CN110754419B - Construction method of mammary external paget disease mouse model - Google Patents

Construction method of mammary external paget disease mouse model Download PDF

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CN110754419B
CN110754419B CN201810836812.8A CN201810836812A CN110754419B CN 110754419 B CN110754419 B CN 110754419B CN 201810836812 A CN201810836812 A CN 201810836812A CN 110754419 B CN110754419 B CN 110754419B
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于政权
宋永利
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Abstract

The invention provides a construction method of a mouse model with an external mammary gland paget disease, which comprises the steps of mating a K14rtta transgenic mouse with a TRE-Msi1 transgenic mouse, feeding tetracycline to a generated progeny mouse, and detecting a mouse with a Musashi1 overexpression gene, namely the mouse with the external mammary gland paget disease. The mouse model for the external mammary gland paget disease established by the invention lays a theoretical foundation for systematic research of the disease from the molecular mechanism in future.

Description

Construction method of mammary external paget disease mouse model
Technical Field
The invention relates to a construction method of a disease animal model, in particular to a construction method of a mouse model of an extramammary paget disease.
Background
Mammary Paget disease was discovered by James Paget in 1874 (Paget J,1874, St Barth Hosp Rep 1874; 10: 87-9). The etiology is unclear, and the diseases are divided into mammary gland Paget disease and mammary gland external Paget disease, and the clinical characteristics of the mammary gland Paget disease are as follows: eczema-like changes appear in the areola, the unilateral attack is more, the symptoms are that the nipple is extremely itchy, the skin at the nipple is red, and the external paget disease of the breast is more frequently encountered in the armpit, the scrotum, the periphery of the anus and other parts. Mammary and extramammary paget diseases share similar clinical features, with large numbers of paget-like cells in the epidermis (GUOHAI SHUI,2010, APMIS118: 777-781). Some common markers are expressed. Such as breast paget, AR (88%), ER (10%), PR (0%). Extra-mammary paget: AR (78%), ER (4%), PR (0%) (Bernadite Liegl,2005, Morden Pathology,18, 1283-. The specific pathogenesis and dissimilarity need further research, the paget-like cells have multinuclei, a large amount of mucus exists in cytoplasm, the nucleus is pushed to the side, the nucleus is not in the center of the cell, the cytoplasm is large and lightly stained, amylase and mucosialoprotein are contained, and hyaluronidase is contained, so that the staining is positive when PAS staining and anaxin staining are carried out. The source of paget cells for breast paget disease and extramammary paget disease remains a matter of controversy.
As for the paget-like disease, no good treatment method exists at present, the surgical operation is the first choice scheme for treating the mammary-like paget disease at present, the pathogenesis of the paget-like disease is still not clear, and the development of a drug target is influenced, so that a mammary-like paget disease mouse model can be established, the method has a very important effect on the research of the pathogenesis of the mammary-like paget disease, and has milestone significance for further researching the drug treatment of the mammary-like paget disease.
Musashi1 belongs to a member of the RNA-binding protein family, is highly conserved evolutionarily (Gunter, K.M,2011, IUBMB LIFE63,678-685), is first discovered in experiments by a neurobiol famous in the 90 th century, Montell, and when studying the developmental process of Drosophila sensory organs, significant results were found, wherein wild-type sensory organ precursor cells (SOP) form a non-neural progenitor IIa cell and a non-neural progenitor IIb cell when asymmetrically dividing, and when Musashi1 is mutated, the dividing mode of SOP cells is greatly changed, and two identical IIa cells are formed after dividing, with the result that the phenotype of Drosophila shows double seta. Due to its phenotypic characteristics, it is thought that Miyamato Musashi, the Japanese famous warrior, was held by the same warrior as a twin sword, and in order to commemorate the warrior, this gene was named Musashi (Fox, RG,2015, Annu Rev Cell Dev Biol 31, 249-267).
