Wfdc2 molecule-deficient non-human animals exhibiting chronic obstructive pulmonary disease-like pathology in adults

Abstract

To provide an animal to be a model of human chronic obstructive pulmonary disease.SOLUTION: A method for producing a non-human animal model of chronic obstructive pulmonary diseases, comprising inducing deletion of Wfdc2 genes by administering an antiestrogen to adult non-human animals which have genes in which at least one exon of CreERT2 is flanked by two loxP sequences in both alleles, which systemically express antiestrogen-dependent recombinant Wfdc2, and which can delete Wfdc2 genes in an antiestrogen-inducible manner. The chronic obstructive pulmonary disease model non-human animal is produced by the method.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to non-human animals lacking the Wfdc2 molecule that exhibit chronic obstructive pulmonary disease-like pathology in adults. [Background technology]

[0002] Based on data from limited countries such as the United States, Europe, and Japan, estimates of the global prevalence of chronic obstructive pulmonary disease (COPD) in humans suggest that there are at least 200 million patients worldwide, with an estimated 6 million in Japan (Non-Patent Document 1). It is likely that even more patients exist in developing countries if accurate data becomes available. According to a WHO report, COPD is the third leading cause of death worldwide, claiming more than 3 million lives per year as it progresses with age (Non-Patent Document 2). COPD is characterized by a mixture of peripheral airway lesions, primarily chronic obstructive bronchitis, and emphysema. Extrinsic risk factors include smoking and air pollution, while endogenous risk factors include genetic predisposition.

[0003] Reported animal models of COPD have included mice, rats, hamsters, pigs, sheep, and monkeys. Mice offer advantages, such as a well-defined genetic background (gene sequence), ease of genetic modification, and abundant antibody resources. However, COPD is a complex disease that simultaneously manifests multiple pathologies, including chronic obstructive bronchitis (COPD), sputum retention, and emphysema. Therefore, few mouse models have been developed that encompass all of these. Many known mouse models have been reported to exhibit emphysema. Examples include acute and chronic cigarette smoking models (Non-Patent Document 3), as well as mice overexpressing neutrophil elastase, MMP-1, IL-13, and IFN-γ, which are secreted by alveolar macrophages and neutrophils in response to smoking (Non-Patent Document 4). Forced expression of these proteases leads to destruction of the lung parenchyma. Conversely, it has been reported that, for example, genetic deficiency of MMP-12 reduces the incidence of smoking-induced emphysema (Non-Patent Document 5). It has also been reported that smoking-induced MMP overexpression can be abrogated in TNF-α receptor-deficient mice (Non-Patent Document 6). However, the phenotypes described in these mice are primarily focused on emphysema, and the reproduction of other COPD phenotypes, such as chronic bronchitis, is poorly demonstrated, making their use in elucidating the pathogenesis of COPD difficult. In fact, nearly 20 years have passed since these findings were discovered, yet clinical trials of biologics for COPD targeting neutrophil-mediated inflammation suppression, including IL-1b and TNF-α, which are thought to be causative molecules of emphysema, have all failed to demonstrate efficacy (Non-Patent Document 7). Clinical studies of neutrophil elastase and MMP inhibitors have either been ineffective or discontinued midway, and to date have not led to the development of therapeutic agents that control protease activity (Patent Document 8). From the above, it must be said that the direct cascade between molecules involved in the activation of alveolar macrophages and neutrophils and the onset and progression of COPD is not simple, and its exact nature remains unknown.

[0004] Currently, smoke-exposed mice are relatively commonly used, but their creation is time-consuming and expensive, and the resulting COPD lesions are mild and do not progress to severe disease. These drawbacks are cited as drawbacks of these mice as a model (Non-Patent Document 9). A model using LPS administration has also been proposed. While reported to resemble COPD, LPS administration induces systemic inflammation, resulting in lung lesions. Furthermore, discontinuing LPS administration resolves the inflammation, suggesting a significant difference from the actual state of COPD (Non-Patent Document 10). [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Safiri S. et al. Burden of chronic obstructive pulmonary disease and its attributable risk factors in 204 countries and territories, 1990-2019: results from the Global Burden of Disease Study 2019. BMJ (2022)378: e069679 [Non-patent document 2] WHO homepage / Newsroom / Fact sheets / Detail / Chronic obstructive lung disease (COPD): https: / / www.who.int / news-room / fact-sheets / detail / chronic-obstructive-pulmonary-disease-(COPD) [Non-patent document 3] Fricker T. et al. Animal models of chronic obstructive pulmonary disease. Expert Opinion on Drug Discovery (2014) 9: p629-645.Doi:10.1517 / 17460441.2014.909805 [Non-patent document 4] Snider GL. et al. Animal models of emphysema. Am Rev. Respir. Dis. (1986) 133: p149-169. [Non-patent document 5] Hautamaki RD. et al. Requirement for macrophage elastase for cigarette smoke-induced emphysema in mice. Science (1997) 277: p2002-2004. [Non-patent document 6] Shapiro SD. Animal models for COPD. Chest (2000) 117: (Suppl. 1) p223S-277S. [Non-Patent Document 7] Hiroyuki Nagase, Future Prospects for Drug Therapy: Biological Agents and PDE Inhibitors (Special Feature: Health Japan 21 and Chronic Obstructive Pulmonary Disease: Current Status and Prospects of Treatment), Journal of the Japanese Society of Internal Medicine (2024), Vol. 113, No. 6, pp. 923-931 [Non-patent document 8] Vlahos R. and Bozinovski S. Recent advances in pre-clinical mouse models of COPD. Clinical Science (2014) 126. p253-265. [Non-Patent Document 9] Tetsumoto, K. et al. Topic 3: Respiratory Diseases and Animal Models (Special Feature: Cellular and Molecular Biology for Tomorrow's Clinical Practice) Journal of the Japanese Respiratory Society (2014) 3: p631-635. [Non-Patent Document 10] Kobayashi S. et al. A single dose of lipopolysaccharide into mice with emphysema mimics human chronic obstructive pulmonary disease exacerbation as assessed by micro-computed tomography. Am J Respir Cell Biol. (2013) 49: p971-977. Summary of the Invention [Problem to be solved by the invention]

[0006] The present invention aims to provide an animal model for human COPD and to develop markers that will be useful in investigating the etiology of COPD, as well as in developing therapeutic agents, early diagnosis, and assessment of the progression of the disease. [Means for solving the problem]

[0007] In 2019, the inventors generated the world's first Wfdc2-genetically modified mice and reported that they died within one week of birth due to respiratory failure caused by factors such as alveolar surfactant abnormalities (Dis Model Mech. (2019) 12;12(11):dmm040139.doi:10.1242 / dmm.040139). These Wfdc2-genetically modified mice were conventional knockout mice, lacking Wfdc2 during early development. Therefore, they generated tamoxifen-inducible Wfdc2 gene knockout mice using the Cre-loxP system, which retains the Wfdc2 gene until adulthood and then induces gene deletion upon tamoxifen administration. As a result, they discovered that systemic deletion of the Wfdc2 gene in adults resulted in a mouse model similar to COPD, leading to the completion of the present invention.

[0008] The gist of the present invention is as follows. (1) A non-human animal that has a gene in which at least one exon of Wfdc2 is flanked by two loxP sequences in both alleles, and that systemically expresses the enzyme CreERT2, which causes recombination in an antiestrogen-dependent manner, thereby enabling antiestrogen-induced deletion of the Wfdc2 gene. (2) A method for producing a non-human animal model for chronic obstructive pulmonary disease, comprising inducing a deficiency of the Wfdc2 gene by administering an anti-estrogen drug to the non-human animal described in (1) after it has become an adult. (3) A non-human animal model for chronic obstructive pulmonary disease, produced by the method described in (2). (4) The non-human animal model of chronic obstructive pulmonary disease according to (3), which is used for screening of preventive and / or therapeutic drugs for chronic obstructive pulmonary disease. (5) The non-human animal model of chronic obstructive pulmonary disease according to (3), which is used for screening biomarkers for chronic obstructive pulmonary disease. (6) The non-human animal model of chronic obstructive pulmonary disease according to (3), which is used to explore the cause of chronic obstructive pulmonary disease.

[0009] The COPD model animal (e.g., mouse) of the present invention caused by a Wfdc2 gene deficiency is 1. Depending on the duration of tamoxifen administration, COPD model animals (e.g., mice) can be obtained that are suited to the purpose, ranging from mild to severe. 2. Both chronic bronchitis and emphysema phenotypes are present. 3. Patients can survive for more than a year while exhibiting symptoms, making long-term observation possible. This is similar to the pathology of COPD in humans, the number of patients of which increases with aging. These may have advantages not found in previous model animals. [Effects of the Invention]

[0010] The present invention provides an animal model similar to human COPD. [Brief explanation of the drawings]

[0011] [Figure 1]The diagram shows the gene structure and production method for the genetically modified mice used. A. The Wfdc2 genome structure is shown. It has five exons. GFPΔWfdc2 mice were created by genetically removing part of Exon 1 and Exon 2 from the Wfdc2 mouse genome and replacing them with a gene encoding EGFP inserted downstream of the internal ribosome entry site (IRES) via homologous recombination. B. The completed diagram is shown. C. Wfdc2 lacZ knock-in mice were created by inserting the IRES-lacZ gene and the hBacP-neo gene into Intron 2 of the Wfdc2 mouse genome. FRT sites (blue) were inserted between these two genes; this region can be removed by crossing with Flipping mice (B6.Cg-Tg(ACTFLPe)9205Dym / J), which express the Flip enzyme systemically. In addition, Wfdc2 lacZ knock-in mice contain loxP sequences (red) inserted between the IRES-lacZ gene and the hBacP-neo gene, 3' to the FRT sequence 3' of Intron 2 of the Wfdc2 genome, and in Intron 4. D. As a result, mice with loxP sequences (red) within Intron 2 and Intron 4 of the Wfdc2 genome can be generated. E. Wfdc2 GFP / F mice and Wfdc2 F / F mice were crossed with Rosa-CreERT2 mice, which express CreERT2 systemically and can excise the gene region between the two loxP sequences in a tamoxifen-inducible manner, to generate Rosa-CreERT2::Wfdc2 GFP / FC mice (F) or Rosa-CreERT2::Wfdc2FC / FC mice (G). In the genotype, F indicates the mouse before excision of the loxP sequence, and FC indicates the mouse after excision. F. Rosa-CreERT2::Wfdc2 GFP / FC mouse. G. Rosa-CreERT2::Wfdc2FC / FC mouse. H. An example of a protocol for inducible Wfdc2 gene deletion using tamoxifen administration. Mice aged 6-12 weeks were given 75 mg / kg BW of tamoxifen intraperitoneally for five consecutive days as the first cycle.The following week, after a one-week drug rest, the same dose as described above will be administered intraperitoneally for five consecutive days in the second cycle. After a two-week drug rest from the following week, the same dose as described above will be administered intraperitoneally for five consecutive days in the third cycle. After another two-week drug rest from the following week, the same dose as described above will be administered intraperitoneally for five consecutive days in the fourth cycle, and the mice will be analyzed 12-13 weeks after the start of administration. [Figure 2]This figure shows that Wfdc2 transgenic mice exhibit COPD similar to that of humans. Mouse weight, lung CT images, lung morphology, pathological morphology, bronchoalveolar lavage fluid, and mucin production were evaluated. A. Data show the ratio of mouse weight 12-13 weeks after tamoxifen administration divided by weight immediately before tamoxifen administration (N = 9 wild-type mice, 14 heterozygous mice, 18 knockout mice). Knockout mice exhibited a significant decrease in weight compared to wild-type and heterozygous mice. Heterozygous mice exhibited similar weight loss as wild-type mice. B. Typical small-animal micro-CT images from 10 weeks after the start of tamoxifen administration. Knockout mice exhibited atelectasis not seen in wild-type or heterozygous mice. Knockout mice also exhibited peripheral bronchiectasis. These imaging findings were highly reproducible in knockout mice. C. Lung volume was measured using CT analysis software from CT images taken before and 10 weeks after tamoxifen administration, and lung volume ratios were calculated for each genotype. While there was no significant difference in lung volume between wild-type and heterozygous mice, knockout mice exhibited significantly increased lung volume. (N = 7 wild-type mice, 7 heterozygous mice, 8 knockout mice) D. Typical examples of macroscopic lung findings 12 weeks after tamoxifen administration are shown. As predicted by the CT scan, the lungs of knockout mice are hyperinflated compared to heterozygous mice. E. Typical examples of HE staining of the bronchial and alveolar regions by genotype are shown 12-13 weeks after tamoxifen administration. In the airway region, unlike wild-type and heterozygous mice, the intrapulmonary bronchi of knockout mice are filled with mucus containing cellular components (top row, Wfdc2 FC / FC). The alveolar spaces of knockout mice are enlarged compared to those of wild-type and heterozygous mice (bottom row). This suggests destruction of the alveolar wall. F. Mean linear intercept (MLI, the average inter-alveolar distance) measurements are shown in the alveolar region of the HE-stained tissue in E. Compared to wild-type and heterozygous mice, the knockout mouse lungs showed a statistically significant increase in MLI. (N = 3 wild-type mice, 3 heterozygous mice, 3 knockout mice) G. Bronchoalveolar lavage fluid (BALF) collected 12-13 weeks after tamoxifen administration is shown.Before centrifugation, BALF from wild-type and heterozygous mice was clear, whereas that from knockout mice was cloudy and foamy. After centrifugation, the cellular components precipitated were greater in knockout mice than in wild-type and heterozygous mice. H. The number of cells in BALF was measured using a counting chamber. The number of cells was significantly higher in knockout mice than in wild-type and heterozygous mice (N = 3, one-way ANOVA; p-value 0.019, Tukey's; p-values, wild-type vs. heterozygous mice 1.0, wild-type vs. knockout mice 0.029, heterozygous vs. knockout mice 0.029). I. The total protein concentration in BALF was measured using the protein BCA assay. Total protein concentration was significantly increased in knockout mice compared to wild-type and heterozygous mice (N=3, one-way ANOVA; p-value 1.19e-05, Tukey's p-value: wild-type vs. heterozygous mice 0.41, wild-type vs. knockout mice 1.67e-5, heterozygous vs. knockout mice 2.96e-5). J. The precipitated cells shown in G were spread on a glass slide and subjected to Diff-Quick staining for cell fractionation. The knockout mice contained numerous neutrophils (red arrowheads), which were not seen in wild-type or heterozygous mice, and macrophages also showed swelling and partial phagocytosis (black arrowheads), suggesting macrophage activation. K. Images of bronchi collected 12-13 weeks after tamoxifen administration, stained with Alcian-blue / HE staining. In the knockout mice, mucin stained light blue was identified on the bronchial surface, suggesting that mucus was accumulated. [Figure 3]We used mice in which GFP was knocked into the Wfdc2 locus to identify Wfdc2-expressing cell types and examine the frequency of gene-expressing cells. Because Wfdc2 is a secreted protein with a transmembrane domain, we feared that antibody detection of WFDC2 protein might identify not only expressing cells but also non-expressing cells with WFDC2 attached to their surface. Therefore, we used mice in which GFP was knocked into the Wfdc2 locus and identified Wfdc2-expressing cells by co-staining with various lung epithelial cell-specific differentiation markers. A. Alveoli from 19-22 week-old mice were co-stained with antibodies against GFP (Wfdc2-expressing cells), ProSPC (type II pneumocytes), and podoplanin (PDPN, type I pneumocytes). Nuclei were stained with DAPI. As a result, a portion of ProSPC-positive type II pneumocytes were GFP-positive, while no cells co-stained with PDPN-positive type I pneumocytes and GFP were identified. B. The frequency of GFP (Wfdc2)-positive cells in type II pneumocytes (ProSPC-positive) was examined using the stained image in A. It was 51.7% in heterozygous mice carrying the GFP allele and 51.7% in knockout mice carrying the GFP allele. These results suggest no difference in the proportion of GFP-positive cells in type II pneumocytes between heterozygous and knockout mice. C. Bronchi from 19-22 week-old mice were co-stained with antibodies against GFP (Wfdc2-expressing cells) and SCGB1A1 (club cells). Nuclei were stained with DAPI. A portion of SCGB1A1-positive cells was found to be GFP-positive. D. The frequency of GFP (Wfdc2)-positive cells in type II pneumocytes was 21% in the stained image in C. E. Bronchi from 19-22 week old mice were co-stained with antibodies against GFP (Wfdc2-expressing cells), neural calcium sensor 1 (NCS1, present in the basal layer of ciliated epithelial cells), and acetylated-tubulin (Ac-tub, present in the ciliated portion of ciliated epithelial cells). Nuclei were stained with DAPI. The results showed that there were very few GFP-positive cells among the NCS1 / Ac-tub-positive cells.F. The frequency of GFP (Wfdc2)-positive cells among all NCS1-positive cells was determined to be 1.5% in the stained image of E. G. Bronchi from 19-22-week-old mice were co-stained with antibodies against GFP (Wfdc2-expressing cells), Keratin 5 (KRT5, basal cells), and p63 (basal cells). Nuclei were stained with DAPI. GFP-positive cells were present in a portion of KRT5-positive and p63-positive basal cells. H. The stained image of G showed expression in 2.7% of KRT5-positive basal cells. Analysis results for two individuals are shown. I. The stained image of G showed GFP expression in 6.9% of p63-positive basal cells. Analysis results for two individuals are shown. These results demonstrate that Wfdc2 is expressed in a portion of basal cells, club cells, and type II alveolar epithelial cells, which have been reported to have stem cell activity in lung tissue. [Figure 4]Phenotypic analysis of the alveolar region in Wfdc2 knockout mice. A. Immunostaining with WFDC2 antibodies confirmed the disappearance of WFDC2 protein in both the airway and alveolar regions of knockout mice (Wfdc2 FC / G) at 12 weeks after tamoxifen administration. In heterozygous mice (Wfdc2 G / +), signals were detected in both the airway and alveolar regions. B. Immunostaining with WFDC2 antibodies compared WFDC2 protein expression levels in the airway and alveolar regions of heterozygous mice at 12 weeks after tamoxifen administration. A stronger signal was detected in the cytoplasm of bronchial epithelial cells than in alveolar epithelial cells. These results suggest that WFDC2 protein is more abundant in bronchial epithelial cells than in alveolar epithelial cells. This result was consistent with the analysis of reporter mice in which GFP was knocked into the Wfdc2 gene region. C. The phenotype of type I pneumocytes was examined in Wfdc2 knockout mice. Alveolar tissue from mice 11–13 weeks after tamoxifen administration was double-stained for HOPX, a marker for type I pneumocytes, and Nkx2.1, a marker expressed in both type I and type II pneumocytes (white arrows indicate HOPX-positive, Nkx2.1-positive alveolar epithelial cells; yellow arrows indicate HOPX-negative, Nkx2.1-positive alveolar epithelial cells). D. The proportion of HOP X-positive cells was calculated using the staining images in C as the denominator, and the knockout mice showed a significantly lower proportion of type I pneumocytes than heterozygous mice (38.5% in heterozygous mice vs. 23.8% in knockout mice, p-value: 0.0049). E. The phenotype of type II pneumocytes was examined in Wfdc2 knockout mice. Alveolar tissue from mice 11-13 weeks after tamoxifen administration was double-stained for ABCA3, a marker for type II pneumocytes, and Nkx2.1, a marker for alveolar epithelial cells expressed in both type I and type II pneumocytes (white arrow: ABCA3-positive, Nkx2.1-positive alveolar epithelial cells; yellow arrow: ABCA3-negative, Nkx2.1-positive alveolar epithelial cells).F. Comparing the ratio of ABCA3-positive cells to Nkx2.1-positive cells in the stained images of E, the proportion of type II alveolar epithelial cells was significantly higher in knockout mice than in heterozygous mice (32.0% in heterozygous mice vs. 41.8% in knockout mice, p-value: 0.021). [Figure 5]Phenotypic analysis of Wfdc2 knockout mice. A. Bronchi were collected from mice 12 weeks after tamoxifen administration and whole-mount specimens were immunostained. Specific antibodies for GFP (green), SCGB1A1 (red), and NCS1 (magenta) were used, and F-actin was detected with fluorescently labeled phalloidin (white). Similar to the results of the sections, GFP was expressed in a subset of SCGB1A1-positive club cells in Wfdc2 knockout mice, but not in NCS1-positive ciliated cells. In Wfdc2 knockout mice, GFP was expressed in a subset of SCGB1A1-positive club cells, similar to the heterozygous mice. Furthermore, GFP-expressing ciliated cells were observed among NCS1-positive ciliated cells, which were absent in Wfdc2 knockout mice (white arrowheads). B. The percentages of club cells and ciliated cells among all bronchial epithelial cells were calculated for heterozygous and knockout mice. The knockout mice showed increased numbers of ciliated and club cells compared with heterozygous mice. C. To examine the distribution of Wfdc2-expressing cells in the airway epithelium, GFP antibody staining was performed on the trachea and bronchi of heterozygous mice 12 weeks after tamoxifen administration. GFP-expressing cells were abundant in the bronchi but almost absent in the trachea. Furthermore, GFP-positive cells were abundant in the more peripheral lung regions of the bronchi, both on the ventral and dorsal sides. This trend was also observed with Wfdc2-specific antibodies. These findings suggest that Wfdc2-expressing cells, including basal cells and club cells, are specific to the bronchial region. D. To observe the morphology of cilia, the tracheal epithelium of mice 12 weeks after tamoxifen administration was observed under a scanning electron microscope. The image shown corresponds to the region close to the bronchi. Cilia were significantly shorter in knockout mice than in heterozygous mice. E. Twelve weeks after tamoxifen administration, the alveolar walls of heterozygous and knockout mice were observed under a transmission electron microscope. Thickened extracellular matrix deposition was observed in the alveolar walls of the knockout mice, as indicated by the yellow dotted line. F. Based on the results of E, the thickness of the alveolar wall was calculated from the electron microscope images (measured area and divided by the length of the long axis).The percentage of alveolar walls thicker than 7 μm was considered a significant increase, and the results are shown below. Compared to heterozygous mice, the knockout mice had significantly increased alveolar wall thickness (N = 3 heterozygous mice, 4 knockout mice). [Figure 6]This figure shows a comparison of lung gene expression between heterozygous and knockout mice, and the application of this genetically engineered mouse to in vitro cell culture. A. Bronchial and lobar tissues were isolated from heterozygous and knockout mice at 7 and 13 weeks after tamoxifen administration, and gene expression was analyzed separately using RNA sequencing. The figure shows a volcano plot comparing expression in heterozygous and knockout mice. Genes whose gene expression in knockout mice decreased relative to that in heterozygous mice are shown to the left of the zero point, and genes whose expression increased relative to that in knockout mice are shown to the right of the zero point. The number of genes corresponding to each group is shown in blue (left) for decreased (down) and red (right) for increased (up). Comparing the number of genes whose expression changed between bronchi and lobes, the bronchial tissue showed a higher number of genes at both 7 and 13 weeks after tamoxifen administration. Furthermore, when observing changes at 7 and 13 weeks, the latter showed a higher number of altered genes in both increased and decreased groups. B. Gene ontology analysis of genes whose expression levels were altered between heterozygous and knockout mice 7 weeks after tamoxifen administration. Inflammatory response, immune system processes, and other inflammation-related terms were significantly more prevalent. Furthermore, COPD patients have been reported to have increased levels of IL-17A in sputum and CD4+ IL-17-producing cells (Th17 cells) in the airway mucosa. The genes highlighted in red are Th17 cell-related genes that are elevated in COPD patients. C. Knockout mice can be used as a COPD disease model mouse for screening pathogenesis and early diagnostic markers. The top panel is an illustration of the air-liquid interface (ALI) culture method established by You et al. (Am. J. Physiol. Lung Cell. Mol. Physiol. (2002) 283: L1315-L1321) using cells isolated from mouse bronchial tissue (white circles). The lower panel shows an example of immunohistological analysis of cells cultured for 27 days by the ALI method using this mouse.In this way, bronchial epithelial cells extracted from this mouse can be used to examine in vitro whether or not there are differentiation abnormalities, and if there are abnormalities, to analyze the mechanism of the abnormalities. DETAILED DESCRIPTION OF THE INVENTION

[0012] The present invention will be described in detail below.

[0013] The present invention provides a non-human animal that has a gene in which at least one exon of Wfdc2 is flanked by two loxP sequences in both alleles, and that systemically expresses the enzyme CreERT2, which causes recombination in an anti-estrogen drug-dependent manner, thereby enabling the Wfdc2 gene to be deleted in an anti-estrogen drug-inducible manner.

