Construction method of mouse model, cell line with functional deletion of edar gene and application

Abstract

This invention relates to the field of biomedical technology and discloses a method for constructing a mouse model. Edar Cell lines with gene loss of function and their applications, wherein the mouse model at least has epithelial cells. Edar A gene frameshift mutation, and the frameshift mutation leads to Edar The mouse model is characterized by the loss of functional expression of a gene-encoded protein; the method includes obtaining the mouse model through conditional deletion or systemic knockout. The method described in this invention successfully constructed a mouse model that highly mimics human diseases and further obtained related cell lines, clarifying its application value in disease mechanism research, drug screening, and therapeutic target validation.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a method for constructing a mouse model. Edar Cell lines with missing gene functions and their applications. Background Technology

[0002] Hypophidrotic ectodermal dysplasia (HED) is a rare congenital genetic disorder, with typical clinical manifestations including sparse hair, abnormal sweat gland development, and congenital tooth absence. Studies show that nearly one-quarter of HED cases are caused by… Edar Caused by genetic defects, patients exhibit symptoms such as dentition, enamel hypoplasia, etc. Edar The detailed mechanisms by which these mechanisms affect the development of teeth and other ectodermal accessory organs have not yet been thoroughly investigated.

[0003] Existing spontaneously mutant mouse strains Edar sleek It explained Edar While the biological functions of Edar have been observed, the phenotypic profile induced by Edar inactivation has not been fully reproduced. The lack of animal models that accurately mimic patient phenotypes and are suitable for mechanistic studies and drug screening also limits the scope of research. Edar A comprehensive study of HED caused by this condition.

[0004] Therefore, developing technologies that can specifically target epithelial cells is necessary. Edar The lack of animal models is crucial for elucidating the pathogenesis of HED abnormalities and developing intervention strategies. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a method for constructing a mouse model. Edar Cell lines with gene loss of function and their applications; this mouse model can accurately simulate... Edar HED caused by this.

[0006] To achieve the above objectives, the first aspect of the present invention provides a mouse model in which at least epithelial cells are present. Edar A gene frameshift mutation, and the frameshift mutation leads to Edar The functional expression of the gene-encoded protein is lost; The method includes obtaining the mouse model by conditional deletion or systemic knockout.

[0007] The second aspect of the present invention provides Edar Cell lines with gene loss of function, wherein the cell lines Edar The gene contains a frameshift mutation, and the frameshift mutation leads to Edar The functional expression of the gene-encoded protein is lost.

[0008] A third aspect of the present invention provides applications of the aforementioned mouse model or cell line, said applications including at least one of the following: (1) Screening or evaluation for prevention, relief or treatment Edar Candidate drugs, gene therapy methods, cell therapy, or other biological agents that target gene expression defects are preferred for screening purposes. Edar Candidate drugs with expression defects; (2) Research Edar The role of signaling pathways in the development, differentiation, or homeostasis of ectodermal organs such as teeth, hair, sweat glands, skin and their appendages or cornea.

[0009] Through the above technical solution, the present invention achieves the following beneficial effects: (1) This invention successfully constructed a mouse model that highly simulates human diseases. Through objective measurement and statistical analysis using micro-CT, tissue staining, sweat gland function tests, etc., it was found that compared with the existing spontaneously mutant mouse strains, this model can more comprehensively cover the phenotypic characteristics of patients and more accurately replicate the multidimensional distribution of their phenotypic spectrum, overcoming the limitations of existing models and having important clinical value for HED-related research. (2) In the cell line of the present invention Edar The presence of frameshift mutations in genes leads to the permanent loss of function of the encoded proteins, providing a stable and homogeneous disease cell model for in vitro studies and a new research platform for in-depth exploration of related molecular mechanisms. Attached Figure Description

[0010] Figure 1 It is a systemic absence Edar Construction and phenotypic characteristics of mouse models of gene expression; Figure 2 It is a systemic absence Edar Construction, breeding, and genotyping of mouse models of gene expression; Figure 3 The study focuses on the construction of a mouse model with a specific absence of Edar gene expression in epithelial cells, as well as changes in tooth morphology and mineralization. Figure 4 yes K14 Cre ;Edar fl / fl Schematic diagram of ectodermal defect phenotypes in mice; Figure 5 It is a specific deficiency in epithelial cells. Edar Detection of the degree of enamel mineralization damage and ameloblast function in mice expressing the gene; Figure 6 When it is P0 Edar fl / fl(Control group; Ctrl) and K14 Cre ;Edar fl / fl (Conditional knockout; cKO) Single-cell transcriptome analysis of the mandibular M1 tooth germ in mice; Figure 7 It is a transcriptional feature of an epithelial subset of a developing mouse mandibular molar germ; Figure 8 yes Edar fl / fl and K14 Cre ;Eda rfl / fl H&E staining images of the mandibular first molar (M1) of mice at 7.5 days (P7.5) and 11.5 days after birth; Figure 9 yes Edar fl / fl (Ctrl) and K14 Cre ;Edar fl / fl (cKO) Quality control (QC) metrics for the molar tooth germ scRNA-seq dataset; Figure 10 These are transcriptional features of different cell types in the mandibular molar tooth germs of P0 mice; Figure 11 yes Eda , Edar and Edaradd Expression in mesenchymal cells; Figure 12 It is different epithelial cell subsets Dlx3 , Shh , Msx2 , Irx5 , Adgrf4 and Bambi Comparison of expression levels between the conditional knockout group (green) and the control group (blue); Figure 13 yes Edar Knock down Dlx3 Expression and impairment of ALC mineralization can be achieved through Dlx3 Over-expression to salvage. Detailed Implementation

[0011] The specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0012] A first aspect of the present invention provides a method for constructing a mouse model, wherein the mouse model has at least epithelial cells. EdarA gene frameshift mutation, and the frameshift mutation leads to Edar The functional expression of the gene-encoded protein is lost; wherein, the method includes obtaining the mouse model by means of conditional deletion or systemic knockout.