Musashi1 is mainly expressed in stem cells and progenitor cells, while expression in differentiated cells is relatively reduced (Sakakibara, S,2001, J.Neurosci.21,8091-8107), which when Musashi1 is knocked out in a nematode, causes a disturbance in male mating behavior, suggesting that Msi1 may regulate the activity of differentiated neural cells (Yoda, A.2000, Genecls 5, 885-895). Musashi1 can bind to (G)/A)UnThe AGU (n-1-3) motif, which maps to the 3' UTR region of the mRNA of the target gene (Ohyama, T,2012, Nucleic Acids res.40, 3218-3231). In vivo, few Msi1 target genes were identified, such as m-numb (Imai, T,2001, Mol, cell. biol), CDKN1A (Battelli, S,2006, Nature395, 124-125). In addition, 64 Msi1 target genes were identified in 293T cells by RIP-ChIP method (de Sousa Abreuet al, 2009) compared to the control group, the mRNA of these 64 target genes was enriched to Msi 1-related subpopulations, which mainly include two classes, one, cell cycle, cell proliferation, cell differentiation and apoptosis, depending on their function for tumor formation; second, protein modification, including ubiquitination, ubiquitin cycle (Raquel de Sousa Abreu,2009, The joural of biological chemistry,284,18, 12125-.
Disclosure of Invention
The invention aims to provide a method for constructing a mouse model of the mammary external paget disease.
In order to realize the purpose of the invention, a skin-specific over-expression Musashi1 mouse is constructed, after Musashi1 is shown in a table, human-like characteristics of the external breast page disease appear, and the phenotype appearing in the mouse is proved to be the external breast page disease-like symptom by combining methods of histology, transcriptomics and immunohistochemical analysis.
The method for constructing the mouse model with the external paget disease of the mammary gland provided by the invention comprises the steps of mating a K14rtta transgenic mouse with a TRE-Msi1 transgenic mouse, feeding tetracycline to a generated progeny mouse, and detecting a mouse with a Musashi1 overexpression gene, namely the mouse with the external paget disease of the mammary gland.
Among these, K14rtta transgenic mice were purchased from Jackson laboratory, cat No. 007678. TRE-Msi1 transgenic mice were donated by professor Chiristopher J Lengners, university of Pennsylvania. Two transgenic mice were constructed as described in Wang S, Li N, Youefi M, Nakauka-Ddamba A, Li F, Parada K, et al. (2015) Transformation of the endogenous epitope by the MSI2RNA-binding protein. nat Commun 6: 6517.
In the method, when the offspring mouse grows to 6 weeks, tetracycline (the final concentration is 0.2g/L) is added into drinking water of the mouse, so that the mouse can drink water freely, sufficient drinking water is ensured, and the gene Musashi1 is induced to be over-expressed. Mice with symptoms like extramammary paget disease, namely extramammary paget disease mice (DTG mice), can appear after being fed with tetracycline doxycyline (Dox) for 48 hours.
In the invention, the specific method for identifying the mice with the external mammary gland paget disease is as follows:
(1) extracting mouse genome DNA as a template, performing PCR amplification (the PCR target is K14 rta gene) by taking F1 and R1 as primers, and detecting an amplification product to generate a 500bp characteristic band;
(2) extracting mouse genome DNA as a template, performing PCR amplification (the PCR target is a TRE-Msi1 gene) by taking F2, R2 and G as primers, detecting an amplification product, and generating two characteristic bands of 300bp and 500 bp;
a mouse identified as an extra-mammary paget disease that satisfies both (1) and (2) above;
the primer sequences used were as follows:
F1:5′-CACGATACACCTGACTAGCTGGGTG-3′
R1:5′-CACGATACACCTGACTAGCTGGGTG-3′
F2:5′-CCCTCCATGTGTGACCAAGG-3′
R2:5′-GCACAGCATTGCGGACATGC-3′
G:5′-GCAGAAGCGCGGCCGTCTGG-3′
in the present invention, the method for diagnosing the external mammary paget disease-like symptom may be selected from any one of the following (1) to (5):
(1) and (5) HE staining. Under microscope observation, the epidermis of the DTG mouse shows large cells which are called paget-like cells, the cytoplasm of the cells is large and slightly stained, and binuclear or multinuclear cells sometimes appear in the cells.
(2) And PAS dyeing. PAS staining is the gold standard for identifying paget-like diseases. Since the paget-like cells contain amylase, mucosialidase and hyaluronidase resistance, PAS staining is positive when the paget-like cells are present.
(3) Immunohistochemical staining, markers include, but are not limited to, Krt7, Her2, Vimentin, Gata3, Podoplanin.