[0014] The Wfdc2 molecule belongs to the WFDC family of molecules. WFDC stands for whey acidic protein four-disulfide core domain. This family of molecules is clustered as a family of approximately 20 proteins on chromosome 20 in humans and chromosome 2 in mice (Bingle et al. Trends in Immunology. (2008) 29: pp. 444-453). These small proteins, approximately 20 kDa in molecular weight, were originally identified as molecules contained in milk and are thought to be involved in antibacterial, anti-inflammatory, cell proliferation suppression, and inhibition of extracellular matrix degradation (Bingle et al. Trends in Immunology. (2008) 29: pp. 444-453). However, information is fragmentary, and only a small portion of the functions of the WFDC family of molecules have yet been elucidated. These family molecules share a characteristic structure: four pairs of disulfide bonds between eight cysteine ​​residues. Biologically, they are classified as serine protease inhibitors. The proteases they inhibit are thought to be diverse, including serine and cysteine ​​proteases and matrix metalloproteinases. Conversely, many protease inhibitors are thought to inhibit a single protease. Therefore, identifying the target protease is difficult. Among WFDC family molecules, the Secretory Leukocyte Protease Inhibitor (SPLI) molecule (Nakamura et al. J. Exp. Med. (2003) 197: 669-674) has been reported to be involved in immune and respiratory functions.

[0015] Accumulating evidence has shown that WFDC2 (clinical name: HE4) is highly expressed in various human tumors. Its association with ovarian cancer was first noted. In particular, the area under the curve (AUC) of HE4 alone was very high (0.92), and when combined with the CA125 marker, the AUC increased to 0.94 (Li et al. Eur Rev Med Pharmacol Sci. (2020) 24:604-610). In addition to ovarian cancer, data from the Human Protein Atlas shows that Wfdc2 stains positively in thyroid cancer, lung cancer, liver / gallbladder cancer, pancreatic cancer, kidney cancer / bladder cancer, breast cancer, cervical cancer, endometrial cancer, and ovarian cancer (https: / / www.proteinatlas.org / ENSG00000101443-WFDC2 / pathology). Numerous papers have been published and are examining its potential use as a biomarker or prognostic predictor for human epithelial cancers, particularly in humans and mice. While Wfdc2 is predicted to be expressed in respiratory tissues and to be involved in innate immunity, its detailed tissue expression and physiological functions have remained unknown. In 2019, the inventors were the first in the world to create genetically modified Wfdc2 mice and reported that they died within one week of birth due to respiratory failure caused by alveolar surfactant abnormalities (Dis Model Mech. (2019) 12;12(11):dmm040139.doi:10.1242 / dmm.040139).Similarly, in 2020, Zhang et al. reported that Wfdc2-deficient mice died of respiratory failure 10 hours after birth, and that apoptosis occurred in the type I alveolar epithelium at that time (Biochem Biophys Res Commun. (2020) 522:456-462. doi:10.1016 / j.bbrc.2019.11.011). Meanwhile, Bingle et al. reported at a conference that Wfdc2-deficient mice also died shortly after birth from respiratory failure accompanied by airway stenosis. Furthermore, they reported that artificially produced WFDC2 protein was unable to inhibit trypsin activity or the growth of Streptococcus (S) mutans, S. gordonii, S. pneumoniae, and Escherichla coli (Eur Respir J, (2020) 56:2775, doi:10.1183 / 13993003.congress-2020.2775).

[0016] The Wfdc2 gene is conserved in animal species such as humans, mice, rats, and monkeys, and its sequence information is registered in a database. Mus musculus: NCBI (Gene ID: 67701) (SEQ ID NO: 1) Rattus norvegicus: NCBI (Gene ID:286888) (SEQ ID NO: 2) Rhesus monkey: NCBI (Gene ID: 710469) (SEQ ID NO: 3) Homo sapiens: NCBI (Gene ID: 10406) (SEQ ID NO: 4)

[0017] The non-human animals of the present invention in which the Wfdc2 gene can be deleted in an anti-estrogen-inducible manner have a Wfdc2 gene in both alleles, with at least one exon of Wfdc2 flanked by two loxP sequences.

[0018] Non-human animals that have established ES cells, such as mice, rats, and monkeys, can be potentially generated. Furthermore, using genome editing technology (CRISPR-Cas9), it is theoretically possible to generate non-human animals lacking the Wfdc2 gene in any animal. Mice are preferred due to the ease of genetic modification, past performance, extensive analytical methods such as drug-induced gene deletion, and the abundance of research resources and literature, including antibodies.

[0019] Mouse Wfdc2 has five exons. Humans have four exons, monkeys have four, and rats have six. The exons flanked by two loxP sequences should preferably include exon 3, which encodes the unstructured linker region separating the two WFDC domains in mice and rats; for example, exons 2 and 3, or exons 3 and 4. In humans and monkeys, the WFDC domains are contained in exons 2 and 3, so these should be flanked by two loxP sequences.

[0020] Furthermore, the non-human animals of the present invention systemically express CreERT2, an enzyme that causes recombination in an anti-estrogen drug-dependent manner, and can cause the Wfdc2 gene to be deleted in an anti-estrogen drug-inducible manner.

[0021] CreERT2 is a fusion protein of the estrogen receptor and Cre recombinase. Cre recombinase is a DNA recombinase derived from bacteriophage P1 that recognizes 34-bp DNA sequences called loxP sequences in the bacteriophage P1 genome and causes recombination. When two loxP sequences are located in the same direction, Cre excises the gene flanked by the loxP sequences. Therefore, the Cre / loxP system can be used to delete genes flanked by loxP sequences. By introducing multiple point mutations into the estrogen receptor, a unique estrogen receptor (CreERT2) can be created that is insensitive to endogenous estrogens but responds only to anti-estrogen drugs (e.g., tamoxifen). CreERT2 is normally present in the cytoplasm, but upon binding to the anti-estrogen drug tamoxifen, it translocates into the nucleus and recombines at the loxP sequences. This can be used to regulate the timing of the Cre-loxP system's nuclear action in an anti-estrogen drug (eg, tamoxifen)-dependent manner.

[0022] Examples of anti-estrogen drugs include tamoxifen, 4-hydroxytamoxifen, and ICI 182,780, with tamoxifen being preferred.

[0023] To generate the non-human animals of the present invention, a transgenic non-human animal is prepared that contains a gene in which at least one Wfdc2 exon is flanked by two loxP sequences in one or both alleles, and is then mated with a transgenic non-human animal that systemically expresses the enzyme CreERT2, which causes recombination in an antiestrogen-dependent manner. By mating the offspring, a non-human animal is born that contains a gene in which at least one Wfdc2 exon is flanked by two loxP sequences in both alleles and that systemically expresses the enzyme CreERT2, which causes recombination in an antiestrogen-dependent manner. By administering an antiestrogen to this non-human animal, the Cre-loxP system is activated, causing deletion of the Wfdc2 gene.

[0024] Transgenic non-human animals carrying a gene in which at least one Wfdc2 exon is flanked by two loxP sequences in one or both alleles can be generated using a targeting vector via homologous recombination in embryonic stem (ES) cells. Briefly, a targeting vector containing loxP sequences flanking at least one Wfdc2 exon is constructed and transfected into non-human ES cells by electroporation. ES cells that have undergone successful homologous recombination are selected and injected into blastocysts. These blastocysts are then implanted into foster mothers to produce chimeric animals. The targeting vector may contain a selectable marker (e.g., the neomycin resistance gene, neo) and a reporter gene (e.g., lacZ) for selecting recombinants. Other elements, such as an IRES, hBacP, or polyadenylation signal (pA), may also be included. FRT sites may be inserted to flank the selectable marker and reporter gene (which may also include an IRES, hBacP, or pA). The resulting chimeric mice (F0 generation) are then mated with wild-type animals to obtain heterozygous mutants (F1 generation). These heterozygous mutants can then be crossed with each other to obtain homozygous mutants (F2 generation). By crossing homozygous mutants with animals expressing the Flip enzyme (e.g., Flipping mice (B6.Cg-Tg(ACTFLPe)9205Dym / J) (The Jackson Laboratory: Strain #:005703)), the region flanked by the FRT sites (the region containing the selectable marker and reporter gene) can be removed. As a result, transgenic animals can be generated that contain a gene in which at least one Wfdc2 exon is flanked by two loxP sequences in one or both alleles. Alternatively, loxP sequences can be inserted into the desired intron using the CRISPR-Cas9 method. Guide RNAs are designed before and after the exon in which Wfdc2 deletion is desired, and a loxP sequence and the genome of the intended insertion region on both the 5' and 3' sides of the sequence are synthesized using oligonucleotides. These are then introduced into fertilized eggs to obtain chimeric mice in which the oligonucleotide has been inserted heterozygously or homozygously.Oligonucleotides containing loxP sequences at the 5' and 3' ends can be introduced into fertilized eggs, or one of the 5' and 3' ends can be introduced first to generate mice with loxP sequences, and then the remaining oligonucleotide can be introduced into the fertilized eggs of those mice. In both cases, mice with loxP sequences introduced into two introns on the same chromosome, one before and one after the exon to be deleted, are ultimately selected. Similarly, the resulting chimeric mice (F0 generation) are mated with wild-type animals to obtain heterozygous mutant animals (F1 generation). These heterozygous mutant animals can then be mated to obtain homozygous mutant animals (F2 generation).

[0025] Transgenic non-human animals that systemically express the enzyme CreERT2, which induces recombination in an anti-estrogen-dependent manner, are available from TACONIC BIOSCIENCES or The Jackson Laboratory (Rosa26-CreERT). 2 Mouse (C57BL / 6-Gt(ROSA)26Sor tm9(Cre / ESR1)Arte Or B6.129-Gt(ROSA)26Sor tm1(cre / ERT2)Tyj Alternatively, it may be prepared by the method described in Nature (2007) 445: pp. 661-665.

[0026] A non-human animal model for chronic obstructive pulmonary disease can be produced by administering an anti-estrogen to a non-human animal that has, in both alleles, a gene in which at least one Wfdc2 exon is flanked by two loxP sequences, and that systemically expresses the enzyme CreERT2, which causes recombination in an antiestrogen-dependent manner, and is capable of causing antiestrogen-inducible deletion of the Wfdc2 gene, after the animal has reached adulthood, thereby inducing Wfdc2 gene deletion.The present invention also provides a method for producing a non-human animal model for chronic obstructive pulmonary disease, comprising administering an anti-estrogen to a non-human animal that has, in both alleles, a gene in which at least one Wfdc2 exon is flanked by two loxP sequences, and that systemically expresses the enzyme CreERT2, which causes recombination in an antiestrogen-inducible manner, after the animal has reached adulthood, thereby inducing Wfdc2 gene deletion. The present invention also provides a non-human animal model for chronic obstructive pulmonary disease produced by the above method.

[0027] For example, when the non-human animal is a mouse, mice aged 6 weeks or older (preferably 6 to 12 weeks for intraperitoneal administration, and preferably 8 to 10 weeks for oral administration) are administered 30 to 100 mg / kg body weight (preferably 75 mg / kg body weight) of an antiestrogen intraperitoneally or 150 to 300 mg / kg body weight (preferably 200 mg / kg body weight) of an antiestrogen orally for 5 to 7 consecutive days, followed by a 1 to 2 week break. This cycle is repeated 2 to 4 or more times. Depending on the duration of antiestrogen administration, COPD model animals can be obtained that are suited to the purpose, ranging from mild to severe. Animals that undergo 2 to 3 cycles of the above-mentioned one cycle can be used as mild COPD model animals. Animals that undergo 4 or more cycles of the above-mentioned one cycle can be used as severe COPD model animals. The administration interval and duration vary depending on the genetic background, weight, and age of the mouse, so they may be changed based on the degree of Wfdc2 gene deletion assessed by PCR analysis of tail genomic DNA.

[0028] In the Examples described below, as an example of a protocol for inducibly deleting the Wfdc2 gene by administering tamoxifen, 6-12 week old mice were intraperitoneally administered 75 mg / kg BW of tamoxifen for 5 consecutive days in the first cycle, followed by a one-week drug rest the following week, at which point the same amount as described above was intraperitoneally administered for 5 consecutive days in the second cycle, and after a two-week drug rest from the following week, the same amount as described above was intraperitoneally administered for 5 consecutive days in the third cycle, followed by a further two-week drug rest from the following week, at which point the same amount as described above was intraperitoneally administered for 5 consecutive days in the fourth cycle, and the mice were analyzed 12 weeks after the start of administration.

[0029] The non-human animal model of chronic obstructive pulmonary disease of the present invention preferably has both chronic bronchitis and emphysema, which is thought to make it a good animal model of human COPD.

[0030] Peripheral airways (peripheral airways) are those with an internal diameter of 2.5 mm or less (fourth-order bifurcation and above). COPD is characterized by increased exudates containing inflammatory cells and accumulation of mucus secretions, which are major factors in airway obstruction. In human clinical settings, the presence or absence of airflow obstruction can be confirmed using spirometry, and morphological findings can be confirmed using computed tomography (multislice CT) and histopathological examination. Chronic bronchitis is a clinical condition characterized by central airway hypersecretion, defined as sputum production occurring for more than three months per year for two or more consecutive years. A diagnosis of chronic bronchitis is made when such sputum production is not associated with other causes, such as tuberculosis or allergies. To identify the cause, blood tests, sputum cultures, bacteriological tests, chest X-rays, and chest CT scans are performed. In mice, chronic bronchitis is diagnosed by histological examination of the trachea and bronchi six weeks or later after tamoxifen treatment, when the following are confirmed: exudation of inflammatory cells such as neutrophils and lymphocytes and the presence of dead cells in the airway lumen, the presence of activated macrophages that have phagocytosed foreign bodies, mucus accumulation by mucus staining, and inflammatory cell infiltration into the tracheal epithelium and subepithelial mucosa. Furthermore, bronchoalveolar lavage is performed, and the diagnosis is made by cytological findings similar to histological findings and an increase in the protein concentration in the recovered fluid.

[0031] Emphysematous lesions are lung lesions characterized by irreversible enlargement of the air spaces distal to the terminal bronchioles, accompanied by destruction of their walls. Furthermore, it has been reported that the total cross-sectional area and number of terminal bronchioles (those with 17th-order branching or higher) decrease before the onset of emphysema due to alveolar destruction (NEJM, (2011) 365:1567-1575). Inflammatory cell-derived and endogenous proteases, such as elastase and alpha-1 antitrypsin, destroy the alveolar walls, leading to an enlargement of the alveolar diameter and emphysema. Patients with severe emphysema exhibit significant infiltration of neutrophils, macrophages, eosinophils, and T lymphocytes. These lesions can be confirmed by computed tomography (especially high-resolution CT) and histopathological imaging in humans, and by microCT and histopathological imaging in mice.

[0032] Furthermore, the non-human animal model of chronic obstructive pulmonary disease of the present invention can survive for more than a year while exhibiting symptoms, allowing for long-term observation, and the pathology is similar to that of human COPD, the number of patients of which increases with aging.

[0033] Furthermore, the non-human animal model of chronic obstructive pulmonary disease of the present invention may exhibit abnormalities in cell differentiation and in the innate immune functions of cells, which may lead to elucidation of the pathogenesis of human COPD, and may lead to elucidation of the fundamental mechanism of disease onset and the development of drugs targeting the mechanism of disease onset.

[0034] First, Wfdc2-expressing cells were found to accumulate in the pulmonary periphery (distal) of the main bronchi in mice, and the region corresponding to this in humans corresponds to the peripheral airways, the central area of ​​abnormalities in COPD.

[0035] Examples of abnormal cell differentiation include irregularities in size and shape of epithelial cells differentiated from basal cells, disruption of tight junctions, and abnormalities in the expression of differentiation markers.

[0036] Examples of abnormalities in the innate immune functions of cells include infiltration of inflammatory cells such as neutrophils and macrophages, increased mucin-positive mucus, and increased expression of inflammation-related molecules such as chemokines.

[0037] Therefore, by analyzing the non-human animal model of chronic obstructive pulmonary disease of the present invention, it is possible to explore the causes of human COPD and the process by which abnormalities develop.

[0038] For example, in mice, Wfdc2-expressing basal cells are rarely present in the trachea, but are abundant in the distal (peripheral) portion of the main bronchi, which corresponds to the primary lesion site in human COPD. Loss of Wfdc2 leads to COPD-like lesions. Cultivation of bronchial-derived Wfdc2-deficient basal cells (using the air-liquid interface culture method) has shown disruption of tight junctions, predicting loss of airway barrier function. Therefore, we hypothesize that Wfdc2-positive basal cells in mice are the target cells responsible for the development of human COPD. We then knocked out the Wfdc2 gene in Wfdc2-positive basal cells to induce COPD, and compared gene expression with that of controls to identify altered genes. We then performed pathway and ontology analyses of the altered genes to identify signaling molecules (receptors, secreted proteins, intracellular signaling molecules) and transcription factors that are specifically altered by Wfdc2 deficiency. By examining whether the genes narrowed down in mice are abnormal in human COPD bronchioles compared to healthy areas, it is hoped that the mechanism of COPD onset will be elucidated.

[0039] Furthermore, the non-human animal model of chronic obstructive pulmonary disease of the present invention can be used for screening of preventive and / or therapeutic drugs for chronic obstructive pulmonary disease, biomarkers for chronic obstructive pulmonary disease, and the like.

[0040] For example, we will use bronchial basal cells and conduct experiments using the air-liquid interface culture method. This culture allows the differentiation of club cells and ciliated epithelial cells from basal cells, forming bronchial pseudostratified epithelium similar to that in vivo. We have already identified abnormal phenotypes such as disruption of tight junctions, cell size anomaly, and high expression of a basal cell marker molecule (keratin 5) when Wfdc2 is deleted. Therefore, we will screen for the ability to resolve these phenotypes by adding a library of small molecule compounds.

[0041] Secretory proteins, signaling molecules including membrane proteins, and transcription factors that are specifically altered by Wfdc2 deficiency could be candidate marker molecules that can be used for the early detection of human COPD and for assessing the severity of the disease, because Wfdc2 deficiency in mice causes COPD-like pathology. If it is possible to detect those molecules that escape from cells in the sputum, serum, etc. of human COPD patients, their increase or decrease could be used as marker molecules.

[0042] The present invention will be described in detail below with reference to examples. [Example]

[0043] [method] 1. Preparation of Mice Rosa26-CreERT systemically expresses CreERT2, an enzyme that recognizes bacterial loxP sequences and induces recombination in an anti-estrogen (tamoxifen)-dependent manner. 2 Mouse (C57BL / 6-Gt(ROSA)26Sor tm9(Cre / ESR1)Arte ) is used under a license agreement with TACONIC BIOSCIENCES. Mice carrying the conditional allele of Wfdc2 (Wfdc2 tm1a(EUCOMM)Hmgu ) purchased and imported ES cells from EUCOMM, and generated chimeric mice by blastocyst injection. These chimeric mice were backcrossed with ICR and C57BL / 6 mice to generate mouse lines. The inventors used ES cells to knock in GFP cDNA into Wfdc2 mice (Wfdc2tm1.1Ohbo ) has already been published and reported in a paper (Dis Model Mech. (2019) 12;12(11):dmm040139.doi:10.1242 / dmm.040139).

[0044] 2. Mice generation and breeding For the experiment, Rosa26-CreERT 2 and a mouse (C57BL / 6-Gt(ROSA)26Sor) carrying GFP in one allele and a loxP site in the other allele. tm9(Cre / ESR1)Arte ::Wfdc2 tm1a(EUCOMM)Hmgu ::Wfdc2 tm1.1Ohbo ) (Fig. 1F), or mice with loxP sites in both alleles (C57BL / 6-Gt(ROSA)26Sor tm9(Cre / ESR1)Arte ::Wfdc2 tm1a(EUCOMM)Hmgu ) (Figure 1G) were used according to the purpose. All experiments using animals were performed with the approval of the Yokohama City University School of Medicine Animal Care Committee and the Yokohama City University School of Medicine Genetic Experiment Committee.

[0045] 3. Tamoxifen administration Tamoxifen was dissolved in corn oil at 40 mg / ml. Mice aged 6-12 weeks (Fig. 1H) were intraperitoneally injected with 75 mg / kg BW (Fig. 1H). Oral administration was performed at a dose of 200 mg / kg BW. Each cycle consisted of five consecutive days of administration, followed by a one- to two-week rest period. Depending on the analysis time, two cycles (experimental group 7 weeks after the start of tamoxifen administration) or four cycles (experimental group 12 weeks after the start of tamoxifen administration) were administered (Fig. 1H).

[0046] 4. Micro-CT scan After anesthetizing mice with isoflurane, chest CT scans were performed using the R_mCT2 (Rigaku) ​​at end-expiration with cardiac synchronization and a tube voltage of 90 kV. Lung volume was measured using the accompanying analysis software.

[0047] 5. Bronchoalveolar lavage fluid (BALF) A 20G Surflow was inserted into the trachea of ​​each mouse, and 500 μl of saline was injected and collected five times. The collected lavage fluid was centrifuged (1500 rpm, 15 minutes) to separate the supernatant and cellular components. The total protein concentration of the supernatant was measured using a protein BCA assay kit (Nacalai Tesque, 06385-00). To examine the cellular fraction, cytospin or cell suspension was placed on a glass slide to prepare specimens. After air-drying, the specimens were stained with Diff-Quick stain (Sysmex, 16920) and observed under a Keyence BZ-X800 microscope.

[0048] 6. Fixation of lung tissue Frozen section preparation method: After perfusion of mice with phosphate-buffered saline containing 15 U / ml heparin (hereafter referred to as heparin-mixed PBS) at a pressure of 25 cmH2O through the right ventricle, 4% paraformaldehyde (PFA) was injected through the trachea, right ventricle, and left ventricle to fix the lung tissue. Tissue from the trachea to the lung was removed and fixed in 4% PFA at 4°C for 4.5 hours. The tissue was then replaced with 10%, 15%, and 30% sucrose in that order, and then embedded in OCT compound (Sakura Fine Tissue Tech) to prepare frozen blocks. These frozen blocks were then sectioned at 8 μm. Paraffin section preparation: After perfusion of both ventricles of the mouse with heparin-mixed PBS, the lung tissue was fixed overnight in Bouin's solution. After dehydration in 70%, 90%, and 100% ethanol, the tissue was embedded in paraffin. The paraffin blocks were sliced ​​at 10 μm to prepare sections. Preparation of tracheal and bronchial whole mount specimens: After perfusion with heparin-mixed PBS solution injected at a pressure of 25 cmH2O through the right ventricle of the mouse, 4% PFA was injected through the trachea, right ventricle, and left ventricle to fix the tissue. The tracheal and bronchial sections were removed and fixed in 4% PFA at 4°C for 4 hours. Alternatively, the same tissues were removed without perfusion and fixed in -20°C methanol (MEOH) for 10 minutes.

[0049] 7. Tissue staining Hematoxylin-eosin (HE) staining: After removing the paraffin from tissues fixed with xylene and ethanol, the tissue was stained with Mayer's hematoxylin solution (Muto Chemicals, 88591) for 6 minutes, followed by 10 minutes of development in warm water. After replacing with 70% ethanol, the tissue was stained with 0.5% eosin Y in ethanol solution (Fujifilm Wako Pure Chemicals, 051-06495) for 5 minutes. The tissue was cleared with ethanol and xylene and mounted with Mount Quick (Daido Sangyo). Alcian blue staining: After removing the paraffin from tissues fixed with xylene and ethanol, the tissue was immersed in 3% acetic acid for 3 minutes and then stained with Alcian blue staining solution pH 2.5 (Muto Chemical, 2012-1) for 20 minutes. The tissue was then immersed in 3% acetic acid for 3 minutes and washed. Nuclei were stained with 0.1% Nuclear Fast Red aluminum sulfate solution (Merck, 1.00121.0500). The tissue was cleared using ethanol and xylene and mounted with Mount Quick (Daido Sangyo). Immunohistological staining: After washing with PBS, specimens were blocked with 2% bovine serum albumin (BSA). For whole-mount specimens, cell membranes were permeabilized with 0.1% Triton-X100 / PBS for 30 minutes at room temperature. Primary antibody incubation was then performed for 2 hours at room temperature or overnight at 4°C. After washing with PBS, secondary antibody incubation was performed for 1 hour at room temperature. After washing with PBS, the specimens were mounted using ProLong Glass (ThermoFisher) or ProLong Gold (ThermoFisher). When staining with mouse host primary antibodies, a MOM detection kit (VECTOR, BMK-2202) was used for blocking. Specifically, slides were incubated with MOM mouse IgG blocking reagent (90 μl / 2.5 ml PBS) for 30 minutes at room temperature before the primary antibody reaction, followed by a PBS wash. The primary and secondary antibodies were diluted with the included MOM Diluent (600 μl / 7.5 ml PBS) and reacted. If antigen retrieval was required, the antibodies were retrieved in HistoVT One (Nacalai Tesque) at 70°C for 20 minutes or in 10 mM citrate buffer, pH 6.0, at 90°C for 10 minutes before staining. The primary antibodies used are listed below.Rabbit anti-WFDC2(1:200, PA5-80227, Invitrogen), Chicken anti-GFP(1:1000, ab13970, abcam), Goat anti-GFP(1:200, ab6673, abcam), Goat anti-CC10(1:10000, ABS1673, Sigma), Mouse anti-Acetylated tubulin(1:24000, T7451, Sigma), rabbit anti-NCS1(1:200, ab116230, abcam), rabbit anti-Krt5(1:800, 905501, BioLegend), rabbit anti-p63α(1:400, 13109, CST), mouse anti-Hopx(1:100,000). sc-398703, Santa Cruz), Rabbit anti-TTF-1(Nkx2.1)(1:2000, WRAB-1231,SEVEN HILLS), Hamster anti-podoplanin(1:3000, 11-033, Angio Bio), Mouse anti-ABCA3(1:200, ab24751, abcam), Rabbit anti-ProSPC(1:2000, WRAB-9337,SEVEN HILLS). The price is 2 months old.Donkey anti-Chicken IgY Alexa488 (1:200, 703-545-155, Jackson ImmunoResearch), Donkey anti-Chicken Alexa647 (1:200, 703-605-155, Jackson ImmunoResearch), Donkey anti-Rabbit IgG Alexa555 (1:200, A31572, Invitrogen), Donkey anti-Rabbit IgG Alexa546 (1:200, A10040, Molecular Probes), Donkey anti-Rabbit IgG Alexa647 (1:200, A-31573, Molecular Probes), Donkey anti-Goat IgG Alexa488 (1:200, A11055, Molecular Probes), Donkey anti-Goat IgG Alexa594 (1:200, A11058, Molecular Probes), Donkey anti-Goat IgG Alexa546 (1:200, A11056, Molecular Probes), Donkey anti-Mouse IgG Alexa647 (1:200, A32787, Invitrogen), Donkey anti-Mouse IgG Alexa546 (1:200, A10036, Molecular Probes), Goat anti-Hamster IgG Alexa647 (1:200, A21451, Molecular Probes), Biotin labeled anti-mouse IgG antibody (1:2000, 715-066-151, JacksonImmunoResearch), Streptavidin-HRP (1:500, FP1047, Akoya Biosciences), Phalloidin Alexa546 (A22283, Molecular Probes). Observation was performed using Keyence BZ-800, Olympus FV1000, Nikon AX, and Leica SP8.