[0013] In this invention, the frameshift mutation refers to... Edar Insertion or deletion of one or more nucleotides (not multiples of 3) in the coding sequence of a gene alters the reading frame of the codon downstream of that position, resulting in a translated amino acid sequence that is completely different from the wild-type protein. In most cases, this introduces a premature stop codon, ultimately causing the translated protein to lose its normal biological function. In this invention, the frameshift mutation specifically refers to a genetic variation that causes complete or partial loss of function of the EDAR protein.

[0014] In this invention, conditional deletion refers to the use of a spatiotemporally specific gene recombinase system (such as the Cre / loxP system) to achieve the deletion of a target gene at a specific cell type (such as epithelial cells) or a specific developmental stage. Edar This method involves targeted knockout of genes. It avoids the embryonic lethality or other systemic side effects that may result from systemic knockout, thus more accurately mimicking the loss of gene function in specific cell types during a disease.

[0015] In this invention, the systemic knockout refers to the introduction of loss-of-function mutations into all cells of a mouse using gene editing technologies (such as CRISPR / Cas9, TALEN, or ZFN), resulting in the target gene ( Edar It does not express or expresses non-functional proteins in any of the body's cells and germ cells.

[0016] In this invention, at least in epithelial cells, there exists Edar Frameshift mutations are... Edar The limiting statement regarding the scope of defects has the following meanings, including but not limited to the following three situations: (1) Exists only in epithelial cells Edar Frameshift mutations in genes are normally expressed in mesenchyme and other tissues; (2) Existing in epithelial cells Edar Frameshift mutations in genes, which are also chimeric or dose-dependently reduced in some mesenchymal cells; (3) Existing throughout the body Edar Frameshift mutation, meaning that all cell types lack functional EDAR protein expression.

[0017] In this invention, the mouse model Edar The gene's fourth exon (Exon4) and / or fifth exon (Exon5) are deleted.

[0018] In this invention, the mouse model can be specifically deficient in epithelial cells. Edar Mouse models of gene expression or systemic loss Edar Mouse models of gene expression.

[0019] In this invention, the specific deletion in the epithelial cells Edar Methods for constructing mouse models of gene expression can include: (1) Edar Flanking the gene LoxP site Edar fl / fl Mouse and epithelial cell-specific expression Cre Mice were hybridized with the gene and screened to obtain the genotype of [gene name missing]. Cre; Edar fl / + Offspring mice; (2) Cre; Edar fl / + mice and Edar fl / fl Mouse backcrossing resulted in the acquisition of specific deletions in epithelial cells. Edar Mouse models of gene expression.

[0020] In this invention, the LoxP The site can be located at Edar Flanking genes Exon4 and / or Exon5.

[0021] In this invention, the Edar fl / fl Mice can be obtained through the following methods: (1) In mice Edar Guide RNA (gRNA) targets were designed at both ends of Exon5 in the conserved gene region, and their activity was tested. Based on the Cas9 / gRNA activity results and target locations, gRNAs with higher in vitro activity were compared and selected, microinjected into mouse zygotes, and embryos were taken to test their endogenous activity. (2) After transcribing the endogenous gRNA with high activity in the embryo and Cas9nickase into mRNA and RNA in vitro, host DNA was added and microinjected into mouse zygotes. (3) Two weeks after the mice were born, their tails were cut off, and genomic DNA was extracted for PCR and sequencing. The genotypes of the mice were detected, and mice that achieved precise insertion of FloxP at both sites were selected. (4) Mice that have achieved precise insertion of FloxP at both loci are cultured to 6-8 weeks of age, and then self-crossed or mated with wild-type mice for several generations before self-crossing to obtain stably inherited mice. Edar fl / fl Mice.

[0022] In this invention, the Cas9nickase is obtained by mutating one of the two domains of the Cas9 nuclease and can be a commercially available conventional product.

[0023] In this invention, the epithelial cells specifically express... Cre Genes can be driven by either the keratin 14 promoter (K14) or the keratin 5 promoter (K5). Cre It is achieved through genes.

[0024] Preferably, the epithelial cells specifically lack Edar Methods for constructing mouse models of gene expression include: (1) Edar Flanking the gene LoxP site Edar fl / fl Mice and K14-driven epithelial cell-specific expression Cre The gene was crossed with mice, and the mice were screened to obtain the K14 genotype. Cre ; Edar fl / + Offspring mice; (2) K14 Cre ; Edar fl / + mice and Edar fl / fl Mouse backcrossing resulted in the acquisition of specific deletions in epithelial cells. Edar Mouse models of gene expression ( K14 Cre ; Edar fl / fl ).

[0025] In this invention, the so-called systemic absence Edar Methods for constructing mouse models of gene expression may include: introducing gene expression into the mouse genome using a gene editing system. Edar Loss-of-function mutations in gene pairs result in systemic deficiency. Edar Mouse models of gene expression.