(4) And (3) detecting by Western blot, wherein the markers comprise Krt7, Vimentin, Gata3 and CAM 5.2.
(5) PCR detection, markers including but not limited to Krt7, Vimentin, Gata3, CAM 5.2.
The invention also provides application of the mouse model for the external mammary gland paget disease in research on pathogenic mechanisms of the external mammary gland paget disease.
The invention also provides application of the mouse model for the external mammary gland paget disease in screening of medicaments for treating the external mammary gland paget disease.
By the technical scheme, the invention at least has the following advantages and beneficial effects:
the mouse model for the external mammary gland paget disease constructed by the invention plays an important role in the aspects of disease occurrence research, drug screening and drug action mechanism, and lays a theoretical foundation for systematic research of the disease from the molecular mechanism in the future.
Drawings
FIG. 1 is a schematic diagram of the construction process of the mouse model of the extramammary paget disease in the preferred embodiment of the present invention. A mice (K14rtta transgenic mice) were crossed with B mice (TRE-Msi1 transgenic mice).
FIG. 2 is the result of mouse model identification of the extramammary paget disease in the preferred embodiment of the present invention. K14rttA, 500 bp; b, Tre-msi1, 300bp and 500 bp.
FIG. 3 shows the result of genotyping a mouse overexpressing the gene Musashi1 in a preferred embodiment of the present invention. A: control, B: msi1 overexpressing mice (DTG mice).
FIG. 4 shows PAS staining results of DTG mice in a preferred embodiment of the present invention. The arrow indicates positive cells.
FIG. 5 is a graph showing the results of immunohistochemical staining of the paget-like disease markers in the preferred embodiment of the present invention.
FIG. 6 shows the result of Western blot detection of DTG mice in the preferred embodiment of the present invention.
FIG. 7 shows the result of the quantitative PCR assay of DTG mouse in the preferred embodiment of the present invention.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise indicated, the examples follow conventional experimental conditions, such as the Molecular Cloning handbook, Sambrook et al (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual,2001), or the conditions as recommended by the manufacturer's instructions.
Example 1 construction method of mouse model for mammary extramammary paget disease (DTG mouse)
Preparation of mouse model of extramammary paget disease (inducible mouse model)
Mating the K14rtta transgenic mouse with a TRE-Msi1 transgenic mouse to generate a progeny mouse, and adding tetracycline with the final concentration of 0.2g/L into drinking water of the mouse when the progeny mouse grows to 6 weeks, so that the mouse can drink water freely, sufficient drinking water is ensured, and the Musashi1 gene is induced to be overexpressed. After being fed with tetracycline for 48 hours, the mice with the symptoms like the extramammary paget disease can appear, namely extramammary paget disease mice (DTG mice).
(II) identification of DTG mouse genotype
1. Extracting mouse genome DNA as a template, performing PCR amplification by taking F1 and R1 as primers, detecting an amplification product, and generating a 500bp characteristic band. And (3) PCR reaction system: mix 6. mu.L (Kangwei century, cat # 01037/30252), F10.6. mu.L, R10.6. mu.L, genomic DNA template 1. mu.L, ddH2O3.8. mu.L. Reaction procedure: 5min at 95 ℃; 30s at 95 ℃,30 s at 61 ℃,30 s at 72 ℃ and 35 cycles; 72 ℃ for 2 min.
2. Extracting mouse genome DNA as a template, performing PCR amplification by taking F2 and R2 as primers, detecting an amplification product, and generating two characteristic bands of 300bp and 500 bp. And (3) PCR reaction system: mix 6. mu.L (Kangwei century, cat # 01037/30252), F20.6. mu.L, R20.6. mu.L, G0.6. mu.L, genomic DNA template 1. mu.L, ddH2O3.2. mu.L. Reaction procedure: 5min at 95 ℃; 30s at 95 ℃,30 s at 56 ℃,30 s at 72 ℃ and 35 cycles; 72 ℃ for 2 min.
Mice identified as extramammary paget disease mice that meet both 1 and 2 above. If other genotypes are present, they are littermate control mice.