[0050] 8. Analysis using electron microscope The tissues were fixed in 2% glutaraldehyde and 2% PFA at 4°C. After washing with phosphate buffer, they were post-fixed in osmium tetroxide for 2 hours at 4°C. They were stained with 4% uranyl acetate at room temperature for 1 hour and then dehydrated using ethanol. For scanning microscopy, the tracheal sections were freeze-dried using t-butyl alcohol and metal-coated. For transmission electron microscopy, the lung lobes were transferred to propylene oxide and embedded in Epon resin. They were sectioned at 75 nm and stained with uranyl acetate and lead for observation.

[0051] 9. Gene expression analysis using RNA-sequencing Two sets of analysis were prepared for the control and knockout sets to allow for more accurate comparison. Two-mm squares were excised from the main bronchial region and peripheral lung region, and total RNA was extracted using Isogen solution (Nippon Gene). Two nanograms of purified total RNA were used to prepare an RNA-sequencing library according to the Smart-seq2 protocol. Specifically, cDNA was amplified by PCR, and tagmentation was performed using the Nextera XT DNA sample preparation kit (Illumina). Pooled samples were sequenced using barcoded primers on a HiSeq2500 and NovaSeq 6000.

[0052] 10. Data Analysis RNA-sequencing data were checked for quality using FastQ v C, trimmed using Trim Galore!, and mapped to the mm10 mouse genome using Hisat2. Read counts were performed using SeqMonk, and gene expression differences between the two groups were analyzed using EdgeR. Gene Ontology (GO) analysis was performed using DAVID (version 6.8).

[0053] 11. Bronchial Epithelial Cell Culture The bronchial section from the tracheal bifurcation to the lobes of the lungs was removed from the mouse. It was opened longitudinally from the dorsal side and placed in HBSS containing 5U of Dispase and 1U of DNase. After incubation at 800 rpm for 40 minutes at 24°C on a horizontal shaker, the bronchial epithelium was manually peeled off. After washing with HBSS, 0.1% Trypsin-EDTA solution was added and incubation was continued at 800 rpm for 20 minutes at 37°C on a horizontal shaker. The enzyme reaction was then stopped by adding 10% FBS / PBS. After washing the cells twice with 10% FBS / PBS, single cells were collected at 1.5-2.0x10 5 pieces / cm 2 The cells were diluted with BEGM BulletKit (Lonza, CC-3170) medium to a concentration of 150 μL per well and plated onto a 24-well Transwell (Corning) plate. During the initial basement cell proliferation period, BEGM BulletKit medium was used, with medium changes every 2–3 days. To induce differentiation, the medium was changed to PneumaCult-ALI Medium (Stemcell Technologies, 05001) on day 7. Thereafter, PneumaCult-ALI Medium was changed every 2–3 days. For immunohistological analysis of differentiated cells, the cultured cells were fixed on the membrane with 4% PFA for 10 minutes at room temperature, then permeabilized with 0.1% Triton-X100 / PBS for 5 minutes, and immunohistological staining was performed. The subsequent immunostaining procedures were as described above.

[0054] [result] As shown in Figures 1 to 6.

[0055] [Consideration] 1. Evaluation as a model mouse As described in the background section, most existing COPD model mice have emphysema, but no single model mouse model can fully encompass the lung inflammation, phlegm accumulation, and emphysema that occur in humans with COPD, primarily due to chronic obstructive bronchitis. The Wfdc2 knockout mice we developed here have a phenotype that encompasses all of the major human COPD pathologies listed above, making them highly likely to represent the true responsible gene for COPD and a groundbreaking model mouse. Furthermore, Wfdc2 knockout mice can be bred to produce mild COPD after a short period of time following induction of the gene deletion with tamoxifen, while severe COPD can be generated by long-term tamoxifen treatment.

[0056] Acute exacerbations of COPD cause lung parenchymal destruction, leading to hospitalization and a decline in respiratory function and quality of life, significantly impacting prognosis. Viral, bacterial, and air pollution infections are known to be causes of acute exacerbations. The mortality rate from acute exacerbations ranges from 6 to 11%, making them a major problem. Viral and bacterial infections, such as rhinovirus and influenza virus, are causative agents of COPD. Bacterial infections, including Haemophilus influenzae, Streptococcus pneumoniae, and Psudomonas aeruginosa, are particularly prevalent in the lower respiratory tract of stable COPD patients. The reason for these bacterial colonization remains unclear. The most distinctive finding of Wfdc2-expressing cells in the respiratory tract is that Wfdc2 is expressed exclusively in the mouse bronchi, which correspond to the terminal bronchioles in the lower respiratory tract (H. influenzae and S. pneumoniae). Because Wfdc2 has been suggested to be involved in innate immunity, its deficiency may be related to the colonization of these bacteria in the lower respiratory tract. Experiments using Wfdc2 knockout mice to expose them to these viruses, bacteria, as well as sulfur dioxide, nitrogen dioxide, and ozone may help elucidate the characteristics of COPD lung susceptibility and injury, identify the centrally affected genes and signaling systems, identify specific exacerbation markers, and develop therapeutic agents.

[0057] Furthermore, we examined abnormalities in gene expression networks in Wfdc2-deficient mice to explore whether molecular expression abnormalities similar to those observed in human COPD occurred. We found elevated expression of molecules such as CXCL10, IL-6, TNFSF10, TLR5, and TLR9 in p63-positive basal cells. CXCL10 is a chemokine induced by IFNg and its receptor, CXCR3, is expressed on CD4+ T cells, CD8+ T cells, NK cells, NKT cells, and dendritic cells, and is known to promote migration of expressing cells to inflamed tissues. TLR9 is known to recognize and activate CpG sequences on bacterial DNA, inducing innate immunity. This in turn induces the expression of interferons and inflammatory cytokines, leading to antiviral and inflammatory responses. Furthermore, its response to self-derived DNA is associated with the pathogenesis of allergic and autoimmune diseases.

[0058] 2. The function of Wfdc2 as inferred from the phenotypic abnormalities caused by Wfdc2 gene deficiency The purpose of this study was to investigate what abnormalities occur in adult mouse lungs when the Wfdc2 gene is deleted in adults who have already developed normal lungs. It was quantitatively demonstrated that deletion of the Wfdc2 gene after adulthood leads to abnormal cell differentiation in lung epithelial cells.

[0059] Among lung epithelial cells, basal cells, club cells, and type II alveolar epithelial cells are known to possess stem cell activity. The Wfdc2 molecule showed consistent gene expression, suggesting that Wfdc2 may be involved in lung stem cell function. Interestingly, Wfdc2 is highly expressed in the most peripheral central bronchi, but its expression decreases toward the trachea, where it is almost completely absent. It may exist as a subpopulation of more immature basal cells expressing p63. Club cells and ciliated epithelial cells differentiated from basal cells were increased in knockout mice. Furthermore, ciliated epithelial cells, which normally do not express GFP, were observed in knockout mice. These results suggest that loss of Wfdc2 leads to abnormalities in differentiation from basal cells. In this experiment, analysis was performed 12 to 13 weeks after the start of knockout. Therefore, it can be interpreted that the GFP-positive basal cells differentiated into ciliated and club cells. Because epithelial turnover is low under steady-state conditions, even if Wfdc2-expressing basal cells are the most undifferentiated cells, their phenotype is unlikely to manifest unless differentiated cells present before tamoxifen administration are replaced. Future studies will require the use of lung injury models to observe the dynamics of Wfdc2-positive cells during the regeneration process of lung epithelial cells. Bingle et al. expressed, produced, and purified WFDC2 protein in E. coli and tested its trypsin protease inhibitor activity and bacterial growth inhibitory activity, reporting that it does not possess either activity. However, the expressed and purified WFDC2 protein may have a structure different from the physiological protein, and determining whether WFDC2 possesses physiological activity as a serine protease inhibitor remains a major challenge.

[0060] Furthermore, analysis of adhesion molecules in Wfdc2-deficient bronchi revealed scattered areas of decreased ZO1 protein, a component of tight junctions, by whole-mount staining of the bronchi. Therefore, if Wfdc2-deficient epithelium expresses chemoattractants for neutrophils and T cells, including CD8, these cells are recruited and activated, initiating an inflammatory response. Furthermore, it is known that these cells produce several proteases, leading to emphysema. Therefore, single-cell analysis of bronchial epithelium from Wfdc2-deficient mice revealed increased expression of chemokines, cytokines, and MMPs in basal, club, and ciliated cells. This revealed increased expression of ADAMTS15, AGBL1, PAPPA, ADAMTS17, TLL1, ADAMTS10, TRABD2B, CPN1, CXCL10, TLR5, and TLR9. In addition to the scenario where Wfdc2 deficiency simply results in the dominance of prostheses, leading to a breakdown of the adhesion mechanism, we also considered a scenario where abnormalities in cell differentiation from Wfdc2-deficient basal cells lead to abnormalities in the adhesion apparatus. Therefore, we established an in vitro basal cell culture system and observed epithelial differentiation without the influence of blood cells. The results showed that the signaling of the tight junction molecule ZO1 was heterogeneously reduced, suggesting that this is involved in the development of COPD-like lesions. [Industrial Applicability]

[0061] The present invention makes it possible to elucidate the pathology of human COPD and screen for preventive and / or therapeutic drugs and markers. [Sequence List Free Text]

