymal cells. GFAP is known to function in maintaining the mechanical strength of astrocytes.

The GFAP promoter is a regulatory region necessary for GFAP expression, and may be a DNA region in which transcription is initiated. The GFAP promoter may be a nucleotide sequence that exists upstream from the GFAP transcription start point, i.e., in the 5 'direction. The GFAP promoter sequence may be a human-derived sequence. The GFAP promoter may be a reactive astrocytic-specific promoter.

The Cre recombinase is a tyrosine recombinase from P1 bacteriophage. Cre recombinase is an enzyme that catalyzes site-specific recombination between two DNA recognition sites (LoxP sites).

The estrogen receptor T2 may be an estrogen receptor with a mutation of G400V, M543A, and L544A.

Wherein the transgenic animal has a transcription termination nucleotide sequence located between two LoxP nucleotide sequences and a second gene cassette comprising a nucleotide sequence encoding a Diphtheria Toxin Receptor (DTR) downstream of any one of the LoxP nucleotide sequences Is transformed.

The term " operable linkage " refers to arrangements in which the components of the gene cassette are operable in an intended manner.

The first gene cassette may be a GFAP promoter from the 5 'end and a nucleotide sequence encoding Cre-ERT2 polypeptide linked thereto.

Wherein the transgenic animal has a transcription termination nucleotide sequence located between two LoxP nucleotide sequences and a second gene cassette comprising a nucleotide sequence encoding a Diphtheria Toxin Receptor (DTR) downstream of any one of the LoxP nucleotide sequences Is transformed.

The LoxP nucleotide is a DNA sequence derived from bacteriophage P1, which may be a site recognized by Cre recombinase to cause site-specific recombination.

The transcription termination nucleotide sequence may comprise a nucleotide sequence comprising a transcription termination signal. The transcription termination nucleotide sequence is, for example, a trimer of the SV40 polyadenylation sequence (tpA).

The Diphtheria Toxin Receptor (DTR) is a receptor that specifically binds to a diphtheria toxin, and can be introduced into cells by receptor-mediated endocytosis.

The second gene cassette may further comprise a ROSA26 polynucleotide. The ROSA26 polynucleotide can be a nucleotide sequence or a locus that can be used for constitutive or ubiquitous gene expression.

The transformation may include transformation, transduction, transfection, and viral infection.

In the transgenic animal, the CreERT2-LoxP system can be activated by the administration of an estrogen antagonist to induce the expression of DTR in a reactive astrocytic cell-specific manner.

The estrogen antagonist may be tamoxifen, 4-hydroxy tamoxifen, clomifene, raloxifene, or a combination thereof. The estrogen antagonist may be administered for 1 day to 2 weeks, 1 day to 1 week, or 1 day to 5 days. The estrogen antagonist may be administered once a day, or once every two days to one week.

The CreERT2-LoxP system recognizes two LoxP nucleotide sequences of a promoter-specifically expressed Cre-ERT2 polypeptide and catalyzes site-specific recombination between two LoxP nucleotide sequences to specifically amplify the gene downstream of the LoxP nucleotide . ≪ / RTI >

The transgenic animal can specifically express DTR by the CreERT2-LoxP system when the Cre-ERT2 polypeptide is expressed by a reactive astrocytic GFAP promoter.

Kg / day to 1 mg / kg body weight / day, 1 占 퐂 / kg body weight / day to 500 占 퐂 / kg body weight / day, 5 占 퐂 / kg body weight / day to 250 占 퐂 / body weight kg / Kg / day to 10 占 퐂 / kg of body weight / day to 100 占 퐂 / kg of body weight / day, 10 占 퐂 / kg of body weight to 75 占 퐂 / kg of body weight / day, or 12 占 퐂 / ≪ / RTI >

The diphtheria toxin may be administered for 1 day to 1 month, 1 day to 25 days, 1 day to 20 days, 1 day to 16 days, or 2 days to 16 days.