[0026] Preferably, the gene editing system is a CRISPR / Cas9 system.

[0027] In this invention, the so-called systemic absence Edar Methods for constructing mouse models of gene expression can include: (1) For mice Edar gRNAs were designed from the Exon4 and / or Exon5 of the gene and the adjacent non-coding regions. (2) The guide RNA and Cas9 protein were introduced into mouse zygotes and gene editing was performed using the CRISPR / Cas9 system to obtain edited zygotes; (3) The edited fertilized eggs were transplanted into pseudopregnant female mice to obtain F0 generation mice. Edar Genetically modified mice; (4) Screening through genotype identification Edar Allele heterozygous mutation ( Edar + / - ) mice; (5) Edar + / - Mice mate with each other to acquire systemic [viruses / conditions]. Edar Homozygous gene knockout Edar - / - A mouse model of ).

[0028] In this invention, the mouse model can exhibit developmental abnormalities associated with hypohidrotic ectodermal dysplasia. These abnormalities may include dental malformations and / or ectodermal organ malformations in other regions.

[0029] Preferably, the abnormal tooth development phenotype includes at least one of the following: missing third molars, reduced number of teeth, reduced tooth size, root fusion, reduced number of cusps, molars exhibiting a bovine tooth-like morphology, enamel hypoplasia, or mineralization defects.

[0030] Preferably, the other ectodermal organ developmental abnormalities include at least one of sparse or absent hair, reduced number or dysfunction of sweat glands, abnormal development of skin and its appendages, or corneal epithelial abnormalities; more preferably, the abnormal development of skin and its appendages includes at least one of hair follicle, sebaceous gland, apocrine sweat gland, and nail structure abnormalities.

[0031] The second aspect of the present invention provides Edar Cell lines with gene loss of function, wherein the cell lines Edar The gene contains a frameshift mutation, and the frameshift mutation leads to Edar The functional expression of the gene-encoded protein is lost.

[0032] In this invention, the cell line can be derived from the mouse model constructed by the aforementioned method, for example, it can be directly isolated from the aforementioned epithelial cells that specifically lack [certain cell lines]. Edar Mouse models of gene expression or systemic loss Edar A mouse model of gene expression. This can also be achieved through isolation. Edar fl / fl Mouse cells, and using Cre The cell line was obtained by in vitro knockout.

[0033] A third aspect of the present invention provides applications of the mouse model constructed by the aforementioned method or the aforementioned cell line, said applications including at least one of the following: (1) Screening or evaluation for prevention, relief or treatment Edar Candidate drugs, gene therapy methods, cell therapy, or other biological agents that target gene expression defects are preferred for screening purposes. Edar Candidate drugs with expression defects; (2) Research Edar The role of signaling pathways in the development, differentiation, or homeostasis of ectodermal organs such as teeth, hair, sweat glands, skin and their appendages or cornea.

[0034] In this invention, the Edar Gene expression defects can manifest as developmental abnormalities associated with hypohidrosis ectodermal dysplasia.

[0035] In this invention, the screening is used for treatment. Edar Approaches to candidate drugs with gene expression defects may include: (1) Treat the mouse model or cell line constructed by the above method with the candidate drug; (2) Compare the changes in mouse models or cell lines before and after treatment to determine whether the candidate drugs have an improving effect; (3) Identify candidate drugs that have an improving effect as potential therapeutic agents.

[0036] In this invention, the changes in the mouse model may include changes in tooth phenotype, changes in the phenotype of other ectodermal organs, and changes in the expression levels of marker genes related to enamel mineralization or cell proliferation. The changes in the cell line may include changes in the expression levels of marker genes related to enamel mineralization or cell proliferation. Preferably, the tooth phenotype may include at least one of the following: number of teeth, tooth size, root morphology, number of cusps, and enamel mineralization density and thickness. The other ectodermal organ phenotypes may include at least one of the following: hair density and morphology, number and function of sweat glands, structure of skin and its appendages, and corneal integrity.

[0037] In this invention, the biomarker gene may include Dlx3 , Amelx , Ambn , Enam , Amtn , Odam At least one of them.

[0038] In this invention, the improving effect may include at least one of the following: (1) Improvement of tooth phenotype in mouse models; (2) Improvement of phenotypes of other ectodermal organs in mouse models; (3) Reverse or alleviate the abnormal expression of the marker genes; (4) The differentiation capacity and function of the ectoderm-derived cells or tissues with impaired function are improved or restored.

[0039] This invention also provides a biomarker in preparation Edar Application in products for the detection, diagnosis, or prognosis prediction of hypohidrotic ectodermal dysplasia caused by genetic defects, wherein the biomarker is Dlx3.

[0040] In this invention, the biomarker refers to a molecular entity that can be specifically identified and quantified in the biological samples of a subject, and its presence or expression level is related to... Edar The pathological state of HED caused by gene defects is statistically significant and is supported by biological mechanisms.

[0041] In this invention, the Edar Genetic defects refer to Edar The abnormal reduction or absence of gene function or expression can be achieved through techniques such as gene editing, conditional gene knockout, or RNA interference. This defect can manifest as dental developmental abnormalities in HED, such as enamel hypoplasia.