The primer sequences used were as follows (SEQ ID NOS: 1-5):
F1:5′-CACGATACACCTGACTAGCTGGGTG-3′
R1:5′-CACGATACACCTGACTAGCTGGGTG-3′
F2:5′-CCCTCCATGTGTGACCAAGG-3′
R2:5′-GCACAGCATTGCGGACATGC-3′
G:5′-GCAGAAGCGCGGCCGTCTGG-3′
(III) sampling
The same sex of the mouse is born in the same litter, 48 hours after Dox administration, the mouse is killed by dislocation of the neck, the hair on the back is removed by an electric hair cutter, a small rectangular back skin is taken by a scissors, the skin is fixed by 4% paraformaldehyde for 24 hours, PBS is washed for 3 times, 20 minutes each time, and the skin is cut into tissue blocks with the size of about 1cm multiplied by 0.75cm by a blade. The hair is cut at the angle (in the direction of the hair) and a small piece is cut off in preparation for dehydration.
(IV) dehydration
The dehydration procedure and procedure are as follows
Figure BDA0001744731640000051
The temperature of the last 3 steps is set to 60 ℃ to dissolve the wax, and the rest is normal temperature.
(V) embedding
Embedding in paraffin, vertically embedding, cutting into 5 μm size, spreading in 39 deg.C water bath, observing under solid microscope whether the film is completely spread, taking out the spread film with adhesive glass slide, and air drying.
(VI) mouse phenotype analysis (HE staining)
Hematoxylin-eosin staining (HE staining) is the most common method used in cytology, histology, embryology and pathology, and can visually observe abnormal changes of tissue cells.
1. Baking slices: 63 ℃ and 1 h.
2. Dewaxing and hydrating.
3. Xylene I:15 min.
4. Xylene II 15min
5. Gradient ethanol treatment:
100% ethanol I, II: each for 5min
95% ethanol I, II: each for 5min
80% ethanol for 5min
70% ethanol for 5min
Distilled water: 5min
And (3) hematoxylin: for 10min
Tap water: 5min
95% ethanol I, II: each for 3min
Eosin: 10s
95% ethanol I, II: each for 3min
100% ethanol I, II: each for 3min
Xylene I:10min
Xylene II:10min
And sealing and taking a picture.
(VII) immunohistochemistry
1. Baking slices: 63 ℃ and 1 h.
2. Dewaxing and hydrating.
3. Xylene I:15 min.
4. Xylene II:15 min.
5. Gradient ethanol treatment:
100% ethanol I, II: each for 5min
95% ethanol I, II: each for 5min
80% ethanol for 5min
70% ethanol for 5min
Distilled water: and 5 min.
6. Antigen retrieval: boiling sodium citrate with pH of 6.0 for 20 min.
7. And (3) natural cooling: about 1 h.
PBS treatment 3 times, 5 min/time.
9.H2O2Light shielding for 20 min.
PBS treatment 3 times, 5 min/time.
11. And (3) sealing: the confining liquid acts for at least 1 h.
12. A first antibody: the prepared primary antibody is added according to the proper proportion and amount and stays overnight at 4 ℃. 13. Rewarming: standing at room temperature for half an hour.
PBS treatment 3 times, 5 min/time.
Solution B: room temperature for 30 min.
PBS treatment 3 times, 5 min/time.
Solution C: room temperature for 30 min.
PBS treatment 3 times, 5 min/time.
19. Distilled water: the treatment is carried out for 5 min.
DAB, stopping color development under the condition of proper color development time according to the characteristics of the antibody, and putting the antibody into distilled water.
21. And (3) hematoxylin: counterstaining for 5 min.
22. Tap water: the treatment is carried out for 5 min.
23. Gradient ethanol treatment:
70% ethanol for 3min
80% ethanol for 3min
90% ethanol for 3min
3min with 100% ethanol
Xylene I:5min
Xylene II:5min
And sealing and taking a picture.
Wherein, the solution B and the solution C are from a China fir Jinqiao kit, and the catalog number is as follows: sp9001, sp 9002. A first antibody: primary resistance to krt7(abcam,1:200), Gata3(abcam,1:200), ERBB2(Her 2): 1:400, available from cell signaling technology. Vimentin: 1:400, available from cell signaling technology. Podoplanin (santa,1: 100).
(eighth) Western blot
1. Extraction of proteins
(1) Cut the skin with proper size and cut into pieces with scissors.
(2) To the above sample was added 400. mu.L of protein lysate (PMSF: IP volume ratio 1:100), and the homogenized tissue was dispersed by a homogenizer and lysed on ice for 30 min.