Reactive astrocytes can be induced by administration of the diphtheria toxin to the transgenic animal. Induction of reactive astrocyte cells can lead to brain diseases. The brain disease may be brain inflammation, degenerative brain disease, dementia, stroke, or a combination thereof. The degenerative brain disease may be Alzheimer's disease, Parkinson's disease, Huntington's disease, multiple sclerosis, amyotrophic lateral sclerosis (ALS), or a combination thereof.

The transgenic animal may have neuronal cell death, memory loss, impairment of memory capacity, decrease in brain size, decrease in survival rate, or a combination thereof. The memory loss may be a loss of existing memory. The impairment of the memory ability may be impairment of the memory ability.

The reactive astrocytes may be induced by monoamine oxidase B (MAO-B), inducible nitric oxide synthase (iNOS), p67 [hpox], or a combination thereof .

Another aspect is a transgenic animal that induces reactive astrocytic cells,

A transcription termination nucleotide sequence is located between two LoxP nucleotide sequences and a gene cassette containing a nucleotide sequence encoding DTR downstream of any one of the LoxP nucleotide sequences is transformed,

Wherein the GFAP promoter sequence and the nucleotide sequence encoding the Cre recombinase are operably linked to a locally infected virus,

The present invention provides a transgenic animal in which the expression of DTR is induced specifically in a reactive astrocytic cell.

Transformant, GFAP promoter sequence, Cre recombinase, and operably linked are as described above.

The virus may be locally infected with the hippocampus. The hippocampus may be a hippocampal CA1 region, a CA2 region, a CA3 region, a CA4 region, or a combination thereof. For example, the hippocampus is a hippocampal CA1 region.

The virus may be an adeno-associated virus (AAV).

Another aspect is a transgenic animal transfected with a first gene cassette in which a GFAP promoter sequence and a nucleotide sequence encoding a Cre-ERT2 polypeptide are operably linked,

A step of obtaining a progeny by transfecting a transgenic transgenic animal in which a transcription termination nucleotide sequence is located between two LoxP nucleotide sequences and a second gene cassette containing a nucleotide sequence encoding DTR is downstream of any one of the LoxP nucleotide sequences, ;

Administering an estrogen antagonist to said offspring to activate the CreERT2-LoxP system; And

And inducing reactive astrocytes by administering a diphtheria toxin to the progeny to which the estrogen antagonist has been administered, thereby inducing a reactive astrocytic cell.

Transformant, GFAP promoter sequence, Cre recombinase, and operably linked are as described above.

The term " offspring " includes products of crossing between the transgenic animals, including germ cells, embryos, and nat- ural organisms.

Another aspect is a method of treating a condition selected from the group consisting of: administering a candidate agent to a transgenic animal according to one aspect; And examining the brain tissue of the animal to which the candidate substance has been administered.

The administration is administered, for example, orally, transdermal, subcutaneous, rectal, intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, topical, intranasal, intratracheal, or intradermal. The administration may be intravenous administration.

According to one aspect of the present invention, there is provided a transgenic animal which induces reactive astrocytic cells, a method for producing the same, and a method for screening a substance for treating brain diseases using a transgenic animal, A model can be manufactured. In addition, it can be used to screen substances for treating various diseases in which reactive astrocytes such as brain inflammation, degenerative brain diseases, dementia, and stroke are involved.