[0042] In this invention, the Dlx3 Distal-less homeobox 3 (DICOM) is a homeobox transcription factor that is highly expressed in the early stages of tooth development and specifically regulates ameloblast differentiation and the transcription of enamel matrix proteins. Dlx3 The housekeeping gene is not universally expressed but is enriched in the tooth germ cervical loop (CL) and pre-ameloblasts. Edar When the signal is intact, it maintains its normal expression level; when Edar When function is lost, single-cell RNA sequencing (scRNA-seq) and immunohistochemical (IHC) staining data confirm the presence of the defect. Dlx3 A persistent, dose-dependent downregulation was observed in CL, early ameloblasts, and secretory ameloblasts. This downregulation occurred earlier than the histological manifestation of enamel mineralization defects, indicating... Dlx3 It has the advantage of serving as a time window for early warning.

[0043] This invention, through the Edar scRNA-seq analysis of biological samples from subjects with HED caused by decreased or absent gene function or expression revealed that, in addition to exhibiting enamel mineralization and impaired ameloblast function, six genes were persistently downregulated in their ameloblasts, including... Dlx3 , Shh , Msx2 , Bambi , Adgrf4 and Irx5 .

[0044] In this invention, the biomarker may further include at least one of Shh, Msx2, Bambi, Adgrf4, Irx5, Amelx, Ambn, Enam, Amtn, and Odam.

[0045] This invention will... Dlx3 Transfection of the overexpressed plasmid into ameloblasts of the subject showed successful transfection. Dlx3 Cells overexpressing the plasmid showed reduced or restored mineralization capacity, and its overexpression also upregulated the expression of specific enamel genes (including...). Amelx , Ambn , Enam , Amtn and Odam ).

[0046] In this invention, the biomarker can be at least one of DNA, RNA, cDNA, and protein.

[0047] In this invention, Dlx3 can be detected using any one of the following forms: its gene coding sequence (NCBI Gene ID: 13393), mature mRNA (containing the complete ORF and 3'UTR), or the DLX3 protein. The DLX3 protein can be detected through... Dlx3 The gene encodes and synthesizes DLX3 protein, which can also be an active form of DLX3 protein that has been modified by phosphorylation or altered in subcellular localization.

[0048] In this invention, the product may include a substance for detecting the biomarker at the gene level. This substance may include at least one of a probe that specifically identifies the biomarker, a gene chip that heterologously identifies the biomarker, and primers that specifically amplify the biomarker. The preparation methods for the probe and chip pairs can both be conventional practices in the art.

[0049] Preferably, the primers for the specific amplification of biomarkers are as shown in SEQ ID NO.1-SEQ ID NO.26, as detailed in Table 1.

[0050] The product may take the form of a reagent, test plate, test strip, kit, chip, device, system, or nucleic acid membrane strip, all of which are conventional product forms in the field.

[0051] In this invention, the kit can be a PCR kit, containing specific amplification... Dlx3 The primer pairs and fluorescent probes for the gene are shown in Table 1. Depending on the testing method, specific recognition may also be included. Dlx3 Gene probes and specific recognition Dlx3Gene chips or antibodies that specifically recognize the DLX3 protein.

[0052] In this invention, the system described herein may refer to the system-related content described later in this invention.

[0053] This invention also provides a screening treatment Edar A method for treating hypohidrotic ectodermal dysplasia caused by a genetic defect, the method comprising screening the drug using a biomarker, wherein the biomarker is Dlx3.

[0054] In this invention, the method may include: treating the aforementioned mouse model or cell line with the drug, comparing the expression changes of biomarkers in the mouse model or cell line before and after treatment, and identifying drugs that can reverse or alleviate the abnormal expression of the biomarker genes as potential therapeutic agents.

[0055] The present invention also provides a pharmaceutical composition for the preparation of therapeutic drugs. Edar Application in products for hypohidrosis-related ectodermal dysplasia caused by genetic defects, characterized in that the pharmaceutical composition comprises a Dlx3 activator.

[0056] In this invention, the Dlx3 activator can be a drug capable of upregulating intracellular... Dlx3 Substances that enhance gene expression levels or increase the activity of DLX3 protein.

[0057] In this invention, the Dlx3 activator refers to any agent that can directly or indirectly cause intracellular... Dlx3 Increased gene transcription levels Dlx3 Substances or combinations that enhance mRNA stability, increase DLX3 protein translation efficiency, or enhance DLX3 protein transcriptional activity. Their core function is to reverse or restore [the effects of certain factors]. Edar Caused by genetic defects Dlx3 Dlx3 activators can express abnormalities, thereby rescuing or improving associated case phenotypes in cell or animal models. These Dlx3 activators include, but are not limited to, gene-level activators, protein-level activators, small molecule agonists, or regulatory pathway agonists.

[0058] In this invention, the gene-level activator refers to an agent capable of activating functional genes. Dlx3Preparations for delivering gene sequences into target cells, including but not limited to nucleic acid vectors, viral vectors, or RNA preparations. The nucleic acid vector contains cDNA or gene DNA sequences encoding the complete DLX3 protein or functionally active fragments thereof (such as those retaining DNA-binding and transcriptional activation domains), which can be cloned into various recombinant expression vectors, such as plasmids, viscera, or artificial chromosomes. The viral vector refers to a delivery vehicle using recombinant viruses, including but not limited to adeno-associated virus (AAV), lentivirus, or adenovirus, which are modified to carry... Dlx3 The RNA agent contains genes and can efficiently transduce target cells. The RNA preparation includes in vitro transcription... Dlx3 mRNA, or mRNA modified with bases to enhance stability and translation efficiency, can be directly introduced into cells via lipid nanoparticles or other delivery systems to supplement functional mRNA. Dlx3 mRNA.