[0062] SEQ ID NO: 1: Nucleotide sequence of mouse (scientific name: Mus musculus) Wfdc2 (NCBI (Gene ID: 67701)) ACACCTGCACCATGCCTGCCTGTCGCCTCTGCTTGCTGGCCGCTGGCCTCCTACTAGGGTTGCTACTGTT CACCCCCATCTCAGCCACAGGTGAGTGCAGCCCAGAGTGCCGGAGTCTCAGAAGCCGGGGTTCTGGGACT AAGGTGAGAGGCCAGTGAATATTTAATTTTTGCAACTCTGGGGCCTACAGCTAGTTATCCTTGAAGCTTA GGGTCCGGGGAATGTACTGGGAAGGGGGGAGGGGGAATCAATGAAGGCTTAAAGTTGGAAGGGCGGGGGC TCTCTCAGCTCTGAAAGCAGAGACCTGATGAAGAGGAGCGGGAAACTCCCGGGATGGCGGAAGCTTCCAA GCAGAGGCTCAAGTCCCAGAGGACTGGGGACTTTGAAGCCAAGCATTGGGGAGTTCTCTAGTGTCAGAGA CCCCTGGCTCGGGGGTGAGGCTCTCTGGGATGATATTATGGGGGCCCCTGGGCCAAAGAATCCGAAATCC TTGGGGTTGAAATTTGGGGATCTCGGAGCAGGGAGAGGATATGCGGGTGGGACTGGGCTCGGTTCTCCCT GACAAGTCTCTGCTCCCACCCACCCATCCCAAGGCACCGATGCAGAGAAACCCGGCGAGTGCCCCCAGCT TGAACCAATTACGGACTGTGTGTTGGAGTGCACTTTGGACAAGGACTGTGCGGACAACCGCAAGTGCTGC CAGGCGGGCTGCAGCTCTGTCTGCTCCAAGCCTAATGGTAGCCCCGGGGTGACCTGGCGGGGACAGGGCG GGGTTGTGAGAGGTGGGAGCGGCCCGGGTTTGGGTTTGGCGAAGAAGGGCATCCCAGATCCAGGGAGCCC GAAAAGCGGTGGAAACCCGATCCACACGTGCCTCCCTTGTCTGGCCTGGGGTAAATGCGAGGGACAAAGG CGTCACAGAGGCGCGGCCCAGGGGAAGGAGCCCCCCTCCCCCCCATTCCTGGCTGGATTCGATTCTCTGG TGCATGAAGGGCTTCCGGGCACGCACAGCGAGACTTTGTTCCGGGACCTGAGAGCAGTGGCTTCCTAGGC TGGAGTTGGAGGGCAAACCCTGGGTGGCGTCAGTGCCGGGACCCGAGCCTTAGGGACCTGTTGCCCCTTG GGGTTCTTCCCGCAGGGCCCTCAACGGTCTGGCCTAGCAAGACTCAAGATTTGTACCTGAGGGAGGGAGA AATCTTTTGCCACATCCTAAGGTTGTGGAAGTGAGCCGCCAAAATAAATGTTCTCGCACAAAGTTACACC GCCATCCTGTGGCTGGCAGAGGTTCCGTTTCTTTCTTTGACACTGGATTTTCCTCTTGTATCTTTATTTC CACAACCTTTAGGACCGAGCGAAGGAGAGCTCTCAGGGACAGATACTAAACTCTCAGAGACTGGGACTAC TACTCAATCAGCGGGCCTTGACCACACTACTAAACCACCGGGAGGTCAAGTCTCCACGAAGCCACCAGCT GTGACCAGGGAAGGCTTAGGTGTCCGAGGTGTGGAATCTAAGCATCTTATTTCCAAGAGGGAAAAGCTGA GGCTAGGAGGTGGGGTGGGGGAACAGAAACCAAAGTTTGTAATTTCTGGTCTTTTGGGGGGGCTACAGCC CCTGCCCCTCTTTTGCCTTTATTATTTTTCTCAAAGATGCCAGGAATGACTGGAAGCAATTTATACTTAG GAGTCCTAAGGTGCTATGAACAGGAAGGTGGAAGGGGGAAATGAGACTTGCCCACGGAGGGCAGATTTTG GCTGTGGATGTGCTATGGACGGACCTGGACAAAGAGGGACCAGGAAAGATGGAGTTGAGCCAACTGGCAC ATCCAGCTGTCAGGTGGATAAAGATATTGCTTCCTTGTGTCAGATGGACAGGGCAGATGGTCTCCTGAGC CCGGGAGGAGCCTGGAGGCTTGAATCATAGCCATTTCCTAAGTGCGTGAGCCTGAATAATCTGCTCTTTG AAACTCTGCCTGCTCATCTGCAAAGTGGGCACGTTAATACCCACCTGTAGGTGTGGGGGCTAAATGCAAT GGTACTCAAGATGCGAAGGTTCTCAAAAGAGTGCGAATTCTGCACAAGATAGCTTGCTTCCTTCTGTACT ACATTCTCTCCTGGTTATTCTCATTCTGTCTCAGGGTAGCCACCAAGAGCACCTGTACTAGCAAGGCCAG GATGCTGTCCACCTCTCCTTTACCTGCCTACACACTTCACTGTATGCATCTTTCCTTTCGCTGGGCCTGC CTCCCACCTGGTACTTCTATGGAATGGAACATGGTGTGGGGTTGGTGTGTGTGGAGTGTGGTGGTGGTTT TTGCATATGCATGGGTGACCATGTACGTATGGAGGGCCGTGGCTCTGAGTGAGTGTACCTGGCGGTGGAC TTATAGGAGTCATTGTGGGGAATGTGTGTCACCTCTCTCATGGGCACTGCACTTTATAGCTTATAAAGTA GCACAGAGCCTGGTGGCAAATGCATACACCCTGGAGCCAGATCTGTGGTTCAAATCCTGCCTCTGTCCTC CATGGCCTTAGGCAAAACACTTAACCCTTTACTGCTTCCGTTTCCAGCCACAATGGAAGGAAGGATTAGC AGAACCATTAGCTGTTTTAAGATTCTTGGGTGAACTGGATGAACTCGGGCCTCATCGGTGTCTGGCTCAC CACAGGAGCTGGTTAGGTGGGGGCACTTATGAATACTCAGACCAAGGAGTTTGATCTTTCTGGGTGGGCT GTGGGGGGGGGGGACAATTGCTTTTGTGACAACCCCACAGTGTGCTGACAGAGATTCCATGCGACATCTA AAGACTGCTGCTCTGGGAGATTTGTACTCTAAGCCACCAGGATAGAAAATGGCTGAGGTGGGACTCAAAC TGAGGTTCTGTGATTTCAAAGTTATCTTGGAGCAGGAATAGAGGTTTGTAGATAGTCTCTCCCTGGGACC CCTGAGAGCAGCTCAGGGTCCCATGATGAACTCAGATAGGCAGCTCTGCTGTTGGTGAACCAGGGTGGTG CTCAGCCGGGCTTTGCTGGTTGTCTCCCTGGATCCCTCAGTTTCTCCTTACTCCCTTGTTTACCTAGAAA AGCAGGGCACCTGCCCCAGCGTGGACATACCCAAGCTCGGCCTCTGTGAGGACCAGTGTCAGGTGGACAG CCAGTGTTCTGGCAACATGAAATGCTGCCGCAATGGATGTGGGAAGATGGCCTGCACCACACCCAAATTC TGAGGTAGGTGGGAACAGGAGAGAAGGTTCAGAAACGCCTAGGTATGGGGCCCTTACAGTAAGTTGAAAA GGAAGAGGCTGATAACAGACCTGTCCACCCATTTCATTAATTTAACTACTATTTATCTACACTATGCGGA TAATGTGCAAGCCGACGAAGCAGACTAGAGAGGCTGGCTGCTAGGTTTTCTTGTCTAGTGGTGAGGAGAG AAAGAAGCAAGCTGTTTCTATAGCGTGTCACAGGAAGGTGCATGCTGTGTGACAGGAGAACAGGAGCGAA GTGTACCCGGGGCAGGGGGACGAAAGGGGCATCACTGTAACTTGTACGTGGGACAGTGGGTGAAGCCTCA CCAAGAAGGCGTGAAACAAAAGAAGGAATGTGAGCACACTCAGGCAGAGGAGGCAGCGATCGAGATGAAG TCAAGAGCATTGCTGGGGCATTTAAGGAAGAACAGGAAGGCTGGCTGGATAGATGTGTAAAAGGAAATCA AAGGTATATGGGGCCTGGGACAGTTATCTTAGAGCCTTAAAGGTCATTGTCTCGAGGCACACCCAGAGAG GTGAGTCCTTAGAAAGACACTCGTCCCAATGTTTCCAAAGGACTCTCCTAGAGTATGCTACCATGTAGCT AGACTGTAGAGGACAGGAGAAGATTCCCAGACTCACCAGAGAACGGACAGATGAGAGGGTCCTCAGGATG GAGGCAGGGAAGAGCAGTGGCCAGTTCACCATTTCATGATGAACAATGGGGGTTTGCTGATAAGTATCAG AACTTCCTTTTTGTTTAACAGGACTTGAGTAAGTTACGATGTGGTAGTGGGTAGCTGTGTCCTAGGGGAG GGGACAATGGCTGGTGGAGTTGAGGAGATGGTATCCCTTTGCAAGGTTAGTGAGAGTCAGAAGGGTCGAT CTGCATTTCCTCCAAAGGCCCACCTTTGTGGACAAATGTGGTCAAGGTCGCTAATGTTGGTTGCATTTAT TAGTTGTGTCTGATAATGGGCTAAGTATTTAATCTGAGATAGCTAGGACTAAATTCAGTACCTTTAGTGT CTCAGGCACTAAAAGGAGAAATTTGTGAGTCACCCTTCACTTTGTATGTGATTAAACTAAATACTAAAAT ACTGTTAATTGGGCTAGAGAGGTGATGTGGGTGCTTAAAAACACTTACTGCTCTTGCAGAGAACATAGGT TTGGTTCCCAGTACCCACCCAGTGGCTGACAACCATCTCTAACTCCCGTTGAGGGGCTCAGATGCCCTCT GCTGGTTTCCAAGGGTACTGCATACACATGAGGTACCCACAGATACACACAGGAAAAACATACATTCACT GAAAATAACCACAGAATAGGAATTATGCCACACGGCAAGTGTTAAAAAGGGAGAAACTGAGACAGATACT TGGTTGTCCCATTAGGTGATCCTGCAGTGTGATTGTCTGAGTTCCTAGGACAGAAAGGTGACCATTCTTC TCCCCTCCCCCCCAGCTTGCAGTGCAGTGATTGGTGACACGGTCTGTGTCATCAATGAGTGCATCAGTGC CCACTATTGTGGGAAACAGATGAGGAAATGTCACCCAGGCTTGAGCGAGGGAGGATGGAGTAGAAGCAGC CAGAGAAAGCTTCCCGGGGGGGGGGGGGTTATCGTGAGCTGGCATCAAAGTGTGAGGCAATGATTGTTGG TTTGGTGAGATTCAGGGCGGGGATGCTGTCTGAGGCTGAGGGAGCAGGTTGAGCAAGAACTTGGACAAGC CTTTGGTTGCAAACACAGAGTGCTGGACTCCCTTAGTGCACACAGAGATAACTGAAACCCAAAGAGGGGG AGCGTGCCCAAGGCCTCATGCCTGTTATTTCATGCCTGATATGCTTTTCAGTGGGTAGGAGAGAGCTATA GTTTTCCTGAACTCTGCTATGAGGCTAATGGTCTCTTATCTCCTTCAATGCCAAACTGCACAGTGGGCAT TGCAAGTAGGATGACCACAATGCAGACTCCAGCTCTGACCCTTACCTAGCCTCTTTATCTCCTTGCCCAC AGTTATGTGCTTTCGGTATGGGGATAAAGAGCATCTATTTGGTCAATTTAACTCAGCTCAATCTAAGGAA TGAAGGCATGGTGGGGCTTTTTGGTGATAGAAGAAAGATTCTTAGGGAAATAAATATTTATGGGTCTCCA GAGAGGCCACAACGGTCATTAGGACCATTGTATATCCAAGTAGGTCCTGGAACAGGTTTGATTTGAAGCA TAAGAGGCAAGCCTGGAACTTGCTGGGAGATGCAGACAGAGGCCACAAGAAGGTAGTGCTTTGCTGTGTG TGCAAGTGTGACAGGCTTGGTGGGAGCTGGGTGACCCTTGGGTCTAGTATGACATGTTATTGATGACAGC GCCATGGTCTCTCCCTCCAGCTTCAGCCTCCAGCAGCCTGAGGAATGGAGAGAGGTTGTTTCTGCCGGAC TGTGCATCTGGAGTCGTTCCTGTGGCCTCCTTTCTCTCTGGTCTTTGCATTTCTTCCTGGTCCGATGAAA GCATCTCCTTTTTCTAACCAATAAAGTGATCGCTTTCAGCAATGGAGAAGCTATAA Exon1:1-90 Intron1: 91-593 Exon2: 594-737 Intron2: 738-1342 Exon3: 1343-1498 Intron3: 1499-3077 Exon4: 3078-3224 Intron4: 3225-5620 Exon5: 5621-5796 loxP insertion site 942-1012 in intron2 3375-3500 in intron 4 Cassette insertion site (The cassette refers to the sequence from the 5' FRT sequence (blue) to the IRES-LacZ-pA sequence, the loxP sequence (red), the hBacP-neo-pA sequence, the 3' FRT sequence (blue), and the loxP sequence in intron 2 in Figure 1C.) 942-1012 in intron2 SEQ ID NO: 2: Nucleotide sequence of rat (scientific name: Rattus norvegicus) Wfdc2 (NCBI (Gene ID: 286888)) TTGGCCACTTAATCCACAGCATCTTGCCTCCCTGCAGCCTTGCCATTTCCGCCTTAATCCTGGACTCTAG TCATTCCCGGGAGGACAACGGACTCATTTAAAAATGAGCCCAGGGACCCTGTATCTGGGCCTTCCACTCC AGCTGCCCTGCCTTGCCCTCAGGCCCTAGAAAGGTAGGAAGAGAGGGAGGGGTGGACAAGTGTCCGGCTG CAGCTGGCTTGGAAGCTGTCTCGGTTCCTGGTTCCCACCTGAGCCAAGAGTGACACCTGATCCCGGGGGA TTGTGAAATGGCTTGAGGTGGCAGGGCGCCCGCACTGCCCGCTGTGCTTCCTACCCGCCTCCTGCACCCC TCACTCGGCCCCGCCCCCGGCGCGAGTGGTTAAATCCCCCCCATTCAGGCTGCAGGTTACACCTGCACC ATGCCTGCTTGTCGCCTCTGCTTGCTGGCCACAGGCCTCCTACTCGGGCTGCTGCTGTTCACCCCCCTCT CAGCCACAGGTGAGTGCAGCCAGGAGTTCAGTCTCAGAAGTGGGGGTTCTGGGACAAAAGTGGGAGGCCA GCGAAAATTTAAATTTTGGATCTCTGGGGCCTACGGCTAGTGATCCTTGAAGCTTAGGGTCTGGGGTCCA GTGTACTGGAGGTGGCGAGACCAATGAAGGCTTAAAAAGTTGGAGGGGCGGAGGCGCTCTCAGCGCTGAA AGCAGAGACCGGATGAAGAGGAGCGGGAAGCTCCTTGGATGGCTGAAGTTTCCAGGCAGAAGCTCAAGTC CCCAGAGGACTGGGGACTCTGAAGCCAAGCATTAGGGATCTCTCTAGTGTCCGAGACCCCTGGCCCAGAG ATGGGGCCCTCTGGGGTGTTATGGGGGCCCTTGGGCCCGATAATCTAAAATCCTTGGGGTTGAAATTTGG GTATCTTGGAGAGGGGAGTGAGTATGCCGGTGGGACTGGGTCCCGTTCTCCCTGAAAATTCTCTGCTGCC ACCCATTCCAAGGCACCAGAGCAGAGAAACCCGGTGTGTGCCCCCAGCTCGAACCAATTACTGACTGTGT GAAGGCGTGCATCTTGGACAACGACTGCCAGGACAACTACAAGTGCTGCCAGGCCGGCTGCGGCTCTGTC TGCTCCAAGCCTAATGGTAGCCCCACCGCAACCTAGCGGGGACAGGGCGGGGCTGCGAGAGGCGGGAGTG CCCGGGTTTGACGAAGATGGGTATCCCAGACCCAGGGACCCCCGAGAAGCGGTGGAAACCTGATCCACAT GTGCCCTCCTTTGTCTGCCCTGGGGTAGATGCGAAGGACAAAGGCGTCACAGAGGCGCGGCCCAGGAGGA GGAGGTCCCCCTCGTTCCTGGCTGGACTCGATTCTCTGGTGCATGACGGGCTTCCGGGCATGCACAGTGA GACTTTGTTCCGGGACCTGCGAGCAGTGGCTTCCTAGGCTGGAATCCAGAGGGCAAACCCTGGGTGGCAT CAGGGCTAAGGACTTCTTGCTTATGGGAGTCCCTCTCGCAGGGTCTTCCACGGCCTGGCAAGATTTGTTC CCGAGGGAGGGAAAATGTTTGCCACATCCTAAGTCCGTGGAAGTGAGCCTCCAAAAGAAACGTTCTTGCA CAAAGTTACACTGCCATCCTGTGGCTGGTAGAGGTTCCCTTTCTTCCTTTGACACTGGATTTCCGTCTTG GATCTTTATTTCTACCCACTTCAGGGCTGAGCGAAGGAAAACTCTCCAGGACCGCGACGGGTACAACGAC TCTATCAGCCGGCCTTGCCCGCACATCTCCTCTATCGAGAGGTCAAGTGTCCACGAAGCCACCCGTTGTG ACCAAAGAAGGAGGTAACGGAGGTATGGCACCTAAATTTCTGATTTCCAGGATGGAAAAGCTGGGGTTAG GTGGTGGGGGTGGGGGCAAGCCGGGAACAATAACCAACGTTTGTCATTTCTGCTCTTTTTCTGGGCTACA GCCCGCTCCCCTCATTCTACCGTTACTATTTTCCCCAAAGAGCCAGGAGAGGCTGGGAGCATGTATAGTT AGGAGCCCTAAGGCGCATGAACAGGAAGATGGGAGGGGTAGGCGAGGCTTGCCCATGGAGGGCAGATTTT GGCTGTGCGGTGCTATGCATGGGCCTGGAAAAGAAGGGACCAGGAACGATGGATGTGTGCCAGCTGGCAT CGAGCTGTCAGGTAGATAAAGATATTGTTTCCTTGTGTCAGATGGACAGACCAGACGGTCTCCTGAGCCA GGGAGGAGCCTGGAGGCTTGAATCCTCTCAAGTGTGAGAGCCTGAATCATTTGCTCTTCGAAACTCTGCC TGTTCGTCTGTAAAGTGGGCATGCTAACGCCCATCTGTAGATGTGGGGGTTAAGTGCAAAGGTACTCAAA AGAGCGGGCGTTCTGTACAAGATGGCTTGCTTCGGTACTACCTCCTCTCCTGGTTATTCTCATTTTGTCT CAGGGTAGCCACCAAGAGCACCTTGGCTAGCCAAGCCAGGACACTGGCAACTGGTCCTTTACCTGCCTAC CCACTTCACTGTCTGCATCTTTCCTTTCACCAGGCCTGGTTCCTGCCTGTTCTATTGAATGCAACATTGT GTGTGTGGTTGGTGCATGGGGTGAGGTGTGTGTATGGTGTGTGGTGTATGGTGGTGGTTTATATATATAC ATGGGTGATGTATAGCATATGTATGGAGGGCCATGGCTTTGAGTGAGTGTAACTGGCAGTGAACCTATGG GAACAATTGTGGGGAATGTGTGTCGCCTCTCACATGTATGCTTCACTTTACAGCTTATACAGTAGCATAG AGCCTGGTGGCAAATGCATACACCCTGGAGCCAGATCTCCTTCGGTTCAAATCCTGCCTCAGTCCTCCAT GGCCTTAGGCAAAACACTTATCCCTCTACTGCCTCAGTTTCCAAGCCTGCAGCAGTAAGGAAATGTTAGC AGAACCATTCGCTTCTTTTAAGATTCTTGTGCAAACTGGATAAACTCGTGCCTGAACAATGCCTGGTTCA CTGCAGGAACTAATTAGGTGGCAGTGCTTACAGTTACTGTTACCAAGTAGTTTGATCTTTCTGGGTGGTG TATGTGGGGGACAGTTGCTTATGTAACAACCCCACAGTGTGGTGACGGGGATTCATTCCATGTGACATCT AAAGACTGGTGCTCTGGGAGATTTGGGCTCTAGGCCACAAGGCTAGAAAATGGCAGGGCTTGGGACTCAA ACTGGGTTCTGTGATTTCAAGTTATCTTGGAGCAGGCATTGAGGTTTGCAGAGAATCTCCCCAGGACCCC TGAGAGCAGCTCACGTTCCCGTGATGAACTCAGAGAGGCAGCTCTACGGCTGATGACCCAGGGCTCCGCT CAACCAGGCTTTGCTGATCCCCTCAGCTTCCCTCCCCTTTCCCTTGTCTACCTAGAAAAGCAGGGCACCT GCCCCAGCGTGGACTTCCCCAAGCTTGGCCTCTGTGAGGACCAGTGCCAGATGGACAGCCAGTGTTCTGG CAACATGAAATGCTGCCGAAATGGCTGTGGGAAGATGGGCTGCACCACACCCAAATTCTGAGGTAGGTGG GGGCGGGAGAGAAGGTTCAGAAACGTCTGGTATGAGGGTCCTTACAGAGACTGGAAAACACCGTGATAAT AGATCTGTTTACACACGTTCATCCACTTAGCACTATGTACCTACAGTGTATGGATAATGTGTGAGGACCA ATGACGCGGACAAGCGAGGCTGGCTGGTAGGTTTCCCTGTCTAGTGGTAAGGGAAGAAAGGAACAAGCTG ATTCTGAAGCGTGTCACAGGAAGGTAGCAGATGTCATGGGACAGGAGAACAGGAGTGACGTGTACCCCGG GGGAGGGGTGGAAAGGGGCATCACTGTAACTTTTACGTGGGACAGTGGGTGAAGGCTCACCAAGAAGGCG TGAAGTGAGAGGAGGAACGTGAGCACGCTCAGGAGAGGAGACAGCGATAGGAAGGCAAGAACACCACTGG GGCCTTTAAGAAAGCACACGAAGACTGAATAGACGTGTGAAGGGAAGTCAAAAGGTATCTGGGGGACTGA CACAGGTCATCTTAGGGCCCTAGAGGTCATTGTCTTGACTCAGAGGCGTGTCCTCAGAAAGACACTCGCC CCAACATCTCCAAAGGACTCTCCCACAGTATGCTGCCAGGCAACTAGACTGTAGAGGTCAGGAGGAGATT CACAGACTTGCCAGTGAGTGGACAGATGAGAGAACTCTGCCCAAGATGGAGACAGAGAGAACAGTGGCCA GTTCGCCATTTCATGAAGAACAGTGGGATTTGCTGATGAGTATCTGAATTCTCTTTTTGTTTAACAGGAA CTTCAGTAGGATTTGGAAGGCAGTAGCTGTGTCCTAGGAGAGGGGACAATGGGCTGGTCGGGTTGAGGAG ATGGTCTCCTTTTGCATGCTTAGTGAGAGACAGAAGGGTTGATCTCCATTTATTCCAAAGGCCGGCTTTT GTGGGCAAATGTTATCAAGGTTGCTAACGTTGCTAGCATTTATTAGTTGCGTCTGAAATGGGCGAAGTAT TTAATCTGAGCCAGCCAGGACTAAATTCAGTACCTTTAGTGCATCAGGCACTGAAAAGAGAAATTTGTAG AGTTACTCTTCCCTTGTGTGTGATTAAAACTAAAATACTATTAAGCTGGGGCTGGAGAGGCGATGTGGGT GTTTAAGAACACTTACTGCTCTTGCAGAGGACAGAGGTTTGGTTTTCAGTACCCATCTGGTGGCTGACAG CCATCTATAACTCCAGTTGAGGGGGCTCAGATGCCCCCTTCTGGCTTCCACGGGCACTGCACACAAATGA GGTGCACACAGATGTGCACAGGAAAAACGCACACTCGCTTACAATAAACACTTAATACGGCATTGTGCCA CACAATGAGTGTTAAAAAGGGAGAAACTGAGACAGATACTTGGTTGTCCCATTAGGTGATCCGGCAGTGT GGCGGTCTGAGATTCTAGGACAGAAAGGGGACCGCTCCCCTGAGATTGCAGTGCAGTGATCGATGACATG GTCTGTGTCACCCATCAGTGCCCACTACTGTGGGAAACGGATGAGAAAATGTTGCTCAGGCTTGAGGGAG GGAGTGAGGATGGAGTAGGAATAGCCAGAGAAAGCCTTGGGGTGTGTGTGTGTGTGTGTGTGTGTGTGTG TGTGTGTGTGTGGTCCTGACCTAGGACGAGGCAATGATTGGTGGTTTGGTGAGATTCGGGGTGGGGATGC TATCTGACGCTGAGGGAGCAGGATGAGCAAGAAGTTGGATAAGCCTTTGGTTGCAGACCCAGCTTGCTGG GCTCCCTTAGTGCACATAGAGAAAACGGAAACCCAAAGAGGGGGAGTGCGGCCCAAGGCTTGCTGTTTCA AAGTATGCTTTTCAGTGGGTAGGAGAGTGCTACAGTCTTCCTAGGACCCTGAACTCTGCTTCAATGCCAT GGTGGGCATTGCAAAAAGGATGACCACAGTTCAGACTTCAGCTCTGACCCCTATCTGGCCTCTTCGTCTC CTTGTCCACAATTATGTGCTTTTAGGATGGGGATAAATATTTGGTCAACTTAACTCAACTTGATCTAAGG AGTGAAGGCATAGTGAGGCTTTTTTGGTGATAGAAGGAAGAGTCTTAGGGAAATAAATGTTTACGGGTCT CTAGAGAGGCCATAACAGTCATGTGGGACCATGGTAGGTCCTGGAACAGGTTTGGTCAGAAGCACAAGAG GCAAGCCTGGAACTTGCTGGTAAATACAGGCAGAGCCCCCAAGAAGGTAGTGTTTTGCTGTGTGTGCAAG TGTGATGGGATAGGTGGGAGCTGGGTAACAGTTAGGTCTGCTATGACAGATGGTATTGATGACAGTGCCA TGGTCTCTCCCTTCAGCTTCAGCCACCAGCAGCCTGAGGAATGGAGAGAGGTTGCTTCTGCTGGACCATG CATTCTGGTGTCGTTCCTGTGGCCTCCTCTCTCTCTGGCCTTTGCATTTCGTCCTGGTCCCACAAAAGCA TCTCCTTTTTCTAACCAATAAAGCGATCACTTTCAGCAATGGAGAAGCTATAACACCTGCTCTCCTCACT TTCTTCTGTTTCTCTTGACTTGGGCTAGGTGGGTGGGACAGTACCGGACAACTGGGTCATCGAGGTACAA CATCCAGGGAGGGATTAGTTCTGAAGCTGAGCTGTAAACAATAATTCAGGTGTGTCGCTGCCATGTTAGC ATTTCTCCAGTGGGAGATAACACGGCCTCAGAGAGGTGTTGGAATGCTTCTGAAATCACTCAGACCCACT GCTCTGTATGTTGCATGTCTATGTGCCAGCCATAGTGCTGGGAGACACACTCATGAGGTTTCCTGACTAC TCGGAGCTCTCAGAGTAAGTAATTCATAGCAATCCAGGGGTCTGGTGAGAAGGCTGTCAGCTCAGTGCCC