FIG. 1A is a schematic diagram showing the production process of an astrocyte-specific toxin receptor animal model ("GiD" mouse), FIG. 1B is a schematic diagram showing an experimental procedure for selectively inducing reactive astrocytes in GiD or Gcon mice (GFAP, blue: DAPI). Fig. 1C shows immunostaining and nuclear staining of GFAP in GiD or Gcon mice.
FIG. 2A is a graph showing immunostaining and nuclear staining images of GFAP according to the induction of reactive astrocytes in severe and mild conditions and analysis thereof, and FIG. 2B is a graph showing the results of sholl analysis.
FIGS. 3A and 3B are graphs showing immunohistochemical images of neurons in a GiD severe model and results of analysis thereof. FIG.
4A and 4B are graphs showing a schematic diagram of the passive avoidance reaction test and a delay time of the mouse, respectively.
FIG. 5A is a graph showing the size of the brain contraction image and brain size in the GiD severe model, and FIG. 5B is a graph showing the survival rate (%) of the mouse.
6A is a graph showing immunohistochemistry of reactive astrocyte cells in a GiD mild model, and FIG. 6B is a graph showing the GFAP intensity (multiple of change) and the intensity (multiple of change) of GABA in GFAP + cells in the serigalline or AAD-2004 treated group .
FIG. 7A is an immunohistochemical image of a GiD severe model, and FIG. 7B is a graph showing GFAP intensity (change multiple), iNOS intensity, p67 [phox] intensity, and lba1 intensity in a GiD severe model.
FIG. 8A is a graph showing the immunohistochemical image of fGID, FIG. 8C is a graph showing the GFAP intensity (AU: arbitrary unit), DAPI staining Cell number (number of 50 탆 x 50 탆 region of interest (ROI)), and CA1 size.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these embodiments are intended to illustrate one or more embodiments, and the scope of the present invention is not limited to these embodiments.

Example  1. Production of experimental animal models capable of specifically inducing reactive astrocytes

1. Production of astrocytic-specific toxin receptor model

A nucleotide encoding a human glial fibrillary acidic protein (hGFAP) promoter sequence and a Cre recombinase-estrogen receptor T2 (Cre-ERT2) fusion protein that specifically function in astrocytes A GFAP-cre / ERT2 transgenic mouse (Jackson Laboratory, stock number 012849) with operably linked sequences was prepared.

An inducible Diphtheria Toxin Receptor (iDTR) transgenic mouse (ROSA26), in which the nucleotide sequence encoding the ROSA26 locus, the STOP cassette, and the Diphtheria Toxin Receptor (DTR) Jackson Laboratory, stock number 007900).

An astrocyte-specific toxin receptor animal model was prepared by crossing the prepared GFAP-cre / ERT2 transgenic mouse with an iDTR transgenic mouse and named this "GiD" mouse (FIG. As a negative control, GFAP-cre / ERT2 transgenic mice were used and named "Gcon" mice.

2. GiD  Selective induction of reactive astrocytes in mouse models

Tamoxifen (Sigma, T5648) was intraperitoneally injected into a GiD mouse or Gcon mouse prepared as described in 1. above at a dose of 100 mg / kg body weight per day for 5 days. After 10 days, diphtheria toxin (DT) was intraperitoneally injected at 50 ㎍ / kg body weight / day for 2 days. One week after DT injection, each mouse was perfused with cryosection (30 쨉 m thickness) (Fig. 1B).

The frozen sections were immunostained with anti-GAP antibody (Millipore ab5541) and stained with DAPI (4 ', 6-diamidino-2-phenylindole). Immunostaining and nuclear staining results are shown in Fig. 1C (green: GFAP, blue: DAPI).

As shown in FIG. 1C, GiD mice were found to be strongly stained for DT-induced GFAP compared to Gcon mice. Thus, it was confirmed that GiD mice can selectively induce reactive astrocytes by DTR specifically expressed in astrocytes.

3. GiD  Establishment of induction conditions of mild and severe reactive astrocytic cells in mouse model

GiD mice or Gcon mice were intraperitoneally injected with tamoxifen at a dose of 100 mg / kg body weight per day for 5 days. After 10 days, diphtheria toxin (DT) was administered for 16 days (severe: s) at a dose of 50 / / kg body weight / day for 2 days (mild: m) or 12 / / Conditions). One week or one month after injection of DT, each mouse was perfused with heart and frozen (30 mu m thick). GiDm condition for mild one week and GiDs for severe one month condition were named.