[0059] In this invention, the protein-level activator includes, but is not limited to, recombinant proteins or protein stabilizers. The recombinant protein refers to a biologically active recombinant DLX3 protein or its functional domains expressed and purified in vitro, which can directly supplement the deficiency of functional proteins in cells. The protein stabilizer can bind to the DLX3 protein, inhibiting its ubiquitination or nuclear localization by small molecule compounds or peptides, thereby prolonging its half-life and enhancing its activity in the cell nucleus.

[0060] In this invention, the small molecule activator refers to an agent that can act on... Dlx3 Gene promoters, enhancers, and other regulatory regions, or by modifying chromatin states (such as histone acetylation and DNA demethylation), can upregulate endogenous genes. Dlx3 Compounds for gene transcription. The small molecule activators also include small molecules capable of directly binding to the DLX3 protein and enhancing its transcriptional activation activity, or promoting its interaction with coactivators.

[0061] In this invention, the regulatory pathway agonist refers to an agent that activates... Edar Downstream effector molecules of the signaling pathway (such as the key kinase IKK or transcriptional subunit p65 in the NF-κB pathway), or inhibitors Dlx3 The negative regulatory pathway, thereby indirectly enhancing Dlx3 Substances that express or activate genes.

[0062] In this invention, the pharmaceutical composition may also contain other therapeutic agents. EdarThe active ingredient for hypohidrotic ectodermal dysplasia caused by genetic defects preferably further includes at least one of Shh activator, Msx2 activator, Bambi activator, Adgrf4 activator, Irx5 activator, Amelx activator, Ambn activator, Enam activator, Amtn activator, and Odam activator.

[0063] In this invention, with Edar Dental developmental abnormalities associated with hypohidrotic ectodermal dysplasia due to genetic defects may include at least one of the following: absence of third molars, reduced number of teeth, reduced tooth size, root fusion, reduced number of cusps, bovine molar morphology, enamel hypoplasia, or mineralization defects.

[0064] The present invention also provides a method for Edar A system for detecting, diagnosing, or predicting the prognosis of hypohidrotic ectodermal dysplasia caused by a genetic defect, characterized in that the system comprises: A detection component for detecting biomarkers at the gene level; Result judgment component: The result judgment component is used to output at least one of the detection result, diagnosis result and prognosis result based on the result of the biomarker detected by the detection component.

[0065] In this invention, the preparation methods of the detection component and the result judgment component are all conventional operations in the art. The biomarker is the aforementioned biomarker of this invention.

[0066] The present invention will be described in detail below through examples. Unless otherwise specified, all reagents and materials are commercially available. Experimental methods not specifically described herein are generally performed under standard conditions or as recommended by the manufacturer. Unless otherwise defined, all technical and scientific terms used herein are intended to be used in the same manner as those familiar with the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be used in the present invention.

[0067] Materials and Methods: Construction and acquisition of mouse models All animal experimental procedures in this invention were approved by the Institutional Animal Care and Use Committee (IACUC) of the Ninth People's Hospital Affiliated to Shanghai Jiao Tong University School of Medicine (SHPH-2025-A1567-1). All mice were housed on a C57BL / 6J background under standard pathogen-free conditions. The CRISPR / Cas9 system was used to construct... Edar Knockout homozygotes ( Edar - / - The mouse model was purchased from Beijing Vitonda Biotechnology Co., Ltd. The mouse strain creation strategy involved knockout... Edar (ENSMUSG00000003227.5) Exon 4 and non-coding region sequence of the transcript region.

[0068] Edar fl / fl The mice were constructed by Beijing Vitonda Biotechnology Co., Ltd. Edar The fifth exon of the gene is flanked by LoxP Site. K14-Cre The mice were a generous gift from Professor Zou Weiguo of the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, and were accompanied by... Edar fl / fl Mouse hybridization was used to generate ectoderm and its derivatives (including skin, oral ectoderm, hair follicles, and salivary glands) cell-specific knockout mice.

[0069] Starch-Iodine Experiment Mice were restrained in a device, and their hind paws were coated with a 3% (w / v) iodine ethanol solution. After drying, a 40% (w / v) starch mineral oil suspension was applied. Images were taken after 90 seconds using a microscope (Leica KL300 LED, Nussloch, Germany). Sweat was detected as dark spots.

[0070] Micro-CT Scan Analysis Mandibles with molars and incisors were obtained from 5-week-old mice for micro-CT scanning (instrument: SCAN 1176, Bruker, Kontich, Belgium). All samples were preserved in 70% ethanol prior to micro-CT scanning. Tooth volume, enamel thickness, pulp chamber height, and distal root length were analyzed using Bruker's CT analyzer software. Tooth morphology reconstruction was performed using Bruker's CT-vox software.

[0071] Scanning electron microscopy (SEM) analysis The mandibles of 5-week-old mice from all groups were dissected and fixed in 2% paraformaldehyde and 2.5% glutaraldehyde prepared in 0.1 M dimethylarsine buffer (pH 7.4). The samples were then dehydrated sequentially with increasing concentrations of ethanol and dried in a critical point desiccator. After gold plating, the samples were observed using a FEI Quanta 250 field emission environmental scanning electron microscope (Hillsboro, OR, USA).