ACTGTACAAGCAAGAGGACATGAGTTTGATCCCCAACCAGCACACGTGTATGAAACAACTAAGTGCAGCC TGGAGCTGGTGCAGGAGACAGGTTAATGCCCACATCTTCTTGGCCAGCCAGGCTAGCCAGACAGCAAGTA CCAGCAAGGTTCAGAAAGAGACAACTTGCAGGAATTGGCTCTCTCTCTCTCTTTTCATTATGTGGATGTT CAGGGATCATGGTCAGGTGGTCAGGATTGGTGGCAAGCAGCGTTACCCACTGAGCCATCTTGCTGGCCTT ACTCTGTGGTTTAGAAAGAAGTCCTTGCCTATTTCTCTGTCTATGCATCCCTGAAATAATGATGGGCCAG CTCCACATCGTCGTCCTCTCCCTTCAGAGGGTAATAAACACTGCTCAAGGCAAACTAAAGGACCCTGGAA CCACAGGTTCTTCAGTTCTCTTCCCGTGGGACAGAAAGACGTGGTCTGACTTTAGTAATAGGTAAAAAAA AATTTCCCTATTTTTAAAATTAAAAGTGGTCTTAAAATAATTAGTTTTTTCCCCCCATGTCCACTCCCTC TTTCTTCCTGTTTCTGTTTACGGTGGAGCCTGACACAAGCATCTCTCACATTGAGGGTCATATTTTTTTA ACTTCGGTGTAGACTTCCAGCTCACCCCCTCCCTTTGAAATTTTTCTCCTCAGGTAGGTGAAATGAGTAA TTCATTCTAAGACCTCGTGAGTGGCACTTCTAAATTTTCCCAATGTACTTCAGTATGCACACTCTGTAAG GAAGGGCTTTTAAACAGGATAACAACATTTATTGATTCCGGTAGACATAATCTTAGGGAAACGTATTT AGAGAAATGCACTTTTTATTCACAAGCAAAAGCCTTCAGGGGTCCCTTAGGGAGCTTGACTCGGTGGAG GGCTGGGATGGCTTGACAAGTCAGGTGCTTGCGCAGACTGTCTGGCACTGGTTTCTGCTCAGAAAGTTGT TCTCGTTGCCTCTGCAGCCTCCATAGTGGAACCAGTCACACTTCTTAGTTTTGGTGTCATAGTACCATCT CACAAAGATGGGCGCACATTCGCCTTTTTCCATGGGAAGTGTGCACATAGACGGTAAGGACTTCCTGGCT ACTTGAAGAGGAAGGTGGTTAAGGAGACCAAATCCATCGCCGTCCCCTCGCTACTCTCTCCTCTACTGG AGGTTTCCATGGCAACAAGGTGCAAGGCTGAAGTCACCCGTGACAGCCATACCTTCCCACCCAGCCTGGT CCATCTCCACAGAAGCCAAGGTTTGTGCTTCTGTAAGTAGCGAAACTCTGTTGCCTAGGATTCGACTCAG GTGGATTCACATCTGGCTCTGCTACCCCACTACCGAACGAGACTGTGGAGTGGTCCCTTCACTTCTTGAAG TCTCCAGTTGGTTGTCTGTGACACAAGGAACTGAGGGCTATATCTCCTTTGGCCCCTCAGGTTTGATTTT CCACAGTCGGACCCTAAAGGCTTCTGCCTCCCACCGCACGGTCTGTTCCAGAATGGACAGACTGCAAAGA AAGGGTGAGCCTAGGTTCTGAGAGCAGGGTTTCAGGTCTGATTCAGTCATTTGCTAGCTGTCTAACCTTG AACAAAGGGCCTATCTCTTCTGTGTTGCGTTTTCCTCACTGTAAAATGGAGTCTAATATTCACACCTTCG GGCTAATTGAGAGTTTAAAGACAACTCAGGAAGGCTGGGTGTGCTGGAACACACTTCTAATCCCAGCATT CAGGAGAGGCAGAGGCAGGAGGATCTCTGGGTTAGGGGCCAGCCTGGTCTACATAGAGAGTGCCAGGTCA GGCAAAGCTACGCAGTGAGACCCCTCTCAACAAAACACCACCACCACTGCAACAAACAGGGAACAAAAAC AAAACAAACAAACAAAAACAACAACCAAGAAGTCAAAAGTCAGTGGATGACTGTGAACTACCAGTAAGAT CCTTTCCTTTTCAAGTAATGAGGCCCAGCAACGAAAACCTTGCCTACCAACCGGCAGCCCTAACCCCAAC ACAGGCCAGCCTCTGCGTCCTCAGCAAACTCTCAACAAAGAATTTGTCTTCGCAATTACTATTTGCCATT CTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTTTCTCTCTCTCTTT CTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTGTGTGTGTGTGTGTGTGCGCGCGCGCGCGCGT GTGTGTGTAGGGGGGAGACTGGCATGTTCCCAAGACATCTCGGTCACAGTAGCATCCTCTTTAACCCCAG GAGACTCCACAGGAGGAGTCCCACCAGTCTTGGTTTTCCTCTTCATGGACTCTATTAACACCAAGAGGAA ATCACTTAATCATTTACTTTTGTATGTAATAGAGATGACCTTCTCCCAGTGTGTGTGTGTGTGTGTGTGT GTGTGTGTGTGTGTGTGTGTGTGTGTTTTAAATGTGGCTAAAGTCTTTCATGAGGAACCATTGAGAACTG TTAGACTAACACATGTATACACATCAGATTTGACCAATAAAGCCGACACAACCATTACTCTCAGTCTTTT AGAGGAAGAAACTGACATTCTGGGAGACCAAACAACACGCCAGAGGTGCACACACACACACACACACACA CACACACACACACACACACACACACACACACGATGGGGTCAACCTCTGAACTCTGATGCTACTGACTTGG GAGCCTGGGGCTTGTGCATGCAGTGCTACCCACCGAGTTATTTCCCAGCTCTGACTGTCTGTAAAGGGAA TTGTACTGTTCACGACCACACCACACTCATTTCCCCACAGCCACCTTTTAAGCTGCAGCAAGCAGAGCCG TGGCCCAGGAAGGAGCCTGGGTCTGTAGAGAGGTCCACTGAGCTAATTCTCTCTATATCCAATGTGAGCT TACATATCTCCACATAGTCTTAACTTTATCCATCACAAATCCAAACTTTAAAGCCTGCTTCCCTACAAAG CCTTTTCTTTTTCCTTCAGTCTTTCACAGTCTACTATGCTATCACAGATGCTATCTGCCTGTCTTCCCCC TCCCCCCTCTACCCCCACCCCCCGCCACCATTAGAGTTTAAACACTGTAAAGGCAGAGGCTTTGCCTGGT GACTGTCACTGAGCTCCTAGAAAGGTGCCTGGCACCCAGCAGATACCTGACCTCGTCTGTCGATGACTTG AACTGGCTCCCTCTCGTGCTAGATCAGAGCCAGACTCTGACTGCAGGGTCTCTACTGGATTCCTGCTCTA CACTGAGACCTCCATTCCATACCCACCTTGTCTTGGTTCTGAGCAAAGCTCTTGGCAGAAGGTAAGGATC AGGAAAAAGGAGAAGGAGGCCTGGAGCTGCATGGTGTCTGCCGGCGGTGGCCCAGCCTGGGACTGTCTTA TTTAGAGAGAAAGGAGACTAAGAGGAGGGGCTGACCCTGAATCCTGCTCACTCGCCCCTAACTGCTGCTG CACACTGGGCTGGCTGCTCCTGGAACAGAGAGTCCCCGGCAAAGCACAGTCCTGTCCCTCCCAGACATAT CTAATCTGGCCTTCAAAGCCAACACACCACCATTTCAGTATAGAAGCCCTTTGAATAGGAACGGGACAGC AGTTACTACAGGCTGTGTGCCTTCCTGCTTCCAGGCGCCACACAAAACACACTTCACACACTGTTTGAAG TTTACACAGGAACCCCGGAAGATTTGGATATGGTGTGAGTGTGTCAACAAGGGGTCCACACCTTGGAAAC TTGATCCCCAAGGCGGCACTGTAGAGAGACGGTCGAACCTTCGGCAGATAGAACTTAGGGCAAGGTACTC TGGAGATGCTGTTCTTGAAAGAGACTAAGGTTCTCATGAGACCCCAGTTAGTTCCCCTGAGAGTGAGTTG GTATGAAAAGAGGAAGCCATGGGGCTGAAGGGGTGGCTCAGCAGTTAAGAGCATAGACGGTTTGGGGTTC TGTCGTGAAGTTTTTATTTTTATGTGCATGAGTGTTTTTCCTGCATGCATGCCTGCAGCACATGCCTGCC TGGTTAACTCATGGAAGTCAGGCTGGATCCCCTAGGACTGGAGTTACAGATGGTGCCATGTGGGTGCTGG GAATTGAACCTAGATCCTCTGGAAGAGCAGCTAGTGCTTTTGACCACTGATTCATCTCTCCAGCCCCCAT GAGCGGATGCTGTTCTTGCGGAAGACCTGACTTGGGTTCTTAGCACCCAAGTTGGGTGGTTGATTGGGTG GGGTGGGGTTCTCATTGAAAGTCAGGCTGATGAGCCACCTCCTCTTGGACTTTCACCTTCCAATATTTTT TTGAACTAAGTAAACCTCTTTTTAAAATAAGGCATCGCGCTTGCCTAGGAAGCGCAAGGCCCTGGGTTCG GTTCCCAGCTCCGAAAAAAAGAACCAAAAAAAAAAAAAAAAAAAAGAACTAAAATAAGGCATCCAAGTCT TAGGCATTTTGTTATAGCAACAGAAAATATACAGAAGCACTCCATTTTTGTAACGAAGCAAGAAGATTAA ACTACTGTGACCACAGTCATGTGGAGAGCAAGTCTCGGAGCCACGGCTGTGTCCCTGTTCTGTTCACTCT ACTGATTCATGCCAGTCATTGACTACAGAGACCATCTCAAGTCTCTTACACTGGGCTGACTCCTGTCAGG AGCTGTCTACACACCTGGGTGCTCCCAGGGAATTATGGGAACAACACGCACTTGGTCTAAAAGTTAGAAA CTGTTGGAGAAGGGGCAAATTCAGTGTATTCAAGCAGAAATAACAAAAGCTCAGTATAAAAACGAGCTGT GGTTTATGCTTGCAATCCTGGTACTTGGTACCAGGATCACAAGTTCAAGGTCACCCTCTGCTAAGTTGTA GGTTAAAGGTCAGCCTGGGCAATATAAAACCCTGTTTGTTTTGGTTTTCTTTTTCTTTCTTTCTTTTTGT TTTTTAAAGAAAAAGATTCCAAACCCAACAGTGTATCCTTTTGTTAAAAAAAAGTTTATTGTTTTTAATT TTGTGTGTATGCGTGTGTTGGGGGACATGTACACATGAATGTGGGTGTCTGGGGAGGCATCGGTTCTCCT GGAGTTACATGTGGTTGTGAGACTCTAACGTGAAGGCTAGAAACTGAACTCAGGTCCTGTGTAAACGCTG TGCGTGCTCTGAACTGCCAGTCCTCCTCCAGCTCCTAAACTTTAGGGAAAATGTCTTTTTGGGTCTTTGC TCATTTTTCCTCTCCTTTCCTCCACTATTTTTTTTTTTTTTTGGTTCTTTTTTTCGGAGCTGGGGACCGA ACCCAGGGCCTTGAGCTTCCTAGATAAGCGCTCTACCACTGAGCCCCCCTCCACTATTTTTTATTCCCTT TATTTTGTGGCATGGACAATTAAATCCAGGTCTCCTGCAGTTTGCCGCCTGAATTATATTCTCAGCCGCA TCTTTTGTGAATCTCTTTACTTCATTTATTTTTGTGTTATTGAAATTCAGGAGTTCTAAATATATCATAC TAATAGATATTATTGATATAAGTTATTGTCTTTATTCATATTTTGGGTAGGGTATAAGCCAGGCTGGCCT GGAACTCCAAGTTCTGCCTACCTCTGCCTCCTGAGTCCGTGGTCAAAGGTCTGTGCCACTATGTCTGGCT ATCGTTTTATTATTTATTTATTTTATTTATTATTTTATTATTTGTCATATATTAATATTTTAATACACAA TTATATTAAACTTATTTTAATGTTTAAGGTTAGTTTAAATAATAAATATTTTATTACTTTTTTTTAATTTA TTTATTTTTAGGCAGGGTATTACTATCTGGCCTTGGCTGGCCTGGAACTTGCTCTGTAGACCATGCTGGC CTCAATCTCACAGAGTTCTCTCTGCCTCCCAACTGCTGGGATTAAGTCTATGCCACTATACCTAGTTA TTTATTTATTTACTCTGAATACTTTGAAACCCATTAGAATTTTTTAAAATGTTGTTCTGGAGAGGGGAT GGATGGAGACAGGTATATATATATATATATATATATATATATATAGAGAGAGAGAGAGAGAGAGAGAGAG ATTTTTAAATATTTTTTTCTTCTTTTCGGAGCTGGGGACCGAACCCAGGGCCTTGCATTTGCTAGGCAAG CGCTCTACCACTGAGCTAAATCCCCAACCCCAATTTTTTAATATTTTTAAGGTGATCTTAACCACGGAGC CATCTCTCCAGCTCCTGTTGTCTTTTTTGAATTCAACATTATTATTTGACAACCTTTTTCCTCTTTAATCA ATTTGATTATATATTCTAATAATTTTGATAACCTCTTGTTTCCGATGAAAATGAAAACATTATTTCTTCC AAAATATTCCTGTGTAAACGCTCTCGGTGCTCTTAACTGCCAGTGAGCTTGCCATTAAAATTACGTAGTG ATTTCTCCATCTACACATTTTTATCCATCGCTAGCCCATCCCTCCATCCATTTATTATTTTATTTATAT TTAATTATTAATTAATTTATAATTAATTTCAACAGTATCTTTCTATATAGTTTTGGCTGTTGTGGAACTT GATCTGAGATGCACCTGTCTCTGCCTCCTGAGGGCTGAGATTAAGGGTTTGGGCTACCACACCCATCCTA GATTAAAAGAATGCATTCATTTGATTAGGAGGTTGAGAGAAGTAGGTGACTCTCCCTAGACATGAATCAA GTAGATCTGAGGGTAGCACACTTCTGACGTCTTCATACCCTTGTATCACAACACTGATTGTGTGTTCATT TTACCTGATAAAGTAACACACTCATGTAGATAATAAAGAAGAAATCAGGCTGGAGAGATGGCTCAGCGG TTAAGAGCGCTGTCTGCTCTTCCAGAGGTCCTGAGTTCAATTCCCAGCAACCACATGGTGGCTCCAACC ACTTATAATCAGGTCTGGTGCCCTCTTCTGGCCTGCAGGAATACACGCAGGCAGAAAGCTGTATGATGTA TACATAATAAATAAATTTATAAAAAAAAAAAAAAGAAGAAGAAATCAGAGGCAAAAGGAAGATATTGT GCATTTGGAAAAAAAAACAGTGAATTGTGTACATAGAACCCTAAGCTAGTTAGCATTTAACAGGAAAAGCA GATATGAAGGGTGGGTCTTCAGAGCAAAAAAAAATAATAAAAAGTCAGGGATCAGTCAAAGAATAAGTGT TGTGAATTAAAGGGGATCTGCTCTGCATTTGATAAAACTTACTGAGAGCATGTCAGAGTAGGGTGTGGTG GCATGTATCAATAACCACAGCACTCAGTGATTGAGTTGGGGGGGTGGTGTCCAAAGAGTTGAGGCCAGCC TAGGCTACACAGAGGATTTTAGAACAGTTTGAGCTAGGTAGTGAGATTCTTATGAAAAATTTCAAAATAT AACACTATTTGAGATTGGAGAGTGTGACTGATCACCTTATTTCCTTCTAGCTGCAAGGGAGGTGGGGAGA GTGAATGTACTGAGTACTTATTTCTTTCTAGCTGCAAGGGAGGCTGGGAGAGTGAATGTGCTGAGTACTT AGCTACTTTCTTTCATCAAGAAGCCCACCTTCTCTGTGGTTTTGGGAATGGGACTCTGTATGTTAAATGT CCCTACACAAGCTCTTTCTGGCATCTTTCAAACCTGGTTCCCTGTGTTTTTTCTTAGATACCATCTCTAA GTAAGCATACCTTTCCACCTAGTAATCCAAAAATCACTAGTATTCCCAGAGGTCTGAGTCCAGACAGGAC ACATTTTAGAAGACAGAGTCCAGGTATACATGTCCCACAGTCTATTCCTCCATGAGAGTCAGCTCCAGTA GTATTTAATGACCCCCATGCTTCCAAACCTAGAAGTAAGAAAAGCTCAACTTCCCCTTCTTCCTCCCACC CTCCAACACTCATGCAACTATAAACACCGTGTTTAAAATACCTTTTGCATGTCTGTGTTCTTTGCTTATT TTCTATGTGTGTATGGTATGTGTGTGTCACACATACTTGAGTGTTCTTAAGTTAAGTATTCTTGCATACA GTCCATGGCACACATGTAGAAGTGAGAGAATAACCTTGGGTTTTGGTCCTGACCTCCGACCTTGTTTGGG ACAGGGTTTCAATCTAGCTGGCCTACAAATTTCCAGGAATTCTCCCATTTCCACTTCCCATCTCTCCCAA GGAGTACTATGATTCCAGGTATGTATTAATATTTTCTACTTTATGTGGATTCTAGGGATTCGAACTGAGC TATCTTTTATCTCTCCAGCCCTGTTGTTTCTGTGTTCTTTACTGGATCCTGACAAACATAGACTAAAAGT CTGGCTTTTTCAGACCTAACACAAATGGTGGTTTTTGACTCTAAGCAAGACTCCTCCAGTAGAGAATTCT TCAACTCCAGGAAAGAGACTTTCATACTGAATATTAAAAGCAAAACAACAGATAGTTCTAAAGTCACCTG CATTTCCAAGGGTTCAGGATTTGGCAATACCTGACCACTCTTCAGTCCGGGGTTTTAGCTCGTTTTGCTA TGACATGCTGCTGTGTGGTTTTGGACAAGGCCATTCTTTATTAGAAGTTTGCATTTATACACACGCAGAA GCAAAGGAAGAGTGGTGAAGTTAGCCCAGATGCTCTGTTTCCAAATTACCCAGCTCCCCCATGCATTTCT GATTGTGGAGGTAGATCCGAGGGGAACATCAGGAGAGAAAGGTAAGTGGTCCTGCACCTTGGAACCAAGG ACTGTCCACCTAGGGTGTCTACTGGGATCTAACGAGGGAATAATGAAGAGATGAAGGCCATTCAGTTCCA CTGAGGTCCTGAGGTTCTCTGTGCACTGCCCTGAAAGCTCCAAGTTCATTCCTGAGCAGAGCTCGTGAGC AGATGTTATCAGCACCCAGCACACATCAGGCACTTCTAAGGCTCCCCTTGCTGCAGGGGCAGGAGCAGTT TGTTCCAGTCACTCATGTATGGCAAACCTGACCGAGGCTAAAGGGTGTAAAGATTCTGAGTGGTAGGTGT TTATCGGATCTGGCAGCATTCCTCCAATGCCTTGTTTTCTCTGAAGTAGAAATACTATTGACATCACTAG CCAATCGCCTTTCCTATTATGAATGGGAAACAGGCCCTGAGAAGGAGATCCCTCCACACTCTTCTGTCCCA GCTAGGGGTGTATACCGTGCTGACCTGGGTGTTGCTAGGGGGCAAGGATGAGGTTTAAATTTGGGGCTTCT GAATCTCATGCATGTGCCCTCTCAACACAATACCAACCCATCATAGCTTCTGGATTGTAAGGACACACAG AGCTGAGATAGGCTTCCTGGGAGAGTCAACCACGCACTGCTGAGAACAAAGAAACAAACACAGACGACAC ACCAGGGCTACACAGCCAGTAATGTTTCCCGGGCTGTGGCCACCACACACCCTGCATCACCATGACCCAG CCTTCTCCTCCCATCCTCGTCAGTGATTGAATAGTTCCTCCAATTTCTTCTGTAATAGGTCAGACATCAA AGACTATCTTTTTAAAAACAAGATGTGAGTGTTTTTCTTGTATGTCTGTGCACCATATATATGCAGTGTC CAAGAAGGCCAGAAGAAGGCGTTGATCTACGGAACAGAGTTACAGATGTTATAACTCACTATATGGGTGC TGGGAATTGAACTCGGATTCTCTGGAAGAGTGTGAGATCGTGTGTGCGTGAATGAGTGTGTGCATGGATG TGAGTGTGTGAGGGTAACTGTGTTCTGTATGTAAATGTATGTGTGAGAGTGTGTTCGTATAAGTGTGTACG TGTGTATGAATAGTGTGTGAGAGTGTGTGTGCATGAATGAGTGTATACATGGGTGTGAGTGCATGTGAGT GTGTTTATATGAGTGTATGTGTGAGAGAGACTGTGTGTTTGAGAAAGGAAAGAGAGGGGAGAGAGGGGAG GGGGAGGGGGAGAAGGAGAGGGAGAGGGAGGGAGGGAGTTTGTTTATTCATGTCTGCCACAGTGCATTTG TGGATCAAAGGGCAGCATTCTGAATTAGTTCTCTCTTCTACCTCGCTGAAGCAAAGGTCTCACCATTTCT GCTGTGTCGTGTTCTTTAGCCCAGCTGACCCATAAGCTTCTGAGTGATCCTCCTGTGTCCCCTCCCATCT CACCACAGGGGTGCTTGCAATACAGATGTGTGCTGCCACACCCGGCACCTTATGGGTCCTGTGGATCGAA CTGCACTCGTCATTCATTCTGTGGCAAATGCTTGGCTTCCAGCCATCCGGCTAGCCCTGTGTCCGTTATT TTAAAAGCATGCCCCATTTTCTAACACTCGCACCAAACATCCTTTCCTTGGAAACTATTTGGGCTTTAGC TGTAAATGTTTTTGTTGTTTAAACAGGTGTATTAAAACACTGTTTACATGAAAACGGCACAAATGCAAGA TGCATTTGCAATGTACCCGTACTGTCACCTCCTGAGTAGTCGACACGCCCTTCACTTTGCACTCTTTGCT TTTGGCGAGGACCCGTGAGATTTATCCTCTTAGCAAATTTCAAGGATAAAAACAAATTTCAAGGATAAAC GATAGTTATACTATTGTATATTAGAGCTCCAGAATTTATGTGTAATAAAACTTTGCAACTGTTCACCAGC GTCCCTCTGGAAATTTTTCTGCTCTGTTGCACTTGTGTCAGTTCTGCTCCAGATTCTACTCGGAGTGAAA GTGTGCAAAATTTGTCTTTCCGTGTCTGACTCGTTTCAGCAAGTGTTTCTTCCTTCTTTGTCAAGGCTGA GGAACACCCCAACCATCCACCGCCACTGCCTAGTTTGTCTGTCGATGGACGTTTAGGTTAGTTATATACC CTGACCCTGCAGTGGTCTTGGATCATCACCAAGCTCTCTTTCCAGGAATCCGTTTAGCTCATTCCTAGGA TGTTAATCCTTTCAAGGTGTCCTTTGTACACCTTGTGAGATAGGAGACCAGCCTTTTTCTTTTACTCCTA GATATTTATTGATTTTCTTTGTACAAACTACTGAAGAGTGGAACTTCTCTCTTTTGTGTGTCCTTTGGCA CTCGGTCATGAATCAGCTGTCCGAGAGGGCGTGTTCTCACGGCCAGAGAGGGCGTGGTTTTAGCAGTCGG AGAGGGCGTGGTTATAGCGGTCCGCTCTCTGTCTTAGTCCTTTAGTCAGTATCTGATTCATGTTAACAAT GCTAAAAACGATGCTATTTTTGCTGACTATGCTTTGGAATACAAGTTAAAGTCAAGAGATGTGATTCTGA CACATTCCTTCTTTTTTTTCTAGATGTGGCTTTGGAGATTCGGAGTCTTTAATGGATCTGTGTGAGTTTT AGGAAGCTTTTCCTTTTTTCAGTTAAAGTGTTTTTATGGTGTGGAATGTAGAGGTGGTTGAAAGTAGTGT GGACACTTTGGCCATGAATTCTTTTTTTTTTTTTTTTCTTTTTTTGGGAGCTGGGGACCGAACCCAGGGC CTTGCGCTTGCTAGGCAAGTGCTCTACCGGTGAGCTAAGTCCCCAACCCCGAATTCTTTTTATTTATGAA CCTAGGATATTTTTCATCTAGATGGTTTTTGTTTTATGAATTTCCTTAATCAATACTTCATGACTTTTGG TACATACATATTTTACCTTTTGGATAATTTGAAGAACTTCGTTTTTTTTTTCTGTTACTTATGGAAATAT GATTGTTTTCTAGATTTCCATTTTGTGTGTAACTTTATTAGTATGTGAATAACGACAGACTTTTAGATAT TTAATTTTTAACCGTAAACTTTAATGAATATACTATTCTGACTCTGTGTGTATGTGTAATCCTTTATAGC TTGCCTCTATATAACATGCAAATATGTATACAGAGGGATATGAACATATGTGAACATCTGCATACAGGTA TGAAGTGGCAATCAGAAGACACGGTTGCATTGAGCCCAGCACTCACGCTTTTTCTGGTTTCCCTGTTTGC CAGAGGATGTGGAAAGGATCCCATCCCCCTTCCACTGGCTGAACGATCTGTTTCTAGGGTGACTGGAGC CGAGCGTTATCCTTGAAATACACAGGACAATGGACAGTTGTGTACATAGAGAACTGTGAGCTGATATGAA CACCTGCAGACAGCAGATGAGATATTCAGACTCTGAGACATCAGGACTCACAGGATCCTTACTAGGAGG AAGTAGAGTCTACACAGGAATAAATTTGAACCCTGAAAGTTGAGGTGATAGGGTCCCCAGACGGAGAGGGG GTCAAAGGTCGAAGATCATGATGTCTGAGGAGCAGCTGGGTTTGTGGGAAGGGAAGGGAATAAGCAGATTCTGG GTTCTGGTTTGGGAACAGCCAAAGTCTCAGTGGCTAAGACTTGGCCGCTGGCAGCCAATCCAGAGCATGT GCTGGGCTCACGGGAAGAAAGCCTCAGATTAGGAGCCAAGACCTGGAGTTTCTTCCAGCTCATCCTTGTC ATATCCAGGAAGACATTTCTGCAAGACACAAGGACAAGGATGGACACGCTCGGCTCCAGTCACCCTAGGA AACAGATCGTTCAGCCAGTGGAAGGGGGATGGGTTCCTTTCCACATCCTCTGGCAAACAGGGAAACCAGA AAAAGTGTGAGTGCTGGGCTCAATGCAACCATGTCTTCTGATTGCCACTTCATACCTGTTGTTTCAGTGA GGTGACCACAACAGGCCTCATGTCCTCCTCTGGATTGGGCTGGGCTGGGCGCCCCCTACTCCTGTTAACT TCACACTCTCATGACTCACCTCAGACTTCCAGGTCCTCTCTGCCGACTGCTTCCCTGCTATTCTCTCCAC CTCAGGTCCTTCCGTTCCCACTCCCAGGACTGCCCAGGTCTTGGAAAGGACCAATGCTCAAATCTCTGTT TGGAATTGGTGAACATGGCTGCTGGGGGATCTTTAGCACTTACGTTTCTTTTTGCAGACAACTGTGCAGA