For frozen sectioned tissue, immunostained with anti-GAPAP antibody as described in 2. < RTI ID = 0.0 &gt; 2, &lt; / RTI &gt; and stained with DAPI. (Green: GFAP, blue: DAPI, AU: arbitrary unit, ***: p <0.001) are plotted in the staining image and the GFAP fluorescence intensity in each group.

In addition, a sholl analysis was performed on the immuno staining result, and the results are shown in Fig. 2B.

As shown in FIGS. 2A and 2B, in GiD mice treated with DT under mild conditions, about one week later, the enlarged GFAP signal returned to its original state after about one month. In GiD mice treated with DT under severe conditions, the reactivity of astrocytes gradually increased to 1 month and the number of glial cells increased. Therefore, we established the conditions that can regulate the reactivity of astrocytes according to the concentration and frequency of DT injection in GiD mice.

4. GiD  Induction of neuronal cell death in a severe model

Gcon mice and GiD severe models were prepared as described in 3.

To the GiD severe model (GiDs), AAD-2004 (2-hydroxy-5- [2- (4-trifluoromethylphenyl) -ethylaminobenzoic acid) (GNTPharma) which is a hydrogen peroxide (H ₂ O ₂ ) From the starting point, the mice were intraperitoneally administered at a dose of 3.3 mg / kg body weight per day for 16 days. Brain tissues of mice were prepared as described in 2. Then immunohistochemistry was performed using anti-NeuN antibody (Millipore mab377) and anti-MAP2 antibody (Abcam, ab5392) to detect NeuN and MAP2, which are biomarkers of neurons. Immunohistochemical images were analyzed to calculate NeuN and MAP2 intensities, and immunohistochemical images and NeuN intensities and MAP2 intensities were shown in Figures 3a and 3b (****, ++++, p < 0.0001, N = number of ROI).

As shown in FIGS. 3A and 3B, the intensity of NeuN and MAP2 in GiDs mice was significantly decreased. In addition, when AAD-2004 was treated, the decrease in the intensity of NeuN and MAP2 was inhibited. Therefore, in the GiDs model, H ₂ O ₂ Dependent neuronal death by severely reactive astrocytic cells.

5. GiD  Loss of existing memory and permanent memory impairment in severe models

Passive avoidance tests were performed on Gcon and GiD mice.

Using the characteristics of a mouse that preferred a darker room than a bright room, a Gcon or GiDs mouse in a bright room entered the dark room and gave electrical shock to the floor, creating a horror memory of the dark room Acquisition). From the next day, the time to enter the dark room (delay time) when placed in a bright room was measured and the memory of the formed fear memory was measured (Retrieval). After the first memory test, DT was injected under severe conditions. For AAD-2004 treated group, AAD-2004 was intraperitoneally administered at a dose of 3.3 mg / kg body weight per day for 16 days from the beginning of DT treatment. On the 30th day, a second memory test was performed. At day 31, mice were given electrical shocks to reinforce fear memory. FIG. 4A shows a schematic diagram of the passive avoidance response test, and FIG. 4B shows the delay time of the mouse (Acq .: memory formation, ret. 1: measurement 1, ret. 2: Ret. 4: Measurement 4).

As shown in FIG. 4B, it was confirmed that the fear memory, which was formed by the decrease in the delay time in GiDs, disappeared. No new memory was formed even after the remodeling of fear memory. In the AAD-2004 group, memory impairment was significantly suppressed and fear memory was well formed after remodeling of fear memory. Thus, when the severely reactive astrocytes are H ₂ O ₂ Dependent memory impairment.

6. GiD  In severe models, brain size shrinkage and Survival rate  decrease

GiDs mice were intraperitoneally injected with AAD-2004 at a dose of 3.3 mg / kg body weight per day for 16 days from the start of DT treatment, and then the brains of the mice were extracted. The results of measuring the size of the brain are shown in FIG. 5A. In addition, the survival rate of the mice was calculated and shown in FIG. 5B (**, ++, p <0.01, *, p <0.05). As shown in FIGS. 5A and 5B, brain size and survival rate of GiDs mice were significantly decreased. On the other hand, when AAD-2004, an H ₂ O ₂ scavenger, was administered, it was confirmed that the brain contraction was suppressed and the survival rate was also restored.