[0072] Hematoxylin and eosin (H&E) staining and picric acid Sirius red (PSR) staining Mouse mandibles were dissected, fixed overnight in 4% paraformaldehyde (PFA; Servicebio), decalcified with EDTA decalcification solution (Servicebio), dehydrated using a gradient of ethanol, embedded in paraffin, and sectioned sagittally (4 µm). H&E staining was performed according to standard protocol to observe tissue morphology. For collagen detection, tissue sections were stained with 0.1% Sirius red in saturated picric acid for 1 hour, and then rinsed in acidified water.

[0073] Tissue preparation and single-cell dissociation The mandibular first molar tooth germ was carefully dissected using microforceps under a stereomicroscope and transferred to MACS tissue storage solution (Miltenyi Biotec) until further processing. Newborn mice were designated day 0 (P0) on the day of birth. When obtaining P0... K14 Cre ;Edar fl / fl mice and Edar fl / fl Tooth germ cells were used for scRNA-seq. Molar tooth germ tissues were obtained from eight newborn mice in each group and then combined.

[0074] Single-cell RNA-seq experiments were conducted by researchers at Novogene Co., Ltd. The tissue sample processing is briefly described below. First, the samples were washed with 1640 culture medium and then chopped into small pieces (approximately 1 mm) on ice. 3 The cells were digested at 37°C for 30–40 minutes with 1 mg / mL collagenase I, 0.5 mg / mL collagenase II, 0.4% 1 mg / mL dispersin, and 50 U / mL DNase I. The digestion was terminated by adding an equal volume of 1640 culture medium containing 10% FBS to the supernatant. After digestion, the sample was filtered through a 70 μm cell filter. After removing the supernatant, the precipitated cells were resuspended in erythrocyte lysis buffer (Miltenyi Biotec) to lyse the erythrocytes. The dissociated single cells were then stained with AO / PI staining, and their viability was assessed using a Countstar fluorescence cell analyzer.

[0075] Single-cell RNA sequencing (scRNA sequencing) and its data processing and analysis scRNA sequencing process: Transcriptome information of single cells was captured using the BD Rhapsody system.

[0076] Single-cell capture was achieved by randomly distributing single-cell suspensions into >200,000 microwells using a limiting dilution method. Magnetic beads with oligonucleotide barcodes were added to saturation, ensuring one bead paired with a cell in one microwell. Cell lysis buffer was added to hybridize polyadenylated RNA molecules to the magnetic beads. The beads were collected into single tubes for reverse transcription. After cDNA synthesis, each cDNA molecule was tagged with a unique molecular identifier (UMI) and a cell tag indicating its cell origin at its 5' end (i.e., the 3' end of the mRNA transcript). A whole transcriptome library was prepared using the BD Rhapsody single-cell whole transcriptome amplification workflow. Briefly, second-strand cDNA was synthesized and then ligated with WTA adapters for universal amplification. The adapter-ligated cDNA product was amplified using 18 cycles of PCR. The whole transcriptome amplification product was used to prepare a sequencing library to enrich the 3' ends of transcripts ligated with cell tags and UMIs. Sequencing libraries were quantified using a High Sensitivity DNA microarray (Agilent) on a Bioanalyzer 2200 and the Qubit High Sensitivity DNA Detection Kit (Thermo Fisher Scientific). All libraries were sequenced at 150 bp paired ends using a Novaseg 6000 (Illumina, San Diego, CA).

[0077] Data processing: scRNA-seq data analysis was performed using the NovelBrain cloud analytics platform by Novogene Corporation. FastP (v0.21.0) with default parameters was used to filter adapter sequences and remove low-quality reads to obtain clean data. UMI-tools was used for single-cell transcriptome analysis to identify a whitelist of cell barcodes. To quantify gene expression in single-cell data, STARsolo (version 2.7.10a) and mouse genome mm10 (Ensembl annotation version 100) were used. Cells containing more than 200 expressed genes and with a mitochondrial UMI rate below 20% passed cell quality filtering, and mitochondrial genes were removed from the expression table.

[0078] Use the Seurat package (version: 4.0.3). https: / / satijalab.org / seurat / Cellular normalization and regression were performed based on the expression table, using UMI counts and mitochondrial gene percentages for each sample, to obtain scaled data. Based on the scaled data, a PCA was constructed using the top 2000 hypervariable genes, and tSNE and UMAP were constructed using the top 10 principal components. Unsupervised cell clustering results were obtained using graph-based clustering methods based on the top 10 principal components of the PCA. Marker genes were calculated using the Wilcox rank-sum test algorithm with the FindAllMarkers function, based on the following criteria: 1. Log2FC > 0.25; 2. p-value < 0.05; 3. min.pct > 0.1. To identify cell types in detail, clusters of the same cell type were selected for re-analysis of tSNE, graph-based clustering, and marker gene analysis.

[0079] Data Analysis: Gene Ontology (GO) Analysis To elucidate the biological significance of DEGs and marker genes, GO analysis was performed. Data from NCBI (…) http: / / www.ncbi.nlm.nih.gov / ), Gene Ontology Database ( http: / / www.geneontology.org / ) and UniProt ( http: / / www.UniProt.org / Download the GO annotations. Apply Fisher's exact test to identify significant GO categories and correct the p-values ​​using FDR.