TACCTTCAGATTGGAAGTTGTTGGGGTTCCCTTGGCAACCACCATAGATGAATTGGGTACAGAGGCCAGT ATCTTCATTATACCACCAGCGTGGGAGGTAGGCCAGGCAGGGGCCAGCATCCTGTGGCAGACTGCATATG TCTGTGGGGAAATCATAGGAGACAGATACTTGGAAGCCTGTGTCCCGAGGGGACAACCCCGCCCCCTTCT GTCCATTGCTCTTTTTGTCTAGCTCAGAGCTGTGCTCCCACTTCAGTGAGAACTGTGACCAGTCACTGGA ATCTCTTTGGTCTCAGTTTTCTCATCTGCAGGATGGTAACACAACTTGCCACCTTGCTTGTTGGGACACG AGGCCTGGAGAGGTGAGGTGGCTCTCCTGATGTCACATAGCTGGCGAGGGAGTTAAATGTAGGTGACCCA GTAGGTGTGCTAGTACGCACTTAGAATACCAGATAGCAGGAGGCTGAGGCAGGAGAATGTCAAAGTTGAG ATCAGCCTCGTCTCCCAGTGATTCCCTCATTCAACAACCAGAACCCAAGCAGACCAGAGCCAGGGGTTGG ATCACGGGGAGCATCAAGGCCCTCCCAGCTCCCTCCTCCATCCAGCCTCACCTACTCCACTTGGCAGCAT TTCCAGGCCTGATCATGGCCCCATATCCCAGGGCCTTCCCATGAGCACTTGTGGGTGTCTGTCAAGCTAC TGCTTTGGTATCTGCGTGCAAACCACCCTCAGTTCCCAAGGCCTGTGTGCCTCTCCCAGGAGAAGCTTTC CTGCCCACAGTGAGTGGGCGGCGCCTTTCAGGGCGATATGAGCACCCCCAAATACTTGTTGACTGGTAAAA TTGGGTGGATAATTTTGATGGTCTTTTGTAGGACTCAAGGATGTGACAGAGAGAAACAAAGTACAGGAGGC CAGAGGTTATCAGAGAGGCAGAGGCCTGGACAAACAAACCAGGAGACACCACACACCTGCCCCCATGCTA CACCCACACCTGCCCCATGCTACACCCACACCTGCCCCATGCTACACCCACACCTGCCCCATGCTACACC TATACCTGCCCCATGTTACACCCACACCTGCCCCCATGTTACACACACACCTGCCCCGTGATACACCCACA CCTGCCCCATGTTACACACACACCTGCCCCATGTTACACCCACACCTGCCCCATGCTACACCCACACCTG CCCCATGCTACACCCACACCTGCCCCATGTTACATCCCCACCCGCCTCAGGGTATCCTACAGCCAGACAC ACTAATCGTGTTTGATGAGTCTTTCATAGCTTCTTGGCATCTCAGAGCAGCACCACAGATCAGCTCAATG GATCAAATGACCCAGCACCTCCATCCATGTTAAGATGGGAAAATGAGGTTCAGAGAGGAAACACCACCAG TCCCACCGTTTGCTGAACGAGAGGAAAACCCTTACAGCACAGATAAAACGAGTCTCTGTTCAGCACCTAC AAGATTTGCATCAAGTAGACAATGCCAGGAACATACTTGAGAAACACTAGATTTGTCGCTCAGGAGAGGA GCGGGACAATGTGAGAGCAGGAGGGCGGGTGGAGCTGTCACAGGACCAGAGAGGTACCTTCCCGGAGGTC CATGCACTTCTTCCCACAGCTGAAGAAGCAGCATCTCTTCTTGTCCGGACACTGTCGGTGCCTCGTACAC TCATTTCTTTCTTCAACCTCACACTTCACTCTTACTCTCGGACATGATCCTATAGGGGGAGGAGGAAGAG GAGGAAGAGGAGGAGATGTGGTCCTGTGAGGTGTTGAAGGGGATACTGTTCTTCCTTCCCAGTCCCAGGG CACCGCTGCAGAGTTCAGAAAGCCTGTTTGTCCCTCACCTGTCACTAGGAGAAGGCAATCCTCAGCAGTT CAGGGTTTCACAACACTGGCCACATCTCCAACCGTAATGACTCTGGCATCTAATTTCTAGTAACTTCTCT ATCCCTCTCTCCACAACCTGTAAGATTCTCCGGAGGGACCCGCCCCTTTCGGATCGTTCTGAGCGTTTCG TTTTGTCCTTGGTTGGTCCTTGTAGCTCTCAGTGTTTGCCTACATTGTTGTCTTGTAGGCTAATGTGACA AAATCTGAAAGGCCTTGGTCAGTGATTCATGCTGAGGAAGTACTTCCAGGAAACAGTGAGTCATACTGAG AAAGTGCTTCCGGGGCACAGCGATTCATACTGAGGAAGTACTTCCGGGGCACAGTGATTCATACTGAGAA GGTGCTTCCGGGGCTTTTGGCCGCTCTCCTCACTCCTCAGTACACAATTATGAGTGGGCCTGGTCAGTGT TGCCCGCACAGAACAGAGGCCCCAGAGCATGGGGAGGCTCCATGGACTCATGCAGAGTCACACAACTCCT GAAGAGTGGACCCAGAATTTGCACCTGTCACGTGACCTCCTAAGCACCCACTTTCTGCTGCTGCCTTCTA GTGCTCAGAATCTCCATGCTCCATCACCCTGAGGGACTCCTGCTCTTTAGCACACAGGGAAGGTAGGTCT CTGTGACACAGGAAAGCTTTGGGGAGCTGCAGTGGGCAGGTTTTGGAGCGGATGGGGTGGTGCTAGGAAT CGCTGGTCCTTCAGTGGAAATCCCTTTGGCTGTTTAATCCTCAAGGCCCATTTAATGTGCACTAGTCCCA GCACCTCCATGAAGAATTCTTTATAACAGGGGAGCCAACCACCGCAGACCGAGGACCTGTGTTCAGCCAG GCCATCTTTCCAGGATATGGAGAAACCCTGACTCCAAACATCACCCCACCCAGGCTCCCGGGCAAGGTGA GGGAGACACTGTCACCTGCAGTGTGCGTTTGTTCTCTAACCCCATCACGTTTCCTCTCTCCATGCTAAGA CCTGGACCCAGTACTTACTGAAAAATAACCCCTCTGTCAGCTCAGGTTTCTGGATGCTCCACAGGAAGAT GAGGGGGACCAGGAATGGCAGAAGCCCCCAGTGTCTCATTTTGGGAAGCTGTAACCTGTTGGCAGGCATT GAACCAGGAGGGGCTGGTCTGCGGCTCTGGCCTCTGCTCTGTGTCCCAACAGTCCAGGAAGCAGCTCTGG ACTTGAGCCAGCTACGTTCCCTCCCCAGAGGCCATCAGTGGACGAGCTGTACCCATGGTGCAAACTGTGG GGGAAAGGGATCTGCCATGCTGGTCTGAGCCTCTGCAAGCGATGGTTCTTGGGCTGTGGCTTACTAGGGA GAGGGGCAGAATTGCTTGGGGTCTTCTAGAATCTCATAGCAAAGCTGTATGAATTCCAGAACCTTCCTTT TCCCAGGGGCAAATTAGAGATCAGAAATTTTCTAGGTTTCTTTTCTGCTGCTGAGACTAAACAGTAACTA AAAGCAACTTGGGGAGGAAAGGGTTTCTTTCAATTTACAGTTCCAGCCCATCAGGGAAGGAAGGCAAGGT AGGACGTGGACGTGGGGTCTAAGGCAGAGAGACCTTGGAAGAATGCTTCTTACTGCTTCCTTCCTCTGGC TTGCTCAGTTACTTTCCTTATACAACCCAGGACCCCCAGCTTCGTCGTGGTGCCACTGCTCACTGTAGGC TAGGACTCCCACCATCAGTTAGCAACCCAGAAAATTCCCCATAGGCATATCCGCAGGCCAATCGGGAGGC AACTCTTGAACTGAGGTTACCTCTTCCCAGTTGTGTCCAGTTGACAACCAAGATCCCCACCGGCTGTCCT CTAGTCCCCTCTTTGGAGTTTCATGCCAAATTCTCATCTTGCTTCTCTGTTTCTGTTCTTCAACCCTCCT CTCTCCTTCCTCCTTTTGGTGCACCTTTCCTTCCTTCCATCCTCACCCCTTCTCCTCATCTCCCCTCGTT TTTTTCTTGAGACAGGGTCTCCTCTCTCCCAGGCTGGTCTCAGACTTGCATCCACACCCACCTGCACTGT ACCCGGATCACAGGTCTGCATCGCCTCACCTAGTGTACGTGGGGCTGCGAGTGGAATCAGGGCTTCACAC ACGCTGGGCAACAAATGGACCGATTGAGCTGTGTACACACCGTCCCGGCCACTGATTCTTCTCAACTCTA ATTATCTGTGGGCTAATTATCTGTGGATTTGAACCTGTGCCTCCCCTTTCTCCTTTTCAATGTCTCTGTC TCGGTCTCTTTCTCCTTCAACTCCCTGTGGATGTTCCCTCTCTTGTCTTTGGCCCAGACTTCATCTAAGT CACCCCCGAGGGTAGAGACTGTTAGGTGGGCATGAACCCACGATTCCCAGTGAGCATCAGGCCAGGAGAT GGCACCTCTAAGGCAGGAGAGAGAGGGAAGCCCAGAGCCTCTGTTCCAAGTGCAGGTTAGCAGGGGCCAT GAGAAGCCATGGCTCAGTGAGCTGCAGGAGGGACTGAAAGTGTCTCTCTGATTGCTCTGGAATATTCCCT CAGCTTAAATCCTGAAGCGTTAGTGAGGGAGCGAGTGCAGTGTGGACACAATGTGAGCCATGCAAGGGTA AACTGAGGCAAGGACTGCTGGCTGCAGCCACGTGTATACACTTGATTCACCCAGGATTTCATCTGCCATT GCAAGAGACAGTCCCATGGTGTCCACTTTGACACACAGTACTAGAATTCCACCAGGATAACATTGGTCAT TTTCAGGTCTTCTTTTCTTGGTGCATTCCTAAGCTTGTGCATCATTGCCAGTTGTGGTGGCACACATCAT CACTCAGGAGGCAGAGGCACACAGATCTCTGGTATTTTAAGGCTAGCCTGGTCTACGAAGTGAATCTAGG ACAGGGCTTTGTTACACGGAGATGCCCTGTTTCAACAAAACAAAAAACAACAAACCAACCCAGACTGTGT GTCCTTTCTACTGGTTTGGAAGTGTTTTTCTTTCCAGCTGCAAACCCTGTCTCAGGAGCAGGAACTCTCT GGCCTCCACACACTCCAACCCCTGAGACTGTTTCCTAGCTTGTAGATTTTCCTGATGTGATGTCTTTTTC TCTTTACTGGTGTTGAGTTCAGTCCTGGGCCTCACTTACATGCTCTACCTCTGAGCTACATCTCTAGCCC CACCCCACCCCCACTTTTTTTTTTTAAGAATTGACATGGGGTTTCACTAACTTGCCCAGGCAGGCCTTTA ACCAGTAATCCTCTGCTTCCTGAGTGTGATTGCAGGCCTTAACCAACACACCCAACATTCTATTTCATGC AAATGGAATTATACAGTACAGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTTGTGCGTG TTGAGGTTTCCATGGCCCACATGTGGAAGTCAGAGGACCATCTCTGAATCCCTGATATGGGATCTCCTCT TCTACCTTGTTTGAGGCAGGGTCTTTTTGGCCTACCTCCATTCGCACACCAACTCTGCTGTACCCCCAGG CTAGCAGGGGCACAAGCTTCTGGGGATTCTTCTGGTTCTTTTCAAGGTAGGAGCATCATAATTACAGGTG TGAGCTACCCTGCCCGACCTTACATAGGTTCTGGGGATTTGAACTCAGGTCCTCATGCACGCCTGTGTGG CAAGCCCTTTATTCCCTTAGCCATCTTCCCAGCTGCTGGTGTATGTCCATCCACAGCAGAGCACCACGTG GCCCGAGGTTTTAGTGGCTGAATAGCATCCTGCCACATGCTAGAGTTTCACTCTCTGTTTATTGGTCCCT CGGAACCTACCCAGCTTGAATGCGAAACATTCTGGGCCATTTGAGGTAGATTTAATATCTCAGGCATCCA GGACGGGGGTTGGGAGGGAGATAGGAAATGCTTTCCTGTCACTGGAAATAGATTAAGAAAATTCAGAGGC GTGTTTTATTGGTATATGTTAAAATTCCCCAGCAGTAACTAACTCAGAAACCACTGCAATTAAAAAGAAA AAAGATTTTTTTTTTTAAAAAAAGGGGGTCATGAAACACATTCTTGCTGTTGCAAGTGAAACTGGGTGGA AGTCAAGTTCGCTGAGCTGTGGCAGCAACAGTCCTTCTACACTTTACCCACCACCCCTACCCTTACCAGC AAATCACTTACCTGGGGGATTTCTGGAGTAAGCCACTTGAACCCCCTTCCTTCCTCCCAGAGTCAGTCTC TGGGACATATAACCATGACATTGCTCACAGGCCTCAGACGGAGGAGAAAAAATTTCCCTGGGCTTCTAGG TTTCAAGGATGTTCCGTACCCACGTCCTTGATTGACGTCACCAGGGGCTCCACTCTCCCAGGACTGAGGA CGTGCAAACTACTCAAGTCTCACAGACACAGGGCGAAGTCAAATACTTTACTGAACCAAGCTGAGAGCTA CATGGAAACAAACTGGGAGCTTTTGACGGAGGAGCTGAGCAGGGTAGCTAAGGGTAGGAGTTAGGAATAG GAACTGGCCGAGTGGGCGGGGAGGCATCCTAGAATCCAGTAACTGGGAAGACCCAAGGCCTTGGAGTGCT CAGACGCCAGCCTGGAGCATCCAGCAGGTCCATAGAGCTTGGAGCAGAAGCCAAATCCTGATGATTTTCC CAGCGGGTCCCCCGTTCAGGTGGAATTGCCTGTTGTGAACAGAGAGATAAGAGATAGCCACACATCTTGG GCCCGGCTTATTTCTGGAACCTGAAGTTATATTTCCACATCTTACCGTGGATGGGGATTCAAAGTGGGGT TTGCCGACGGGAGCAGAAAGAGTTAATTGTGAGCAAAGTGCAGCTGTCTCGGCATAAAAACGTAGTGGAA CTTGCCACTGTATATGCTCACTCCAAAGAACAGTCGAACAAAGTCAGTTCGCTCGCTCAAAGAGAAATGG ATAGAATTGGCCTGGAGGCCAAAGTAAGGAAGGAGTTGTTATGCTTGGGGTGGTTCCTTGGTAGGAGTGT GCATGTAGGGCTGGACACTCTTTGACCGGTTTGCAGATGGTCTCGGGGACCCTTCTACATCCCCCTTCAT CCCTCCTAAATCCGTCCTTGTTTGTTACCCGCCCCCATATCTTCCAGCTTCACTTTCTAGGAGGTCCTGT CTCCCCTCCTGATCAAATGGAGTCCTGTCAGGTCTGAGAAAGACACAGCTGCCTTGGGACAGGCCAACCC ACACCAATGCTTCCCCCGAACAGTGCTTGCCCAGACAGCTTTGGTAAAGATGTTAGATGCGGTTGACAGG GATGAAGGGTCATGTGGGGGGTGGGGCGTGTGCGGGGGTGGGGGAGGCATGTACGGGTTGGGGGACGATT GTGCGGGGGCGAGGCATGTGCAGGGTGGAGCGTGTGCGGAGGTGGAGGCAGGAGGAGGTGTGTGCGGGGT GGGGAGTGTGCAGGGTGGGGTGGGGCGTGTGCAGGGTGGGGTGGGGGAGGCGTGAGAGGGGACAGCGTGT GCGGGGGGGGGGGGCGGGGAGGAGGCATATGCGGAGTGAGGTGTAGGGGGTACGGGGGAGGTGGGGCGGG GGCGGGGCGTGTGCAGGGTGGGGCGTGTGAGGAGTGAAGCGTGTGCGGGGTGGGGCGTTGCGGGATGGGG GCGGGAGGCGTGGATCCAAGACTCACTTTTTTTTTCACAGGCATTCTGGCATATACTTTGGGACTGGAAG TTGTTATTGTTTCCCTGGCAACCACCATAGATGAAGAGTTGGCACGTGCCGTTTTTCTTATTATACCACC AACGAGGGAAGTAAGCCATGCAGTAGCCAGAGTCTTTTGGTAGGCTGCAAATATCTGCGGAGGAACCCCA GAGTTGAGAGCTCAAGAGTGCACAACCCGAAACAAAATAATGAGAGCCACCTTCCCCTTCTCCTTCCCCT TGGAGCAAACTCTTTCTCCCCCTCCCGCTCCAGTGGTTCAACACCTGAAACATTTCGTGTACTTGTGGAA GATGCTACGGAGGATATCCGCGTCCCTGGAGGTAGGCATCCTTGTGGATGGTTATGGTCTGAACATGAAA TGTCCCCTCACAGGCTTACATTCCGAAGACTTGTCACCAGCTGGGGGTGACATTTTTGGAGGCTGTGGGA CCTGTTAGGAGGCGGAGCCTGGGGTTGGGGATTTAGCTCAGCGGTAGAGTGCTTACCTAGCTAGCGCAAG GCCCTGGGTTCGGTCCCCAGCTCCGGGTCCCCAGCTCCGAGAAAAAAAAAAAAAAAAGGAGGCGGAGCCT GGCTGGAGGAAGGATGTCAATGGGAGAGGACTCTTTAAGGTTATAGCATAACTCTGGTTCCTGGACTGAA GCGGTTTTAGGAGTTCCCATCACATGTCTGACACCATGAACTCCGCCATGCTGTTTTCATGGTGGGATAC AGAGGCTTCTTAAAACCGCAGGCCATAAGAAACCTTTAGTATGCATGTTGTTTCTCTTGAGTATCTAACA CACAGGGGACACTCGCTTTTAGGTTTTAGGGTACTAAGAATGGACTGGGGGCCTCAGGCCTGCTAGGCAA GCACTCTGTTATTGAGCTGTATCTTCAGTCTGTAAATGCAGTCAGTTAAGGTGGTTGCATGTGGGAGCCT CTAATCCAATACGGCTGATGCTCTGACAAAGGAGTAAATGTGTATCTATCTCCCTGAGATACCCACACAG GGAAGATGCCGTGTGGACTTGAAGGCAGAGATCAGAACAATGTATCTACAAGCCAAGGAATGCCAAAGAT TGCCAGCAATCTACCCAGTATTAATACAGAGCTGTATAATAAAGTTCTCACTCATGAGCCAGAGAGATGG CTCAACAGATAAAGACACTGTGTAACCCCGATGACCTGAGTTCAATTCCCAGAGACCACGTAAAGGTGGA AGAAGAGAACTGGAGTCTTCAGAGTTGTCCTCTAACCTCCCTCTCCTTCCCTCCCTCCCTGCCCCTCCCC CCACACACACTGGGCCTCACTGGCACCCTAACCTCATCCTTTTAGCCTACAAAGCTGTCAGACAACACAT TGCTGTCCCTCTATTGGAGTGACACTCACAAACCCACACAGTCTCCAGCTAGCCAAGCTCCATGACCGTC AGTCTCCACAGTGTCAGTGGCTTCCCCCTCCTCACACTCATCTCTCCACCAGGCTCTTGGAGCCTGAGTT TCACACCCAGGTTATTGAGGCAGCCTCCAGTTCTTCCTTCTGCCTTCATCCCTAGCACCTCCCACCAAAT CAGTCATTGATCCACCAGGGAGGAGGACATAATGGATCTGAAAATGAGGCAGTTGTGTCGTTCCATACAT AAGACCTCGGCGTCATCACAAGCTCCTTCCCTGTCTGGCTCCTATGCCTGGCCCTGGCCTCTTTTGCCAT GAGATCTCTTTAAGCCCCACACTGTAGCGATTTTCAGTTCAGCACAGCAGGCTCCCCGGCACCTCCAGAC CTCTGCGCATCCACACGCACCATCGAGTGCTAGCAGGATGTTTATTCTTCCTTCAAAAGCTTTCTCAAAA ATCAGTTCCTCTGGGGCTGGACAGATGGCTCAGTGGTTAAGAGCACCGACTGCTCTTCCAGAAGTCCTGA GTTCAAATCCCACACAGTAGCTCACAACCATCTGTAATGAGGTCTGATGCCCTCTTCTGGTGTGTCTGAA GACAGCTACAGTGTACTCATATATAATAAATAAATAAATACATCTTTAAAAAAAA Exon1: 385 - 499 Intron1: 500 - 932 Exon2: 933 - 1136 Intron2: 1137 - 1288 Exon3: 1289 - 1396 Intron3: 1397 - 1704 Exon4: 1705 - 1842 Intron4: 1843 - 3415 Exon5: 3416 - 3562 Intron5: 3563 - 5892 Exon6: 5893 - 6063 Accession No. 3: Nucleotide sequence of Rhesus monkey (scientific name: Macaca mulatta) Wfdc2 (NCBI (Gene ID: 710469)) TCCCGCGCGCGCGGTTAAATCCCCGCACCTGAGCAGCGGCTCACACCTGCACCCCGCCCCGGCATAGCAC CATGCCTGCCTGTCGCCTAGGCTCGCTAGCCGCGGCCCTCCTTGGCCTGCTGCTATTCGGCTTTACCCTA GTCCCAGGTGAGTGGGGCGGGGAGACGCCCAGCGCCCAGAGGGGCCGAGCCACGAGCTCCAGGATGGAGC CAGGCGGAGGCGTAGCCTCTGGAGGCCGGGGAAGTGGGAATTCCTGGGTCGGAAGTGGCGGAGACCCTTG GAGCCTAGGGTGTGGGGTCCAATGTGCTGGAGGTGGAGAGACCACTGACGGCTGAAATTTGGGAGGGTTC TGTGCTGAACGAAGACACTCCGTGGCTGCAGTGGGCTGGGGGTGGGGGCTGCTCTGCCTCGACCGCGGAA GCTCTTAGACACTCAGCTGTGCCGGGTCCCCTAGGGCTGAGATCCGAGGGCCCCGACGCCAAGGGTTAGG AAGCTCTGGTATTCCCAGGGGCTAGGGACTCCTCCTGGTCTGGAAGTGGGAGTCTCTGGGGGCAGTAAGG GGACTCCTAGGGCCAGAGACTGAGAATTCCTTGGAGTTAAGCTTTGGAGCAGGAGGTGGCCATCCTCTGG GGCTGGCGCTACGCCCCACCCTCCACTGTCACGGGCCTGCCCTTCCAGGCACAGGAGCAGAGAAGACGGG CGTGTGCCCCAAGCTCCAGGCGGACCAGAACTGCACGCAGGAGTGCGTCTCGGACAGCGAATGCGCGGAC AACCTCAAGTGCTGCAGCGCGGGCTGCGCCACCTTCTGCTCTCTGCCCAATGGTAACTCCACCACGGCCG AGCGGGGATGGGGCGGGGCTGCGCTGGGCTGGGAGGAGGCGGGAGGGCCTGGGTTCCGGGAACAGGGGCG CCCCAGGACCCGGGGACCCCCGGGAAAGTCAAGACGGATGAAACCAGATCCGCCAGTGCTCTCCCTCGCA CGGTCCCGGGGGAGACAAAGGCGTCGTTGAAGCGCAGCCAAGTGGGGGTCCCCACCCCTAGCTGGATTCG AGTCTCTGGTGCATGACGGGCTTCCGGGCACGCACAGCCGAGACATTGTTCCCCGCGGCCTGTGGACCCG GGGCCGCAGTTTCCTTCTCGGACTGGCCGGAGCCTGCCCTGCGGGAAAGCCCGGAGCCTGGGGCTCTCAC ATCTCCTCTTGGAGTCCCTCCCTGGGGGAAAGACGGGGAGGGCCTGGCCTGGCAAGATTTTCCCCAATTC CTCTGTCCAGGTGGAAGGGAACTTTACAGATTTAGGAAAGTGTCCCGCTCATCTTAAAGATGTGGAAGAG AGCATCGGCGAGAAAAAATTGTTCTTGCCCCAAGGTCACACGGCCATCCCATGGCTGCAGGAGTCTATAGT GAGGGTTCAGCTTTTGCCTTTCGGGCCAGCTGGCTGAGTGATTTGAAGAAGCGAGGAATCCTCCCCGGA CACCGTATCGCCCTTTGTTGTCTTTCAGTCAATCGCTTCCACTCTAAGGATTGAGTGAGCGCGAGCTGGG GACTCCTTCAAAGGTGCTGCTGGAGCTGCAGTGTCTCCACCAGGCTGATCAGAGCAGGGGGTGGCTTAG GCAGCATGGAACCTCAGCTTCTCGTTCTCCAGAGGGAACTGCTGAGTATTAGAGGCAGGGATAGGACAGTG GAGAGAACCAAGGTACTGTAATGATGTTCTTTCCCAGAATACAGTCCCTGTGACTTTTCCTCTTTGTTAT TTCCCTGAAGATGTCAGCACAGGCTGGGAAAGTTTGTAATACGGTGGCCCCCAGACACCGACCAGCAGGG AAGTGGTAAGTGGAGGGGGGACTGGCAGCCCACCAAGGGCAGCTTCAAGGTCAGGAGTCCCCTCCTCTGGGG CCAGGGAGGAGGAGGACCAGAGGAGGAGGAAGATTTGGAGAGAAAGCAGGCAGCAGGCAGTGAGAGAGGGT GGAGGAGCAAAGGAGGGAGTCTTTGCTGTTTGATGGATGAAGAATTGTTCCCTTATCTCAGATGGACAG ACTGAGGCAGGAAGAGGGAACAAGGTCAGATGGCCTCCTGTGCCAGGAGGAAGCTTGGAGGTTTTGAATCC TAGTTCTGCCATTTTCTAGTTGTGTGATCCTGAGCATGTTGTTTACCTCTCTGAGCCTCTGCTCTAAAG TGGGAATAATTCCAAATTCTATAGGATTGTCATGACTAAATGAAATAATGCATGTAAAGTGTCTATTAGT GGGAAGATACTCCATACACAGTGACTTGTTTTGATCATAAATGGTTCCCTATTTATTCCCAGGACAGCCC AATATTGAAGGCTCCTACTCCTTTCAAGAGAATGAATTTCTTTGTATTAGGAAGTCAAGACTGTCGGCAA CTTTTTAATTTTTTTTTTTTTTGTATTATACTTTAAGTTCTGGGGTACATGTGCAGAACGTGCAGGTTTG TTACATAGGTATACATGTGCCATGGTGGTTTGCTGCATCCATCAACCCGTCATCTACATTAGGTATTTCT TCTAATGCTATCCCTCCCCTAACCCCCAACCTCCCTGACAGGCCCTGGTGTGTGATGTTCCCCTCCCTGT GTCCATGTGTTCTCATTGTTCAACTCCCACTTATGAGAACATGCAGTGTTTGGTTTTCTGTTCTTGTGTT AGTTTGCTGAGAATGATGATTTCCAGCTTCATCCATGTCTCTGCAAAGGATGTGAACTCATCATTTGTTA TGGCTGCATAGTATTCCATGGTGTATACGTGCCACATTTTCTTTATCCAGTCTATCATTGATGGGCATTT GGGTTGGTTCCAAGTCTTTGCTATTGTGAACAGTGCTGCAATAAACATACATGTGCACGTGTCTTTATTG TCAAATGATTTATAATCCTTTGGGTATATAGCCAGTAATGGGATTGCTGGGTCAAATGTTATTTCTTGTC CTAGATCCTTAAGAATCATCACACTCTCTTCCACAATGGTTGAACTAATTTACACTCCCACCAACAGTGT AAAAGCGTTCCTATTTCTCCACATCCTCTCCAGCATCTGTTGTTTCCTGACTTTTTAAGGATCTCCATTC