7. Mechanism for induction of reactive astrocytes

To determine whether reactive astrocytes were induced by MAO-B-induced reactive oxygen species, serogyline, a selective MAO-B inhibitor, and AAD-2004, a hydrogen peroxide scavenger, were used.

GiDm mice were intraperitoneally injected with selegiline (Sigma) at a dose of 10 mg / kg body weight / day for 7 days and AAD-2004 at a dose of 3.3 mg / kg body weight per day from the DT treatment starting point. Thereafter, an anti-GAPAP antibody (Millipore ab5541) and anti-GABA antibody (Millipore

abl75) was used to perform immunohistochemistry.

An image of immunohistochemistry is shown in Fig. 6A, and a graph showing the GFAP intensity (multiple of change) and the intensity (multiple of change) of GABA in GFAP + cells is shown in Fig. 6b.

As shown in Figs. 6A and 6B, GFAP and GABA were significantly increased in GiDm mice. Treatment with serigallin inhibited the increase of GFAP and GABA. In the case of AAD-2004 treatment, the increase of GFAP was suppressed while the increase of GABA was maintained. Thus, it was confirmed that structural changes of reactive astrocytes were mediated by MAO-B-induced reactive oxygen species.

8. Severe reactive astrocytic cells Nitrifying  stress

To determine whether nitrosative stress was induced in severely reactive astrocytes, inducible NO synthase (iNOS) and p67 [phox], enzymes that produce reactive oxygen species, were detected.

GiDs mice were intraperitoneally injected with AAD-2004 at a dose of 3.3 mg / kg body weight per day for 16 days starting from the DT treatment start point. Mouse brain tissue was obtained and immunoprecipitated using anti-iNOS antibody (BD, 610332), anti-p67 [phox] antibody (BD, 610913), anti-lbal antibody (Wako 019-19741) Immunohistochemistry was performed and the nuclei were stained with DAPI. The staining image is shown in Fig. 7A and the GFAP intensity (multiple of change), iNOS intensity, p67 [phox] intensity, and lba1 intensity are shown in Figure 7b (AU: arbitrary unit, ****, p <0.0001; ** *, +++, p < 0.001)

As shown in FIGS. 7A and 7B, the intensity of iNOS and p67 [phox] in GiDs mice was significantly increased. Expression of iNOS in reactive astrocytes and p67 [phox] was specifically increased in activated microglia. Furthermore, it was confirmed that when AAD-2004 was treated, the increase in expression was suppressed. Therefore, it was confirmed that expression of iNOS and p67 [phox] was induced in the GiDs model in an H ₂ O ₂ -dependent manner.

9. Establishment of Local Reactive Astrocyte Induction Model

AAV-GFAP-Cre virus (KIST virus facility) was injected into the hippocampal CA1 region of an inducible diphtheria toxin receptor (iDTR) mouse (Jackson Laboratory, stock number 007900) (50 ㎍ / kg body weight / day or 12 ㎍ / body weight kg / day, respectively) for 2 days or 16 days, respectively. ) GiD) and fGiDs (severe localized GiD) for 16 days with a dose of 2 ㎍ / kg body weight / day.

One week or one month after DT injection, each mouse was sacrificed as described in 2. and immunohistochemistry was performed.

(Green: GFAP, blue: DAPI), the result of staining is shown in Fig. 8B, and the GFAP intensity (AU: arbitrary unit), DAPI (**, p <0.01; ***, p < 0.001, ***) in the number of stained cells (number of 50 탆 x 50 탆 region of interest *, p < 0.0001)

The intensity of GFAP gradually increased in fGiDm and fGiDs, and the number of glial cells in fGiDs was also increased. Atrophy of the CA1 region was also confirmed in fGiDs. Therefore, by applying the GiD system locally, we have established conditions that can regulate the responsiveness of astrocytes to each brain location.