[0080] Gene enrichment analysis For gene enrichment analysis, Fisher's exact test was used to calculate the p-value for each gene set. The original p-values ​​were corrected for multiple hypothesis testing using the Benjamini and Hochberg methods. This enrichment analysis was applied to databases including GO (v2.5.13), Kyoto Encyclopedia of Genes and Genomes (KEGG, 201900613), and Hallmark (h.all.7.0.). https: / / www.gsea-msigdb.org / gsea / msigdb / index.jsp ), chromosome location gene set ( https: / / www.gsea-msigdb.org / gsea / msigdb / index.jsp Annotations, including custom gene sets, were included. Forty-one custom gene sets containing terms related to immunity, cytokines, and neurobiology were collected from the CellphoneDB database. QuSAGE (2.16.1) analysis was performed to characterize the relative activation of a given gene set.

[0081] Quasi-time series analysis Using Monocle2 ( http: / / cole-trapnell-lab.github.io / monocle-release / Single-cell trajectory analysis was performed, and significant transformation relationships between cell types and clusters were determined using the DDR-Tree and default parameters. Prior to Monocle analysis, marker genes from Seurat clustering results and raw expression counts of filtered cells were selected. Based on pseudo-temporal analysis, branch fate-determining gene analysis was performed using branch expression analysis modeling (BEAM analysis).

[0082] CytoTrace The application of cell trajectory reconstruction analysis uses the computational method of gene counting and expression (CytoTRACE, v0.3.1) to predict the differentiation state of cells in single-cell RNA sequencing data by calculating the number of genes expressed in each cell.

[0083] Differential gene expression analysis To identify differentially expressed genes among samples, the FindMarkers function and the Wilcox rank-sum test algorithm were used, with the following criteria: 1. Log2FC > 0.25; 2. p-value < 0.05; 3. min.pct > 0.1. This is more conducive to obtaining genes with different functions among samples.

[0084] Cell culture and differentiation induction Ameloblastic lineage cells (ALCs) were kindly donated by Professor Gao Yuguang (Binzhou Medical University, Binzhou, China). ALCs were cultured in DMEM / F-12 (Gibco, Grand Island, New York, USA) supplemented with 10% fetal bovine serum (Merren) and 1% penicillin / streptomycin complex, and in a humidified incubator at 37°C and 5% CO2. The mineralization induction medium consisted of 50 mg / L ascorbic acid, 10 mM β-glycerophosphate sodium, and 10... -8 M DEX (Sigma) growth medium. When the confluence reaches 60%-70%, replace the growth medium with differentiation medium.

[0085] Edar Construction of knockdown cells Stable knockdown in ALCs was achieved using an shRNA-mediated RNA interference system combined with lentiviral transduction. Edar Genes. In short, synthetic targeting. EdarThe short hairpin RNA (shRNA) sequence of the mRNA (5'-CCGGGCACTAAGAGACGACGACTATGCTCGAGCATAGTCGTCTCCTTTAGCGCTTTTT-3') was subcloned into the pLKO.1-puro vector. The recombinant plasmid was then co-transfected with packaging plasmids (PSPAX2 and PMD2.G) into HEK-293FT cells to generate lentiviral particles. Forty-eight hours later, the viral supernatant was harvested, filtered, and used to infect ALCs in the presence of 8 µg / mL polygel. Two days after infection, ALCs were selected for stability after one week using 5 µg / mL puromycin. Edar Cells were knocked down. Total RNA and protein were extracted, and the knockdown efficiency was confirmed by qRT-PCR and Western blotting. Edar Knockdown cells and control cells (infected with non-targeting shRNA lentivirus) were named respectively. Edar Knock down (shEdar) and Edar Compare (Ctrl).

[0086] Alizarin Red S (ARS) staining Change the mineralization induction medium every two days. After incubation for 12-14 days, fix the cells with 4% paraformaldehyde (PFA) and then stain with 1% Alizarin Red S according to the manufacturer's instructions (Cyagen).

[0087] Quantitative real-time PCR Total RNA samples were extracted using the RNeasy® Plus Mini Kit (QIAGEN). First-strand cDNA was synthesized using 1 µg of total RNA using the PrimeScript RTReagent kit (RR036A, Taraka, Osaka, Japan). qRT-PCR was performed using a LightCycler 480 II system (Roche, Basel, Switzerland) and a ChamQ Universal SYBR qPCR Master Mix (Q711-02, Vazyme). Primers used for real-time PCR (CCIN-qPCR-F, CCIN-qPCR-R) are listed in Table 1. Gapdh Genes were used as internal reference genes to compare quantity and quality. Finally, through 2 -ΔΔCt The method determines the relative expression level of the gene to be tested.

[0088] Table 1

[0089] Western blot analysis Total protein was extracted from cells using RIPA lysis buffer (Beyotime). Following sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the proteins were electroblotted onto a polyvinylidene fluoride membrane. The membrane was then blocked with 5% skim milk and incubated overnight at 4°C with primary antibodies (listed in Table 2). The membrane was then incubated with secondary antibodies and developed using Immobilon Western HRP substrate (Merck Millipore).