TAACTGGTGTGAGATACTATCTCATTGTTGTTTTGATTTGCATTTCTCTAATGACCAGTGATGATGAGCT TTTTTCGTTTGTTGGCTGCATAAATGTCTTCTTTTGAGAAGTGTCTGTTCATATCCTTTGCCCACATTTT GATGGGGTTTTTTTCTTGTAAATTTGTTTGAGTTTCTTGTAGATTCTGTATATTAACCCTTTGTCAGATG GATAGATTGCAAAAATTTTCTCCCAATCTGTAGGCTGCCTGTTCGATCTGATGCTAGTTTTTTTTTTTTT TTTTTTTTTTTTTTTTTTGTTTTTTTTTTTTTGCTGTGCAGAAGCTCTTTAGTTTAATTAGATCCCATTT GTCAATTTTGGCTTTTGTTGCCATTGCTTTTGGTGTTTTAGTCATGAAGCCTTTGCCCATGCCTATGTCC TGAATGGTATTGCCTAGGTTTTCCTCTAGGGTTTTAATGGTTTTAGGTCTTACATTTAAGTCTTTAATCC ATCTTGAGTTAATTTTTGTATAAGGTATAAGGAAGGGGTCCAGTTTAACTTTTCTGCGTATGGCTAGCCA GTTTTCCCAACACCATTTATTAAATAGGGAATCCTTTCTCCATTGCTTGTTTTTGTCAGGTTTGTCAAAG ATCAGATGGTTGTAGATGTGTAGTGTTATTTCTGAGGCCTCTGTTCTGTTCCATTGGTCTATATATCTGT TTTGGTACCAGTGTCATGCTGTTTCAGTTACTGTAGCCTCGTAGTATAGTTTGAAGTCAGTTAGCGTGAT GCCTCCAGCTTTGTTCTTTTTGTTTAGGATTGTCTTGGCTATGTGGGCTTTTATTCCATATGAAATTTAA AGTCGTTTTTTTCTAATTCTGTGAAGAAAGTCAGTGGTAGCTTGATGGGGATAGCATTTAATCTATAAAT TACTTTGGGCAGTATAGCCATTTTCATGATATTGATTCTTCTTATCCATGAGTATGGATTGTTTTTCCAT TTGTTTGTATCCTCTCTTATTTCATTGAGCAGTGGTTTGTAGTGCTCCTTGAAGAGGTCCTTCACCTCCC TTGTAAGTTATATTCCTGGTATTTTATTCTCTTTGTAGCAATTGCGAATGGGAATTCACTCATGATTTGG CTCTGTTTGTCTATTATTGGTGTATAGGAATGCTTGTGATTTTTGCACATTGATTTTGTATCCTGAGACT TTACTGAAGTTGCTTATCAGCTTAAGGAGATTTTGGGCTGAGACAATAGGGTTTTCTAAATATACAATCA TTTCATCTGCAAACAGGGACAATTTGACTTCCTTTTTTCTTAATTGAATACCCTTTATTTCTTTCTCTTG ACTGATTGACCTGGCCAGAATTTCCAATACTATGTTGAATAGGAGTGGTGAGAGAGAGCATCCCTGTCTT GCATCAGTTTTCAAAGGGAATGCTTCCAGCTTTTGCCCATTTGGTATGATGCTGGCTGTGGGTTTGTCAT AAATAACTCTTATTATTTTGAGATACATTCCATCAACACCTAGTTTATTGAGTGTTTTAGCTTGAAGTCT TGCATCCCAGGGATGAAGCCAACTTGATCGTGGTGGATAAGCTTTTCGATATGCTGCTGGATTCGGTTTG CTAGTATTTTATTGAGGATTTTCGCATTGATGTTCATCAGGGATATTGACTTGAATTTTTGTTTTTGTTT TTGTTGTGTCTTTGCCAGGTTTTGGTATCGGGATGATGCTGGCCTCATAAAATGAGTTGGGGAGGAGTCC CACTTTTTCTGTTGTTTGGAATAGTTTCAGAAGGAATGGTACCAGCAACTCCTCTTTGTACCTCTGGTAG AATTCGGCTGTGAATCCATCTGGTCCCAGACTTTTTTTTTACTGCCTCAATTTTGGAACTTGTTATTGGT CGATTCAGGGATTCGACTTCTTCCTGGTTTAGACTTGGGAGGGTGTATGTGTCCAGGAATTTATCCATTT CTTCTAGGTTTCTAGTTTATTTGTGCAGAGATGTTCATAATATTCTTTGATGGTAGATTGTATTTCTGT GGGATCAGTGGTGATATCCCCTTTTGTCATTTTTTATTGTGTCTCTTTGATTCTTTTCTTTTTTTCTTCTTT ATTAATCTGGCTAGCAGTCTATTTTGTTGATGTTTTTCAAAACCAGCTTGTGGATTCATTGATTTTTT TTTTTTGAAGGATTTTTCATGTCTCTATCTCCTTCAGTTCTGCTCTGATCTTAGTTATTTCTTGTCTTCT GCTAGCTTTTGAATTTGTTTGTTCTTGCTTCTCTAGTTCTTTTAATTGTGATGTCAGGGTGTCGATTTTG GATCTTTCCTGCTTTCCCTTGTGGGCATTTAGTGCTATAAATTTCCTTCTAAGCACTGCTTTAGCTGTGT CTCAGAGATTCTGGTATGTTGTGTCTTTGTTCTCATTGGTTTCAAAGAACTTATTTATTTCTGCCTTAAT TTTGTTATTTACCTAGTAGTCATTCAGGAGCAGGTTGTTCAGTTTCCATGTAGTTGTGCAGTTTTGAGTG AGTTTCTTAATCCTGAGTTCTAATTTGATGGCACTGTGGTCTGAGAGACTGTTTGTTATGATTTCCGTTC TTTTGTATTTGCTAAGTAGTGTTTTACTTCCAATTATGTGGTCAGTTGTAGAATAAGTGCGATGTGGTGC TGAGGAGAATGTATATTCTGTTGATTTGGTGTGGAGAGTTCTGTAGATGTCTATTAGGTCCACTTGGTCC AGAGCTGAGTTCAAGTCCTAGATATCTTTGTTAATTTTCTGTCTCATTAATCTGTCTAATATTGACAGTG GGGGTTAAAGTTTTCCACTATTATTGTGTGGGAGTCTAAGTCTCTTTGCAGGTCTCTAAGAACTTGCTTT ATGAATCAGGATGCTCCTGTATTGGGTGCATATATATTTAGGATAGTTAGCTCTTCTTGTTGCATTGATC CCTTTACCATTGTGTAATGCCCTTCTTTGTCTCCTTTGATCTTTGTTGGTTTAAACTCTGTTTTATCAGA GAATAGGATTGCAACTGCTGCTTTTTTTTTTTTTTTTTTTTTTTTTGCTGTCCATTTGCTTGGTTAATGTTA CTGCATCCCTTTATTTTTAGCCGATGTGTCTTTGCACGTGAGATGGTCTCCTGAATACAGCACACCGATG GGTCTTGACTCTTTATCCAATTTGCCAGTCTTTGTCTTTTAATTGGGGCATTTAGCCTGTTTACATTTAA GGTTAATATTATTATATGTGAATTTGATCCTGTCGTTATGATGCTAACTGGTTATTTTGTCCATTAGTTG TTGCAGTTTCTTCATAGCGTTGATGGTCTTTACAATTGGGTATATTTTTGCAGTTGCTGATACCAGTTGT TTCTTTCCATGTTTAGTGCTCCCTTCAGGAGGCCTTGTAAGGCAGGCCTTTTGGTAACAAAATCTCTCAG CATTTGTTTGTCTATATAGGATTTTATTTCTCCTTCACTTATGAAGCTTACTTTGGCTGGATATGAAATT CTGGGTTGAAAATTCTTTCCTTTAAGAATATTGAATATTGACCCCCACTCTCTTCTGGCTTGTAGGGTTT CTGTAGAGAGATCTGCTGTTAGTCTGATGGGCTTCCCATAGTGGGTAATCCGACCTTTCTCTCTGGATGC CCTTAACATTTTTTTCTTCATTTCAGCCTTGGGGAATCTGATGATTATGTGTCTTGGGGTTGTTCTTCTC AAGGAGTATCATTGTGGTGTTCTCTGTATTTCCTGAATTTGAATGTTGACCTGTCTTGCTAGGTTGGGGA AGTTCTCCTGGATAACATTCTGAAGAGTGTTTTCCAACTCGGTTCCATTCTCCCCGTCACTTTCAGGTAC ACCAATCAAACATAGGTTTGGTCTTTTCACATAGTCCCATATTTCTTGGAGGCTTTGTTCATTCTTTTTC ATTATTTTTTCTCTAATCTTGTCTTCATGCTTTATTTCATTAAGTTGATCTTCAATCTCTGATATTCTTC TTCCGCTTGATCAATTCGGCTATTGATACTTGTGTAGGCTTCATGAAGTTCTTGTGCTGTGTTTTTCAGC TCCATCAGGTCATTTATGTTCTTCTCTAAACTGTTTACTGTAGTTAGCAATTCCTCTAACCTTTTTCCAA GGTTCTTAGCTTTCTTGCATTGGGGTTAGAACATGCTTCTTTAGCTTGGAGGAATTTGTTATTACCCACC TTCTGAAGCCTGCTTCTGTCAGTTCGTCGAACTCATTCTCCATCCAGTTTTGTTCCCTTACTGGCGAGGA GTTGTGATCCTTTGGAGGAGAGGAGGTGTTCTGGTGTTTGGAATTTTCAGCCTTTTTGTGTGGTTTCTCC CCATCTTCATGGATTTATCTACCTTTGGTCTTTGATGGTGATGACCTTTGGATGGGATTTCTGAGTGGAC GTTGTTTTTGTTGATGTTGATGCTATTCCTTTCTGTTTGTTAGTTTTCCTTCTAACAGGCCCCTCAGCTG CAGGTCTGTTGGAGTTTGCTGGAGGTCCACTCCAGACCCTGTTTGCCTGGCTATCACCAGCGGAGGCTGC AGAACAGCAAAGATTGCTGCCTGTTCCTTCCTCTGGAAGCTTTGTCCCAGAGGGACACCTGCCAGATGCC AGCTGGAGCTCTCCTGTATGAGGTGTCAGTCAGCCCCTGCTGGAAGGTATCTCTCAGTCAGGAGACACAG AAGTCAGGGACTCACTTGAGGAGGCAGTCTGACCCTTAGCAGAGCTCGAATACTGTGCTGGGAGATCATC TGCTCTCTTCAGAACCGGCAGGCAGGCTGAAGTCATGCCCACAGCTGCCCCTTCCCCCAGGTGCTCTGTC CCAGGGAGATAGGAGTTTTATCTATAAGCCCCTGACTAAGGCTGCTGCCTTTTTTTCAGAGATGCCCTGC CCAGAGAGGAGGAATCTAGAGAGGCAGTCTGGCTACAGCGGCTTTGCCAAGCTGCACTGGGCCCTGCCCA GTTTGAATTTCCGGGTGGCTTTGTTTACTCTGTGAGGGGAAAACTGCCTACTCAAGCCTCAGTAATGGTG GATGCCCCACCCCCAACCAAGCTCAAGAGTCCCAGGTTGACTTCAGATTGCTGTGATGGCAGCGAGAATT TTAAGCCAGTGGATCTTAGCTTGCTGTGCTCTGTCGGGGTGGGATCCACTGAACTAGACCACTTGGCTCC CTGGCTTCAGCCCCCTTTCCGGGGGAGTGAATGGTTCTGTCTCACTGGCATTCCAGGCGCCACTGGAGTA TGAAAGAAAAAAAACTAAATCCTGCAGCTAGCTCAGTGTCTGCCCAAATGGCCACCCAGTTTTGTGCTTG AAACCCAGGGCCCTGGTGGTGTAGGCACCCGAGGGAATCTCTTGGTCTGTGGGTTGCGAAGACTGTGGGA AAAGCATAGTATCTGCTCCAGAGTGCACCATTCCTCATGGCACAGTCCCTCACAGCTTCCCTTGGCTAGG GGAGGGAGTTCCCTGACCCCTTGGGCTTCCCAGGTGAGGTGACACCCTACCCTGCCTCTGCTTGCCCTCT ATGGGCTGCACCCACTGTCTAACCAGTCCCAATGAGATGAGCCATGTACCTCCGTTGAAAATGCAGAAAT CACCTGCCTTCTGCGTTGATCTCACTGGGAGCTACAGACTGGATATCCGGCCATCTTGAAAGCTGAACCT AAATCTTTTTTTTTTTTTTTTTTTAAGATGGAGTCTCGCTCTGTCGCCCAGACTGGAGTGCAGTGGCATG ATCTTGGCTCACTACAACCTCCATCCTCCTGGGTGCAAGAGATTCTTCTGCCTCAGCCTTCCAAGTAGCT GGGATTACAGGTGCATGCCACCATGCCCAGCTAATTTTTTTGTATTTTTAGTACATGGGGGGGGTCTCGA AGTCCTGACTTCCAATGATCCACCCGCCTCAGCTTCCCAAAGTGTGGGATTACAGGCCGTGAGCCACCACA CCCGGTGATCTGTCGGCCACTTTCATTGTCCTCTGTAGCTAATCACCCAACTCTGTGCCTGTGTTTTACA ACAAACCTGCACGTTTATAGCAGCTTTATTTATAATTGCCGAAAGTTGGAAGCAACCAAGATGTCCCT CAGTTGACGAATGGATTTGCCTGGGCCTGGCTCCCCTCTATCTAACCAGGAAGTGGAGTGTGATTGTGTG TGTGTGTGTGTGTGTGTGTGTGTGTGTTAGTGGAAGGATGTGTGGTTTCTCTCTGCATGGATGAATGAGT GTATTTGGAGGGTCACAGTGCTGAGAGACTGTAACTGGGATTAGGTTTATGGGTATGAGTGAAGATTATA AGTGTGTAACCCCTCACATGTGTACTGTACTTTATACTTTAAGTGGCAGCATAGTGCTTAACCATGGCAG CAGCGGTTAATGCATAGACTCTGGAGCTAGATTTCTTGGGGTTCAAATTCTGCCTCAATTCTGTGTGACC TTAGGCAAATTACTTAACTTCTCAGTGCCTCAGTTTCCTCACTTGTAACACGAGGAAATATTAACAGATC CCTCCCCCTCTTTTAGGATTATTTTGAGAATTAGAAGAGTTAATATATGTTAGGGGCTTAACATAATACC TGGCATGTAGTAAGAGCTAATTAAGTGTTCACTGTTATAATTTCTCATTATAACTACAGAGTATTTTGAT ATTTCAGGGAGAAATAATATGTTTGTTTCCACTGCTTTCACAACCCTGTGTTGTAATTAGCACTGGTTCC GCTGACACCCAAAGACACAGAGGCTCTGGGAGATTTCTGGCTCTAGGCCATAAAGTCCCACAAAGACAGA GTTGGGACTCAAGCTGAGGTCCTGTGATTCCATTGGGGAGCAGGAGGAGGGATTTGCAGGTGATCTTTCT GGGCCTCCTTAGGTGCCCAAGATGGACTCAGGCAGGCAGCTCTGCTGTATGTGAAGCCAAGTGAGGGGCA GTAGGGGGAGCATGCTGCAAGTACAACTTAACCTCCCTGTATCCCCTCTGCCCACTTACCCTTACTCCTT TTTCTACCCAGATAAGGAGGGTTCCTGCCCCCGGGTGAACAGGAACTTCCCCCAGCTCGGCCTCTGTCGG AACCAGTGCCAGGTGGACAGCCAGTGTCCTGGCCGGATGAAATGCTGCCACAATGGCTGTGGGAAAGTGT CCTGTGTCACTCCCAATTTCTGAGGTAGGTGAATGGGAGACAGAAAGCACATTGATGGCCAGGTGTTGTG GGAAACAGGAGAAAACGTCACCCAGGCAGAGGTGAAGGGAGGTTAGTTGCAGAGAGAGTTGGGAAAGGTA TACCAGAGGGGTGACCCTCAGCCTGTTCTAAAAGAAGAGTGAAAGGTGGGCAGTCTGATGAGCTGCATGG GGAGGCTGTCTCAGGTGGAGGGAACAGAATAAGCAAGAGCTTGGCCAGGTATGGTGGCTCACGCCTGTAA TCCCAGCACTTTGGGAGGCTGAGGTGGGCGGATCACCTGAGGTCGGGAGTTCAAGACCAGCCTGACCAAC ATGGATAAACCCCATCTCTACTAAAAATACCCCCCCAAAAAAACAAAAAAAAACAAACAAAAAAAAAACC AAAAAACCTTTATGGGCTTGGTGGTGGGTGCCTGTAATCCCAGCTACTCAGGAGGCTGAGGCAGGAGAAT TGCTTGAACCTGGGAGACAGAGGTTGTGGTGAGCCGAGATGTCACCATTGCACTCCAGCCTGGGCAACAA GAGCGAAACTCCGTCTCAAATTTAAGAAAAAAAAAAAAAAAGCAAGAGCTTGGTGGTACCCTTGGGCTGA AATCTTGTGGTTGTCATGTGCTACTTTTAACAATTTCTCAGCAAAGGCATCTGAAGAGAACTGATAGAAT TCAATAGTGCACAGGTGAGCAACTGAAGCCCAGAGAGGGGGTGTGCTCTTGGTGCCCGAGAGGGAACCGT GGTTTCTCTAAACTAAGCTATGGTCTCCCTGAAGCCAGAGCTCTCAGACGCCAACTGCATGGATAGGCTT TGCAGTCAAGAAGACCCAGGTTCAAATGTTAGCTCTGACACTTACCTAGTCTTTCACCTCCCTGAGTCAC AGTTATCTTGGTTATAAAATGCAGCTAAATAAGATCTAATGGATCAGATAATCCCATTTAAACCTAAGGA ATGAAGGCATGGCAGAACTTCCTAATAGTAGAGGGATTCTTAGGGAAAGAAATGGCCAAGGTCCCTCAGG AAGGCCACAGTGGACATCCGGGACCACAGTATACCCAGGTGCCTGCTAGGGATGGTCTGAAGGAGAAGAG TATGTCTAGAGTCTGTAGAAAATGCAAGCAGGCCCCCCCCAGAAGGTGAGGCTTTGCTGCATGGGTGTGT GGGATGGGGTGGGGTCAAGGAGCTGGTCTAGGATTTAAGCTGCAGGTACTCTTGCCTAATCTAATGGATA TTGTTGTCGATGGTGTTGTTGTGATTTTCTTCCTCCAGCTCCAGCCACCACCAGGCTGAGCAGTGGGGAG AGAAAGTTTCTGCCTGGCCCTGCATCTGGTTCCAGCCCACCTGCCCTCCCCTTTTTCTGACCTCTGTATT CCCTCCTGGGCTGACCACAGCTTCCCCCTTTTCCAACCAATAAAGTAACCACTTTCAGCA Exon1: 43 - 147 Intron1: 148 - 678 Exon2: 679 - 822 Intron2: 823 - 10091 Exon : 10092 - 10244 Intron3: 10245 - 11518 Exon4: 11519 - 11679 Accession No. 4: Nucleotide sequence of Human (scientific name: Homo sapiens) Wfdc2 (NCBI (Gene ID: 10406)) CCTGGGAGGAGCAAGGCTGGGCAGTTTCCTGGCCCAGGGAGCTGAATAGGGCCCGGAAGAAAGCTGTTGG CAGCAGTGCCTGGGGCTGAATGTACAGTAGCCAGCTGGGTCCACGGGCTAAAAACTGGCTGCTCACCCCA GTGTTCTCTTTGTGCCCTAGACTGGGTTTCACTAGCCTGTTATGGGCTGTGTTCCCACCTGGCTCCTGCC CTCATTGTTGACTGAGCTCAGAGTAAATAGCCCTGGCCCCTCCCAATAACGTCCCCAGTCCCCAAGTTGA Note: There seems to be a typo in your original text where "Exon3: 10092 - 10244" is written as "Exon : 10092 - 10244" in the provided text. I've translated it as "Exon3: 10092 - 10244" in the translation for consistency with the original data structure. If this is not what you intended, please correct the original text for a more accurate translation.GCATGAGAAGCTGGGGGAGGTGTTCTCAGCATCCATTTTGGGGACAGTGGCCAGATGTACTCACATCACC AAACCCTACTCACCTGTCACTATCATCCCAGTGTTCACTGCCCCAGGGCCCCTCTAGCCTCCAGCAGCAA AGACCTCCTGGGGCTTCTAGAACCTCAGATCATTGGAGCAGGAAGCACTTCTTGTCTAACTCCCTGGGTA ACTCTGTTTTGGCACTTAACCCCCTGGATTCTAAAGCTTCATTTATACTTATTGGTCTCCTTCGTCACAG GAGACACTTATCTGTGGAGCACTCATGGCCATGTATCCAGTACCTATCATGGGTCAGGCCTGGAGTCCCT GTGTCAATCAGTGTGGATCTACTCTCAATGCTCCCAGTGAGTACATGGTTGTTCCGTGGCAGGAACGTTG CATTTGTGTTCTCACTGAAGTCCCATATAGGTGTCATTTTCACTGATGATGTGGCAGCTCAGATCCAGGA AAGGATTCATTCCAGGGCCCTCAGGTGGGAAGTGGTGGAGGTGGGAGTCAGACCCCAGACCCTGTCACCC CTTCAAGACCAGTGCTGGAGGGCCCCTTTTGACGACTTTCTCTTCACCAAGCGTCTTGCCCTGCATATGT CACACAGTAGGCACCCAGTGAATATTTACGACGGAATGGAAAAAACAAGGGCCTGGAGGGAGAGACCTGG GTTCAAAACCTTATTTGTACAGCTTTGCAGGGGACCTTGTGGTGGAGGTGCGGTCCTAAATCCCTAAACA CTCAGTTCCTTTGTCATATACAGCTGATAACAGTAACTCCCCGGTCAGAGGACCGGCGACAATTCCTTGA GACAAGGAGCACAGAGCCTGGCACAGTCAGCCCTGATCAGGGGCCGGTAGGACAAAGAGAGGGAGACAGA AACACACACCCAAACGGCTTGAGACTGCTAGGTGCTTCACCTTCCTGGGCCTGTTTCTGATGAGGAGGGT TTGTTTATTAATAATAAGTGCTATTTCTGTAGCCCCTACTATGTGCTCTGTATCACTACCCTAGCCCTCC ATCAAGAGGGATCTGCCCTCTCATCTGGCTGACCCCAAATCGAACCCTTTAACCCGCATGCTCTACTGCC TCCCTGCGGGCCCTGCCTTATCCTCCTCAACCGCCCACTGGGGACACTCTGGAGAAACCTCGGAGTCTTG GAATAGAAACCCAGGAGCCCTGAGCCTTGGCCCTCAGGCCCCAGGAGGGAGGTCAGGAGGGAGGCGTGGA CAAGGGTCCGGCCGTAGCTGGTCCCGTCCTGGAAGCTGCTTAAGTTCCTGGTTCCCACCGGGGCCAAGAG TGATACCTGATCCTGGGGGATTGTGAAATGACCTCATGTGGCAGCCCGCCCGCGGTGCCCGCAAAGCCCT CCACCCTCCCCTTCCCCGCTCGGCTCCACCCCTACCCCACGCCCCCTCCCGCGCGCGCGGTTAAATCCCC GCACCTGAGCATCGGCTCACACCTGCACCCCGCCCGGGCATAGCACCATGCCTGCTTGTCGCCTAGGCCC GCTAGCCGCCGCCCTCCTCCTCAGCCTGCTGCTGTTCGGCTTCACCCTAGTCTCAGGTGAGTGGGGCGGG GAGAGGCCCGGCGCCTAGAGGGGCCGAGCCACGAGCGCCAGGATGGAGCCAGGCGGAGGCGTAGCCTCTG GAGGCTGGGGAAGTGGGAATTCCGGGGGCGGAAGTGGCGGGGACCCTTGGAGCCTAGGGTGTGGGGTCCA ATGTGCTGGAGGTGGAGAGACCACTGATGGCTGCAATTTGGGAGGATTCTGTGCTGAACGCGGACACTCC GTGGCTGCAGTGGGCTGGGGGTGGGGGCTGCTCTGCCTCGACCTCGGAAGCTCCGAGACGCTCAGCTGTG CGGCGTCCCCTAGGGCTGAGATCTGAGGGCCCCGACGCCAAGGGTTAGGGAGCTCTCGTACTCCCAGGGG TCAGGGACTCCTGGTCTGGAAGAAGGAGTCTCTGGGGGCTGTAAGGGGACTCCTAGGGCCAGAGACTGAG AATTCCTTGGGGTTAAGGTTTGGAGCAGGAGGTGGGCATCCTCTGGGGCTGGCGCTACGCCCCACCCTCG ACTGTCCCGGGCCTCCCCTCCCAGGCACAGGAGCAGAGAAGACTGGCGTGTGCCCCGAGCTCCAGGCTGA CCAGAACTGCACGCAAGAGTGCGTCTCGGACAGCGAATGCGCCGACAACCTCAAGTGCTGCAGCGCGGGC TGTGCCACCTTCTGCTCTCTGCCCAATGGTAACCCCACGGCGGCCGAGCGGGAACGGGGCGGGGCCGCGC TGGGCTGGGAGGAGGTGGGAGGGCCCGGGTTCCGGGAACAGGGGCGCCCCCGGACCCGGGGACCCCCGGG AAAGTCAAGGCGGTTGAAACCAGATCCGTCAGTCCTCTCCCTCGCACGGCCCAGGGGTAGACAAAGGCGT CGTTGAAACGCAGCCAAGGGGGGGTCCCCACCCCTAGCTGGGATTCGAGTCTCTGGTGCATGACGGGCTT CCGGGCACGCACAGCCGGGACATTGTTCCCCGCGGCCTGGGGACCCGGGGCCGCAGTTTCCTTCTCGAAC CGGCCGAAGCCTGCCCTGCGGGAAAGCCCGGAGCCTGGGGCGCTCACCTCTCCTCTTGGAGTCCCTCCCT GGGGGCCTCCCCCAGCCCTGGGGAAAGACTGGGAGAGCCTGGCCTGGCAAGATTTTCCCCAATTCCTCTG TCCAGGCGGAAAGGAACTTTACAGATTTAGGAAAATGCCCCGCTCATCTTAAAGATGTGTAAGGGAGCAT CGGTGAGAAAAAAATGTTCTTGCCCAAGGTCACACCGCCATCCCATGGCTGCTGGAGTCATAGTGAGGGT TCAGCTTTTGCCTTTCAGGCCAACTGGCTGAGTGATTCGAAGAAAGTGAGGAATCCTCCCTGGACACTGT ATCGCCCTTCGTCGTCTTTCAGTCAATCTCTTCCACTCTAAGGATTGAGTGAGCGCGAGCTGGGGACTCT CTCAAAGGTGCTTGCTGGAGCTGCAGTCTCCACCAGGCTATCAGAACAGGGGGTGGCTTAAACAGCATGG AATCTCAGCTTTCCGTTCTCCAGAGGAAAATGCTGAGGATTAGAGGCAGGGATAGGACCTGGAGAGAACC AAGGTACTGTAATGATGTTCTTTATCCAGAATACAGCCCCTGTGACTTTTCCTCTCTGTTATTTCCTTGA AGATGCCAGCACAGGCTGGGAAGGTTTGTAATACGGTGACCCAAGACACCGATCAGCAGGGAGGTGGTAA GTGGAGGGGGACTGGCAGCCCACCAAGGGCAGCTTCAAGGTCATGAGTCCCCTCCTCTGGGCCAGGGAGA GGAGGACCAGAAGGAGAGAGGAAGATTTGGAGAGAAAGCAGGCAGCAGCCAGATGAGAGAGGTGGAGGAG CAAAGGAGGGAGTCTTTGCTGTTTGATGGATGAAGAATTTGTTCCCTTATCTCAGATGGACAGACTGAGG