(3) Centrifuge at 12000rpm for 10min at 4 ℃.
(4) And (3) sucking the supernatant, taking a part of samples to detect the concentration, using the samples in a Western blot experiment, and storing the rest samples at-80 ℃.
2. Detection of protein concentration
Protein concentration was measured using Bycyanus kit BCA, 50. mu.g of each lane.
3. Electrophoresis
Concentrating the glue: 60V, separation gel: 80V.
4. Rotary film
And (5) wet-rotating, and rotating the film for 1h at 330 mA.
5. Sealing of
Blocking with 5% skimmed milk powder for 1 h.
6. A primary antibody
According to the molecular weight of protein, the membrane with proper size is cut, sealed and added with proper amount of primary antibody. 4 ℃ overnight.
7. Rewarming
Shake on a shaker for 30 min. Recovering the primary antibody.
8. Washing membrane
TBST 3X10 min/time
9. Second antibody
Secondary antibodies from pelagic days were incubated for 1h at room temperature.
10. Washing membrane
TBST treatment was carried out 3 times at a rate of 10 min.
11. Color development
Primary antibodies for immunohistochemistry: pS6(cell signaling technology, 1; 400, # 4858). CAM5.2(1: 50; ZSGB-BIO, ZM-0316 histochemical secondary antibody and DAB color development kit (China fir gold bridge kit).
Primary antibody for Western blot: pS6(cell signaling technology, dilution ratio 1; 1000). Beta-tubulin (Shanghai assist saint Biotech Co., Ltd., dilution ratio 1; 4000,30101ES50), secondary antibody for Western blot (Biyun, dilution ratio 1: 10000, cat # A0208 (rabbit source), A0216 (mouse source).
PAS staining kit: purchased from Regen Biotechnology Ltd, Beijing (cat # DG 0005).
(nine) quantitative PCR detection (the detection target gene is marker highly expressed in paget disease)
1. Extraction of RNA
(1) A sample of mouse skin was shaved, and cut into skin pieces of 1.5cm long by 1cm wide, and 1mL of TRIzol reagent (sample volume not more than 10% of TRIzol volume) was added to the skin pieces, and the tissue was disrupted with a tissue homogenizer.
(2) 0.2mL of chloroform was added to 1mL of TRIzol, and the mixture was vigorously shaken and mixed for 15 seconds, and then allowed to stand at room temperature for 2 min. Then 12000g, 4 degrees C centrifugal 15min, carefully absorb the supernatant 400 u L RNase-free centrifuge tube.
(3) 400. mu.L of isopropanol was added to the supernatant, the mixture was turned upside down and mixed, and the mixture was left at room temperature for 10min, then 12000g was centrifuged at 4 ℃ for 10 min.
(4) Carefully discard the supernatant, add 1mL 75% ethanol (in DEPC water), gently bounce the pellet up and down, centrifuge at 12000g for 3min at 4 ℃, discard the supernatant, and empty for 2 min. Excess ethanol was aspirated.
(5) Adding 30 μ L DEPC water to resuspend the precipitated RNA, standing on ice for 30min, allowing a portion of the sample to undergo reverse transcription for quantitative PCR, and storing the remaining samples at-70 deg.C.
2. Reverse transcription to synthesize cDNA
Reverse transcription system:
Figure BDA0001744731640000081
70 ℃ for 5 min. Cooling on ice for 3 min.
The cDNA was synthesized as follows:
Figure BDA0001744731640000091
42 ℃ for 60 min. Then, cDNA was obtained at 70 ℃ for 15 min.
3. Quantitative PCR reaction
And (3) PCR reaction system:
Figure BDA0001744731640000092
the quantitative PCR primers were as follows:
MMP9 F:GCAGAGGCATACTTGTACCG
R:TGATGTTATGATGGTCCCACTTG
PODOPLANIN F:ACCGTGCCAGTGTTGTTCTG
R:ACCATGCCGTCTCCTGTACC
KRT7 F:AGGAGATCAACCGACGCAC
R:GTCTCGTGAAGGGTCTTGAGG
GATA3 F:AAGCTCAGTATCCGCTGACG
R:GTTTCCGTAGTAGGACGGGAC
myD88 F:TGCCAGCGAGCTAATTGAGA
R:GACACCTGGAGACAGGCTGA
N-cadherin F:ATCATTCGCCAAGAGGAAGG
R:CCAGTTGGCAGGATCAGACA
V-EGFA F:TGTACCTCCACCATGCCAAG
R:TCTCAATCGGACGGCAGTAG
HIF1A F:TCGACACAGCCTCGATATGAA
R:TTCCGGCTCATAACCCATCA
and (3) PCR reaction conditions: 10min at 95 ℃; 95 ℃ for 10s, 60 ℃ for 10s, 72 ℃ for 10s, 45 cycles.