Claims (18)

As a transgenic animal that induces reactive astrocytic cells,
A first gene cassette operatively linked to a nucleotide sequence encoding a glial fibrillary acidic protein (GFAP) promoter sequence and a Cre recombinase-estrogen receptor T2 (Cre-ERT2) polypeptide; And
A transcription termination nucleotide sequence is located between two LoxP nucleotide sequences and a second gene cassette comprising a nucleotide sequence encoding a Diphtheria Toxin Receptor (DTR) downstream of any one of the LoxP nucleotide sequences is transformed,
Wherein the CreERT2-LoxP system is activated by administration of an estrogen antagonist to induce expression of DTR in a reactive astrocytic cell. 3. The transgenic animal according to claim 1, wherein the GFAP promoter sequence is a human-derived sequence. The transgenic animal of claim 1, wherein the second gene cassette further comprises a ROSA26 polynucleotide. The transgenic animal according to claim 1, wherein the estrogen antagonist is tamoxifen, 4-hydroxy tamoxifen, clomifene, raloxifene, or a combination thereof. The transgenic animal according to claim 1, wherein the transgenic animal is caused by brain disease by administration of a diphtheria toxin. The transgenic animal according to claim 5, wherein the diphtheria toxin is administered at a dose of 0.1 μg / kg body weight / day to 1 mg / kg body weight / day. The transgenic animal according to claim 5, wherein the diphtheria toxin is administered for 1 day to 1 month. The transgenic animal according to claim 5, wherein the brain disease is brain inflammation, degenerative brain disease, dementia, stroke or a combination thereof. 9. The transgenic animal of claim 8, wherein said degenerative brain disease is Alzheimer's disease, Parkinson's disease, Huntington's disease, multiple sclerosis, amyotrophic lateral sclerosis (ALS), or a combination thereof. 6. The transgenic animal of claim 5, wherein the transgenic animal has neuronal death, memory loss, impairment of memory capacity, decrease in brain size, decrease in survival rate, or a combination thereof. The method of claim 1, wherein the responsive astrocytic cells are selected from the group consisting of monoamine oxidase B (MAO-B), inducible nitric oxide synthase (iNOS), p67 [hpox] Gt; transgenic animal. As a transgenic animal that induces reactive astrocytic cells,
A transcription termination nucleotide sequence is located between two LoxP nucleotide sequences and a gene cassette containing a nucleotide sequence encoding DTR downstream of any one of the LoxP nucleotide sequences is transformed,
Wherein the GFAP promoter sequence and the nucleotide sequence encoding the Cre recombinase are operably linked to a locally infected virus,
A transgenic animal in which the expression of DTR is induced specifically in a responsive astrocytic cell. 13. The transgenic animal of claim 12, wherein the virus is locally infected with a hippocampus. 13. The transgenic animal according to claim 12, wherein the hippocampus is a hippocampal CA1 region. 13. The transgenic animal of claim 12, wherein the virus is an adeno-associated virus (AAV). A transgenic animal transformed with a first gene cassette in which a GFAP promoter sequence and a nucleotide sequence encoding Cre-ERT2 polypeptide are operatively linked,
A step of obtaining a progeny by transfecting a transgenic transgenic animal in which a transcription termination nucleotide sequence is located between two LoxP nucleotide sequences and a second gene cassette containing a nucleotide sequence encoding DTR is downstream of any one of the LoxP nucleotide sequences, ;
Administering an estrogen antagonist to said offspring to activate the CreERT2-LoxP system; And
And inducing reactive astrocytes by administering a diphtheria toxin to a progeny to which said estrogen antagonist has been administered to induce reactive astrocytic cells. 17. The method of claim 16, wherein the transgenic animal is a brain disease. Administering a candidate agent to the transgenic animal of claim 1 or 12; And
And examining the brain tissue of the animal to which the candidate substance is administered.
A method for screening a substance for treating brain diseases.

Images