CAGGAAGAGGGACAAGGTCAGATGGCCTCCTGAGCCAGGGAGGCAGCCTGGAGGTTTGAATCCTAGTTCT GCCATTTTCTAGTTATGTGATCCTGAGCATGTTGTTTACCTCTCTGAGCCTCTGCTCTAAGATGGGAATA ATTCCAAACTCTATAGGATTGGCATGAGGACTAAATGAAATAATGCATCTAAAGTGTCTATTAGTGAGAA GATACTCCATACACAGTGGCTTGTTTTGATCATAAATGGTTCCCTATTTATTTCCAGGGCAGCCCAATAT TACAGGTTCCTACTCCTTTCAAGAGAATGAATTTCTTTGTATTAGGAAGTCAAGACTGTTGGCAACTTAA CAACATTTTTTTTTGTATTTATGCTTTAAGTTCTGGGGTACACGTGCAGAACGTGCAGGTTTGTTACATA GGTATACATGTGCCATGGTGGTTTGCTTCACCCATCAACCCGTCATCTACATTAGGTGTTTCTCCTAATG CTATGCCTCCCCTAATTCCCAACCCCCCGACAGGCCCTGGTGTGTGATGTTCCCCTCCCTGTGTCCATGT GTTCTCATTGTTCAACTCCTACTTATGAGTGAGAACATGCGGTATTTGGTTTTCTGTTCTTGTGTTAGTT TGCTGAGAATGATGGTTTCCAGCTTCATCCATGTCTCTGCAAAGGACATGAACTCATCATTTGTTATGGC TGCATAGTATTCCATGGTGTAGACGTGCCACATTTCCTTTATCCAGTCCATCACTGATGGGCATTTGGGT TGGTTCCAAGTCTTTGCTATTGTGAACAGTGCTGCAATAAACCTATGTGTGCATGTGTCTTGGGCATTTG CGTTGGTTCCAAGTCTTTGCTATTGTGAACAGTGCTGCAATAAACCTATGTGTGCATGTGTCTTTATTGT AGAATGATTTATAATCCTTTGGGTATATACCCAGTAATGGGATTGCTGGGTCAAATGGTATTTCTTGTTC TAGATCCTTGAGGAATGGCCACACTCTCTTCCACAATGGTTGAACTAATTTACACTCCCACGAACAGTGT AAAAGCATTCCTATTTCTCCACATCCTCTCCAGCATCTGTTGTTTCCTGACTTTTTAAGGATCTCCATTC TAACTGGTGTGAGATGTTATCTCATTGTGGTTTTGATTTGCATTTCTCTAATGACCAGTGATGATGAGCA TTTTTCATGTTTGTTGGCTGCATAAATGTCTTCTTTTGAGAAGCATCTGTTCATATCCTTCGCCCACGTT CTAATGGGGTTTTTTTCTCGTAAATTTGTTTGAGTTCTTGTAGATTCTGTATATTAGCCCTTTGTCAGAT GGATAGAGTGCAAAAATTTTCTCCCATTCTGTAGGCTGCCTGTTCACTCTGATGATAGTTTCTTTTGCTG TGCAGAAGCTCTGTAGTTTAATTGGATCCCATTTGTCAATTTTGGCTTTTGTTGCCATTGCTTTTGGTGT TTTAGTCATGAAGTCTTTGCCCATGCCTATGTCCTGAATGGTATTGCCTAGGTTTTCTTCTAGGGTTTTT ATGGTTTAGGTCTTACATTTAAGTCTTTAATCCATCTTGAGTTAATTTTTGCATAAGGTGTAAGGAAAG GGGTCCAGTTTCAGTTTCTGCATATGGCTAGCCAGTTTTCCCAACACCATTTATTAAATAGGGAATCCT TTCCCCATTGCTTGTTTTTGCCATGTTTGTCAAAGATCAGGTGGCTGTAGATGTGTAGTGTTATTTCTGA GGCCTCTGTTCTGTTCTGTTGGTCTATATATCTGTTTTGGTACCAGTACCATGCTGTTTTGGTTACTGTA GCCTTTGTAGTATAGCTTGAAGTCAGTTAGCGTGATGCTTCCAGCTTTGTTCTTTTTGCTTAGGATTGTCT TGGCTATGTGGGCTTTTATTTGGTTCCATATGAAATTTAAAGTCGTTTTTTCTAATTCTGTGAAGAAAGT CAGTGATAGCTTGATGGGGATAGCATTGAATCTATAAATTACTTTGGGCAGTAGCCATTTTTCACGATA TTGATTCTTCTTATCCATGAGCATGGATTGTTTTTCCATTTGTTTGTATCCTCTCTTATTTCGTTGAGCA GTGGTTTGTAGTTCTCCTTGAAGAGGTCCTTCACCTCCCTTGTAAGTTGTATTCCTAGGTATTTTATTCT CTTTGTAGCAATTGTGAATGGGAATTCACTCATGATTTGGCTCTCTGTCTATTATTGGTGTATAGGAATA CTTGTGATTTTTGTACATTGATTTGTATCCTGAGACTTTACTGCAGTTTCTTATCAGCTTAAGGAGATTT TGGGCTGAGATAATGGGGTTTTTCTAAATATACAATCATTTCATCTGCAAACAGGGACAATTTGACTTCCT CTTTTCCTAATTGAATACCCTTTATTTCTTTCTCTTGACTGATTGCCCTGGCCAGAATTTCTAATGCTAT GTTGAATAGGGGTGGTGAGAGAGAGCATGCTTGTCTTGTGCCGGTTTTCAAAGGGAATGCTTCCAGCTTT TGCCCATTCAGTATGATATTGGCTGTGGGTTTGTCATAAATAGCTCTTGTTACTTTGAGATACCTTCCAT CAATACCTAGTTTATTGAGTGTTTTAGCATGAAGGGTGTTGAATTTTATCAAAGGCCTTTTCTGCATCTA TTGAGATAATCATGTGGTTTTTGTCATTGATTTTGCTTATGTGATGGATTACGTTTATTGATTTGTGTAT GTTGAACCAGCCTTGCATCCCAGGGACAAAGCCAACTTGATCATGGTGGATAAGCTTTTTGATGTGCTGC TGGATTTGGTTTGCCAGCATTTTATTAAGGATTTTCGCACTGATGTTCATCAGGGATATTAACCTGAAAT TTTTTTGTTATTGTGTCTCTGCCAGGTTTTAGTATCAGGATGATGCTGGCCTCATAAAATGAATTAGGGA GGAGGCTCTCTTTTTCTATTGTTTGGAATAGTTTCAGAAGGAATGGTACCAGCAACTCCTCTTTGTACCT CTGGTGGAATTCGGCTGTGAATCTGTCTGGTCCCAGACTTTTTTTGGTTGGTAGGCTATTAATTACTGCC TCAATTTTGGAACTTGTTATTGGTCGATTCAGGGATTCGACTTCTTCCTGGTTTAGACTTGGGAGGGTGT ATGTGTCCAGGAATTTATCCATTTCTTCTAGGTTTTCTAGTTTATTTGCGTAGAGGTGTTTATAGTATTC TCTGATGGTAGATTGTATTTCTGTGGGATCAGTGGTGATATCCCCTTTGTCATTTTTTATTGTGTCTATT TGATTCTTCTCTCTTTTCTTTATTAGTATGGCTAGCAGTCAGACTATTTTTTGTTGATGTTTTTCAAAAACC CAGCTTGTGAATTCATTGATTTTTTTTGAAGGGTTTTTCATGTCTCTATCTCCTTTAGTTCTCTCTGATC TTAGTTATTTCTTGTCTTCTGCTAGCTTTTGAATTTGTTTGTTCTTGCTTTTCTAGTTCTTTTAATTGTG ATGTTAGGGTGTCAATTTTGGATCTTTCCTGCTTTCCCTTGTGGGCATTTAGTGCTATAAATTTCCTTCT AAGCACTGCTTTAGCTGTGTCTCAGAGATTCTGGTATGTTATGTCTTTGTTCTCATTGGTTTCAAAGAAC TTATTTATTTCTGCCTTGATTTTGTTATTTACCTAGTAGTCATTCAGGAGCAGGTTGTTCAGTTTCCATG TAAGGTTATGCGGTCTTGAGTGAGTTTCTTAATCCTGAATTCTAATTTGATGGCACTGTGGTCTGAGAGA CTGTTAGGATTTCCGTTCTTTTGCATTTGCTAATTAGTGTTTTACTTCCAATTATGTGGTCAGTTTTTAGA ATAAGTGTGATGTGGTGCTGAGGAGAATGTATATTCTGTTGATTTGGGGTGGAGAGTTCTGTAGATGTCT ATTAGGTCTGCTTGGTCCAGAGCTGAGTTCAAGTCTTGGATATCTTTGTTAATTTTCCGTCTCATTGATC TGTCTAATATTGACAGTGGGGGTTAAAGTTTCCCACTATTATTATGTCGGAGTCTAAGTCTCTTTGTAGG TCTCTAAGAACTTGCTTTATGAATCAGGGTGCTCCTGTATTAGGTGCATATATATTTAGCATAATTAGCT CTTCTTGTTGCATTGATCCCTTTACCATTGTGTAATGCCCTTCTTTGTCTCTTTTGATCTTTGTTGGTTT AAAGTCTGTTTTATCAGAGAATAGAATTGCAACCCCTGATTTTTTTTTGCTTTTCCATTTGCTTGGTAAAT ATTCCCTATCCCTTTATTTTGAGCCTATGTGTCTTTGCACTTGAGATGGGTCTCCTGAATACAGCACAC CAATGGGTCTTGACTCTTTATCCAATTTGCCAGTCTTTGTCTTTTAACTGGGGCATTTAGCCTGTTTACA TTTAAGGTTAATATTGTTATGTGTGAGTTTGATCCTGTCATTATGATGCTAGCTGGTTATTTTGTCTATT AGTTGATGCAGTTTCTTCATAGTGTCGATGGTCTTTACAATTTGGTATGTTTTTGCAGTTGCTGATTCCA GTTGTTCCTTTCTATGTTTAGTGCTCCTTTTCAGGAGACCTTGTAAGGCAGGCCTGTTGATGAGAAAAATC CCTCAGCATTTGTTTGTCTATAAAGGATTTTATTTCTCCTTCATGTATGAAGCTTACTTTGGCTGGATAT GAAATTCTGGGTTGAAAATTCTTTAAGAATATTGAATATTGGCCCCCACTGTCTTCTGGTTTGCAGGGTT TCTGCAGAGAGATCTGCAGTTAGTCTGAAGGGCCTCCCATAGTGGGTAACCCAACCTTTCTCTCTGGGTG CCCTTAACATTTTTTTCTTCATTTCAACCTTGGTGAATCTGATGATTATGTGTCTTGGGGTTATTCTTCT CAAGGAGTATCTTTGAGGTGTTCTCTGTATTTCCTGAATTTGAATGTTGGCCTTTCTTGCTAGGTTGGGG AAGTTCTCCTGTATAATATCCTGAACAGTGTTTTCCAACTTGGTTCCATTCTCCCCATCACTTTCAGGTA CATCAATCAAACATAGGTTTGGTCTTTTCACATAGTCCCATATTTTTTGGAGGCTTTGTTCATTCATTCC TTTTCATTATTTTTTCTCTAATCTCATCTTCATTCTTTATTTCATTAAATTGATCTTCAATCTCTGATAT CCTTCTTCTGCTTGATCAATTCGGCTATTGATACTTGTATAGGCTTCATGACGTTCTCGTGCTGTGTTTT TCAGCTCCATCAGGTCATTTATGTTCTTCTCTAAACTGGTTAATCTAGTTAGCAATTCCTATAACCTTTT TTCAAGGTTCTTACCTTTCTTGCATTGGGGTTAGAACATGCTCCTTTAGCTTGGAGGAGTTTGTTATTAC CCACCTTCTGAAGCCTACTTCTGTCAATTTGTCAAACTCATTCTCCGTCCAGTTTTGTTCCCTTGCTGGC GAGGAATTGTGATCCTTTGGAGAAGAGGTGTTCTGGTTTTTGGAATTTTCAGCCTTTTTGTGCTGGTTTC TCCCCATCTTCGTGGATTTATCTACCTTTGGTCTTTGATGTTGATGATCTTTGGATGGGCCTTCTGAGTG GACGTCCTTTTTGTTGATGTTGATGCTATTCCTTTCTGTTAGTTTTCCTTCTAACAATCAGGCCCCTCAG CTGCAGGTCTGTTGGCGTTTGCTGGAGGTCCACTCCAGACCCTGTTTGCCTGGCTATCACCAGCAGAGGC TGCAGAACAGCAAAGATTGCTGCCTGTTCCTTCCTCTAGAAGCTTTGTCCCAGAGGGGCACCCGCCAGAT GCCAGCCAGAGCTCTCCTGTATGAGGTGTCTGTCAGCCCCTGCTGGAAGGTGTCTCCCAGTCAGGAGACA CGGAAATCAGGGACTCACTTGAGGAGGCAGTCTGACCCTTAGCAGAGCTCAAATGCTGTGCTGGGAGATC TGCTGCTCTCTTCAGAACCGGCAGGCAGGCTGAAGCCACATCCACAGCTGCCCCTTCCCCCAGGTGCTCT GTCCCAGGGAGATGGGAGTTTTACCTATAAGCCCCTGACTAGGGCTGCTGCCTTTTTTTCAGAGATGCCC TGCCCAGAGAGGAGGAATCTAGAGAGGCAGTCTGGCTACAGCGGCTTTGCCGAGCTGTGTTGGGCTCTGC CCAGTTTGAACTTCCTGGTGGCTTTGTTTACTCTGTGAGGGGAAAACTGCCTACTCAAGCCTCAGTAATG GTGGACGCCCCTCCCCCTACCAAGCTTGAGAGTTCCAGGTCAACTTCAGACTGCTGTGCTGGCAGTGAGA ATTTCAAGCCAGTGGATGTTAGCTTGCTGGGCTCCATGGGGGTGGGATCCGCTGAGCTAGACCACTTGTC TCCCTAGCTTCAGCCCCCTTTCCAGGGGCGTGAATGGTTCTGTCTCACTGGCATTCCAGGCACCACTGGG GTATGAAAAAAAAATCCTGCAGCTAGCTCAGTGTCTGCCCAAATGGCCACCTAGTTTTGTGCTTGAAACC CAGGGCCCTGGTGGTGTAGGCACCCAAAGGAATCTCTTGGTCTGTGGGTTGTGAAGACCGTGGGAAAAGT GTAGTATCTGCTCCAGAGTGCACTATTCCTCACGGCACAGTCCCTCATGGCTTCCCTTGGCTAGGGGAGG GAGTTCCCTGACCTCTTGTGCTTCCCAGGTGAGATGATGCCCTACCCTACTTCTGCTCGCCCTCCGTGGG CTGCACCCACTGTCTAACCAGTCCCAATGAGATGAGCCACGTACCTCATTTGAAAATGCAGAAATCACCT GCCTTCTGCATTGATTCACTGGGAGCTGCAGACTGGAGCTGTTACTATTCGGCCATCTTGAAAGCTGAAC CTAAATCTTTTTTTTTTTTGAGATGGAGTCTCGCTCTGTCGCCCAGGCTGGAGTGCAGTGGCATGATCTC AGCTCACTACAACCTCTGTCTCCCAGGTGCAAGCGATTCTCCTGCCTCAGCCTCCCAAGTAGCTGGGATT ACCGGTGCATGCCACCACGCCCAGTTAACTTTTTTGTATTTTTAGTAGAGCGGGGGGTCTCGAGTCCTGA CCTCCGATGATCCCACCCGCCTGAGCCTCCCAAAGTGTGGGATTACAGGCATGAGCCACTGCACCCGGTG ATCTGTCAGCCACTTTCCTTGTCCTCTACAGCTAATCACCCAGTTCTGTGCCTGTGTTTTACACACAAAA ACCTGCACATATTTATAGCAGCTTTATTTATAATTGCCAAAAGTTGGAAGCAACCATGATGTACCTCAGT TGATGAATGGATTTGGCTGGGCCTGGCTCCCCTCTATCTAACCTGGGAAGTGGAGTGTGATTGTGTGTGT GTTTGTTAGTGGAAGGACGTGTGGTTTCTCTCTGCATGGATGAATGAGTGTATTTGGAGGGTCATGGTGC TGAGAGACTGCAACTGGGATTCGGTCTGTGGGTATGAGTGAAGATTATAAGTGTGTAACCCCTCACATGT GTACTGTACTTTACACTTTAAGTGGCAGCATAGTGCTTAACCATGGCAGCAGCAGTTAACACATAGACTC TGCAGCTAGATTTCTTGGGGTTCAAATTCTGCCTCAATTCTGTGTGACCTTAGGCAAATTACTTAACTTC TCAGTGCCTCAGTTTCCTCACTTGTAACACAAGGAAATATTAACAGATCCCTCCCCTTCTTTTAGGATTA TTGTGAGAATTAGAAGAGTTAATATATGTTAGGGGCTTGATACCTGGCATGTAATAAGAGCTAATTAAGT GTTCACTGTTATAATTTCTCATAACTACAGAGTAGTTTGATATTTCAGGGAGAAATAATATATGTTTGTT TCCCACTGCTTTCACAACAACCCTATGTTGTAATTAGCACTGTTCCACTGGCACCTAAAGACACGGAGGC TCTGGGAGATTTCTGGCCCTAGGCCACGAAGGCCCACTTGGGACTCAAGCTGAGGTCCTGTGATTCCATT TGGGAGCAGGAGGAGGGATTTGCAGGTGATCTTCCTGGGCCTCCTGAGAGCAGCTCAGGTGCCCAAGATG GACTCAGGCAGGCAGCTCTGCTGTATGTGAAGCCCAGTGAGGGGCAGTGGGGGGGCCATGCTGCAGGTAC AAGTTAATCTCCCTGTATCGCCTCTGCCCACTTACCCTTACTCCTTTTTCTACCCAGATAAGGAGGGTTC CTGCCCCCAGGTGAACATTAACTTTCCCCAGCTCGGCCTCTGTCGGGACCAGTGCCAGGTGGACAGCCAG TGTCCTGGCCAGATGAAATGCTGCCGCAATGGCTGTGGGAAGGTGTCCTGTGTCACTCCCAATTTCTGAG GTAAGTGAACGGGAAAGAGAAAGTGCATTGATGGCCAGGTGTTGTGGGAAACAGGAGAAAACGTCACCCA GGCGGAGGTGAAGGGAGGTTAGTTGCAGGGACAGTTGGGAAAGGTATACCAGTGGGGTGACCCTCAGCCT GTTCTAAAAGAAGAGTGAAAGGTGGGTAGTCTGATGAGCTGCATTGGGGAGGCTGTCTCAGGCAGAGGGA ACAGAATAAGCAAGAGCTTGGCTGGGTGTGGTGGCTCATGCCTATAATCCCAGCACTTTGGGAGGCTGAG GTGTGCGGATCACCTGAGGTCGGGAGTTCAAGACCAGCCTGACCAACATGGAGAAACCCCGTCTCTACTA AAAATACCAAAAAAAAAAAAAAAAATTTAATGGGTGTGGTGGCGGGTGCCACCCACCCGCTACTCAGGAG GCTGAGGCAGGAGAATTGCTTGAACCTGGGAGACAGAGGTTGTGGTGAGCTGAGATCACGCCATTGCACT CCAGCCTGGGCAACAAGAGCGAAACTCCATCTCAAATTAAAAACAAACAAACAAAAAACAGCTTGGTGGT ACCCTTGGGTTGAAATCTCGTGGTTGTCATGTGCTACTTTTAACAATTTCTCAGCAAAGTAACCTGAAGA TAACTGATAGAATCCAATAGTGCAGAGATGAGCAATTGAAATCTAGAGAGGGGGTGTGCTCTTGGTGCCC GAGAGGGAACTATGGTTTCTCTAAACTAAGCTATGGTCTCCCTGGAGCCAGGGCTCTCAGATGCCAACTG CATGGATAGGCTTTGCAGTCAAGAAGACCTTGGTTCAAATGTTAGCTCTGACACTTACCTAACCTTCTCA CCTCCCTGAGTCACAGTTATCTCGCTTATAAAATGTAGCTAAATAAGATCTAATTGATCAGATAATCCCA TTTTAACCTAAAGAAGAAGGCATGGCAGAACTTCCTAATAGTAGAAGGATTCTTGGGGAGAGAAATGGCC ATGACCCCTCAGGAAGGCCACAGTGGACATCTGGGACCACAGTATACCCAGGTGCCTGCTAGGGATGGTC TGACCACAGTATACCCAGGTACCTGCTAGGGATGGTCTGAAGGAGAAGAGTATGTCTAGAACCTGTAGAA AATGCAAGCAGAGCCCCCAGAAGGTGAGGCTTTGCTGTGTGGGTGTGTGGGATGGGGTGGGGTCAAGGAG CTGGTCTAGGATTTAAGCTGCAGGTACTCCTGCCTAGTCTAATGGATATTGTTGTTGATGGTATTGTTAT AATTTTCTTCCTCCAGCTCCAGCCACCACCAGGCTGAGCAGTGAGGAGAGAAAGTTTCTGCCTGGCCCTG CATCTGGTTCCAGCCCACCTGCCCTCCCCTTTTTCGGGACTCTGTATTCCCTCTTGGGCTGACCACAGCT TCTCCCTTTCCCAACCAATAAAGTAACCACTTTCAGCACGTTCTGTCTCTGTCTCCCCTGACTTGGGTTG AGGTGGGTGGGCAAGTGTTAAGGGTGTGACCAGATCATCCAGGCATGAAGTCCAGAGAAGGATCATCTGG TAGGTGTTAGCATTCTCCTGGTGGATCCCTGCATCAACCCTATGAATCTGGTCATTATTCCTCCAGGAGG ATGGATGAGAAAAACGAGGCTCAGACAGGTGGTGGGGTGCTTCCAAAGTCACTCAGATTCATTGCTGGGG GTGCTGATAAGCAGCAGCATGCACCAGGTGCCAGACACGGTGCTGGGAGGTTTGCTTATGAGATCCTCTG GGTCCTCAAAACAATCCCCAGAGCAGTTAACAACATACCCCTGTCATAGACCTGGACTCCATGAGGTTAC TTGCCAGTGGCCACACAGAAAGATGGCAATCCAGGATCCTCTCTTGTTGATACTTTAAGTCAAAGTTTTA TTTTATATAAGTAATATAGTCATATGCATCCAAACTCAAAAGATTAAAAAAAGATATACAATGAAAAGTG TCTTTCACCCTTTATCCCCTGTCTCCACCTTCCACCCCAGCCACCAAGTTGTTTTCTATAAAGACAAAAA ATGTTCTCAGTTTCTTGGGTTTCTTTTCAGATATGTATCCCCAGCTCTCAATCCTTTCTCTCTCCCTTCT CTATTTCTTCCTCTTTTCCTTTCTATTCCCCCTCTATGCCTCACTCTATTCTCTTTCTCTCTCCTCTATT CCTTCCTCTCCATCCTCCCCCTCTCCATACTCCCTCTCTTAATGCCCCTCTCTCTATGTACTTTCCTCTC TCTCCATAGTGTCCCCTTCTACTTCTCTCCCCACTCCCTATCTCTATCATCTCTTTCCATCTGTTAGAGT AGGTAGTTAGACATGAGCAGGACAGGAGAGGGCTCCCCCCTCACACGCCCAGGAATGTCAGGTGACGATC AGGTGATGGTCAGGTAGTGGTTAAACTGTCTCTCTAAAATGATCACTGGGCCGGGTGCAGTGGCTCACGC CTGTAATCCCAGCACTTTGGGAGGCCGAGGCGGGTGGCTCACCTGAGATCAGGAGTTTGAGACCAGCCTG GCCAACATGGCCAAACCCCATTTCTACTAAAAATACAAAAATTAGCCAGGCATGGTGGCATGCACCTGTA ATCCCAGCTACTTGGGAGGCTGAGGCAGGAGAACTGCTTGAACCCGGAAGGTGGAGGTGGAATGAGCTCA GGTCACACGACTGCACTCCAGCCTGGGCAACAGAGAGAGACTGCATCTTATTTTTATTTTTTTAAAGTTAC AAATAAAAGTTTTCTTGGCTCTGAAAAACAAAGATCAGCAATGTTTTAAGTAAAAAGTCATAGAAGGGTT ATTTTAGTCTTCTATTAGTTCAGTCTATGCAGTTAACACCTGTTCTTGATATTCATAAACATTTCAG CTCTCCATGAGAGTCCTGAAAGTTTTTTCCTCGATTCTAATGTCACAATCTCCAAAGGTATCAGAAACCT GCATTCAAGAGCACCTGTCAAAGTCCTATAGCTGATTATAAACCACCTTTTGAAGAGAATCAAAACAAGA CAATAATTGTGGGTGACAAAGGTCTTAGGACAGCCACTACTAAAGCCACATTTGACAATTTTGGTTACT TCTGTGGCATACAATTTTACACAACAATTATAATTATTAATTACATACACTAAGCCATATCAGAATTACA GGACTTTCCAGTAATTTTGGAACATATACTAATAACATATTTATACAAATACAG Exon 1:1769-1876 Intron1: 1877-2404 Exon 2: 2405-2548 Intron2: 2549-11957 Exon3: 11958-12110 Intron3: 12111-13386 Exon4: 13387-13548

Claims

1. A non-human animal that has a gene in which at least one exon of Wfdc2 is flanked by two loxP sequences in both alleles, and that systemically expresses the enzyme CreERT2, which causes recombination in an antiestrogen-dependent manner, enabling the Wfdc2 gene to be deleted in an antiestrogen-inducible manner.

2. A method for producing a non-human animal model for chronic obstructive pulmonary disease, comprising inducing a deficiency of the Wfdc2 gene by administering an anti-estrogen drug to the non-human animal of claim 1 after it has become an adult.

3. A non-human animal model for chronic obstructive pulmonary disease, produced by the method of claim 2.

4. The non-human animal model of chronic obstructive pulmonary disease according to claim 3, which is used for screening of preventive and / or therapeutic agents for chronic obstructive pulmonary disease.

5. The non-human animal model of chronic obstructive pulmonary disease according to claim 3, which is used for screening biomarkers for chronic obstructive pulmonary disease.

6. The non-human animal model of chronic obstructive pulmonary disease according to claim 3, which is used to investigate the cause of chronic obstructive pulmonary disease.

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