FIG. 1 is a schematic diagram of the process of constructing a mouse model of the extramammary paget disease. A mice (K14rtta transgenic mice) were crossed with B mice (TRE-Msi1 transgenic mice).
FIG. 2 shows the results of the mouse model for the extramammary paget disease. K14rttA, 500 bp; b, Tre-msi1, 300bp and 500 bp.
FIG. 3 shows the results of genotyping the mice overexpressing the gene Musashi 1. A: control, B: msi1 overexpressing mice (DTG mice).
FIG. 4 shows PAS staining results of DTG mice. The arrow indicates positive cells.
FIG. 5 is the results of immunohistochemical staining of paget-like disease markers.
FIG. 6 shows the result of Western blot detection on DTG mice.
FIG. 7 shows the result of quantitative PCR assay for DTG mice.
The results show that the invention successfully constructs the mice with the external mammary gland paget disease and provides a good experimental animal model for further researching the pathogenesis of the external mammary gland paget disease.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Sequence listing
<110> university of agriculture in China
Construction method of <120> mammary external paget disease mouse model
<130> KHP181114375.1
<160> 5
<170> SIPOSequenceListing 1.0
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
cacgatacac ctgactagct gggtg 25
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<213> Artificial Sequence (Artificial Sequence)
<400> 2
cacgatacac ctgactagct gggtg 25
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<213> Artificial Sequence (Artificial Sequence)
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ccctccatgt gtgaccaagg 20
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<213> Artificial Sequence (Artificial Sequence)
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gcacagcatt gcggacatgc 20
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<213> Artificial Sequence (Artificial Sequence)
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gcagaagcgc ggccgtctgg 20

Claims (5)

1. The method for constructing the mouse model of the external mammary gland paget disease is characterized in that a K14rtta transgenic mouse is mated with a TRE-Msi1 transgenic mouse, and a generated progeny mouse is fed with tetracycline to detect a mouse with a Musashi1 overexpression gene, namely the mouse with the external mammary gland paget disease;
the specific method for identifying mice with external mammary gland paget disease is as follows:
(1) extracting mouse genome DNA as a template, performing PCR amplification by taking F1 and R1 as primers, and detecting an amplification product to generate a 500bp characteristic band;
(2) extracting mouse genome DNA as a template, performing PCR amplification by taking F2, R2 and G as primers, detecting an amplification product, and generating two characteristic bands of 300bp and 500 bp;
mice with external mammary gland paget disease which simultaneously meet the requirements of the (1) and (2);
the primer sequences used were as follows:
F1:5′-CACGATACACCTGACTAGCTGGGTG-3′
R1:5′-CACGATACACCTGACTAGCTGGGTG-3′
F2:5′-CCCTCCATGTGTGACCAAGG-3′
R2:5′-GCACAGCATTGCGGACATGC-3′
G:5′-GCAGAAGCGCGGCCGTCTGG-3′。
2. the method of claim 1, wherein tetracycline is added to the drinking water of the mouse when the offspring mouse grows for 6 weeks, the mouse is allowed to drink water freely, sufficient drinking water is ensured, the overexpression of the gene Musashi1 is induced, and the final concentration of tetracycline added to the water is 0.2 g/L.
3. The method of claim 2, wherein the mice that develop the symptoms of extramammary paget disease after 48 hours of tetracycline administration are extramammary paget disease mice.
4. Use of a mouse model of an extra-mammary paget disease constructed by a method according to any one of claims 1 to 3 in the study of the pathogenesis of the extra-mammary paget disease.
5. Use of the mouse model of external paget disease of breast constructed by the method of any one of claims 1 to 3 in screening a medicament for the treatment of paget disease of breast.
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