[0090] Table 2

[0091] Immunofluorescence (IF) and immunohistochemistry (IHC) detection Paraffin-embedded sections were dewaxed and hydrated. They were then boiled in sodium citrate buffer (10 µM, pH 6.0) for 15 minutes, blocked in blocking buffer (ZSGB-BIO) at room temperature for 1 hour, and incubated overnight at 4°C with primary antibody against E-cadherin (1:100; ab76319, Abcam, Cambridge, UK). After washing three times with 1x phosphate-buffered saline (PBS), sections were incubated with secondary antibody (ZSGB-BIO) at room temperature for 1 hour. Cell nuclei were stained using DAPI.

[0092] For IHC, when anti- Dlx3 After incubation with the primary antibody (1:100; sc-514094, Santa Cruz Biotechnology, CA, USA), the secondary antibody was incubated, and the slides were developed using DAB according to the instructions (Gene Tech, GK500705). Images were taken (40x magnification) using a pathological slide scanner (KFBIO, KF-FL-020, China).

[0093] plasmid transfection shCtrl and shEdar ALCs were seeded in 12-well plates and transfected with 500 ng of the empty vector or Dlx3-HA expression plasmid using Lipofectamine 3000 (Invitrogen) according to the manufacturer's instructions. Cells were collected for subsequent analysis 48 hours after transfection.

[0094] Statistical analysis All quantitative data are expressed as mean ± standard deviation based on at least three independent samples. Statistical comparisons between two groups were performed using Student's t-test. Statistical comparisons among multiple groups were performed using one-way ANOVA. p < 0.05 was considered statistically significant.

[0095] Experimental results: whole body missing EdarGene expression leads to ectodermal dysplasia and tooth loss in mice. Through hybridization Edar + / - Mouse Edar - / - Mice, in which the CRISPR / Cas9 system was specifically used to delete Edar Exon 4 and non-coding region sequences of genes (such as...) Picture 1 A and Picture 2 (As shown). Western blot analysis of the tooth germ further confirmed this. Edar - / - The successful establishment of mice (e.g.) Picture 1 (As shown in B). Homozygous simulated clinical characteristics of HED patients. Edar The mutant mice exhibited severe ectodermal defects, including hairless tails and abdomens, curved tail tips, underdeveloped eyelids, and bald patches behind the ears.

[0096] In comparison, wild-type (WT) and heterozygous ( Edar + / - Mice do not have these defects (e.g.) Picture 1 (as shown in C).

[0097] Then we focus on Edar Dental manifestations in mutant mice. Compared with WT and Edar + / - The three roots of the first and second maxillary molars (M1 and M2) in mice are typically observed to be different. Edar - / - M1 and M2 in mice are single roots (e.g.) Picture 1 (As shown in D). Furthermore, in Edar - / - Two distinct mandibular dental phenotypes were observed in mice: phenotype A involved the absence of the third molar (M3), while phenotype B was characterized by the M1 becoming a single root. These two phenotypes could be observed simultaneously in the same mouse or occur independently, with incidence rates of approximately 33.33% and 66.67%, respectively (e.g., ...). Picture 1 (As shown in E). Interestingly, in phenotype B, when M1 is very small, M2 and M3 are larger and may exceed the size of these teeth in phenotype A. Three-dimensional reconstruction of M1 shows that the mutant has an increased pulp chamber height, similar to bovine dentition (as shown in E). Picture 1 (As shown in F). Statistical analysis shows that, compared with WT and Edar + / - Compared to the group, Edar - / - At 5 weeks, the tooth volume and enamel volume of group M1 decreased, while the enamel thickness showed no significant difference (e.g., Picture 1(As shown in G). Subsequently, pseudo-color imaging revealed defective enamel mineralization in the mandibular incisors of the mutant mice. Statistical analysis further showed that both tooth volume and enamel volume of the incisors were reduced (e.g., as shown in G). Picture 1 H, Picture 1 (As shown in Figure I). The schematic diagram illustrates... Edar Two phenotypic variations in the molars of knockout mice, as well as mineralization defects and round morphology in the incisor region (e.g., Picture 1 J、 Picture 1 (as shown in K).

[0098] These findings suggest that, Edar Knockout mice do indeed mimic the phenotype of HED patients and exhibit developmental dental abnormalities. However, Edar Complete knockout of the gene leads to embryonic lethality, with less than half of homozygous mice surviving (e.g., ...). Picture 2 (As shown). Furthermore, it remains unclear in which cell types EDAR plays a key role. Therefore, we adopted... Edar The conditional knockout model was developed to study its biological function in more detail.

[0099] Picture 1 It is a systemic absence Edar Construction and phenotypic characteristics of mouse models of gene expression, among which; A is a systemic deficiency. Edar Strategies for constructing mouse models of gene expression; B is a wild-type (WT) at day 0 (P0) of birth and Edar - / - Western blot analysis of EDAR protein expression in the first mandibular molar (M1) of mice; C is male. Edar - / - Severe ectodermal defects in mice include bald patches behind the ears (yellow arrows), underdeveloped eyelids, complete alopecia on the chest (yellow arrows), a twisted tail tip, and reduced sweating. WT and heterozygotes ( Edar + / - The mice were normal; D is the WT at 5 weeks of age. Edar + / - and Edar - / - Representative sagittal and corresponding axial micro-CT images of the maxillary molars of mice; E is the WT at 5 weeks of age. Edar + / - and Edar - / - Micro-CT reconstructed images of the mandibular teeth of mice, in the middle of the mouse EdarThe lack of a third molar leads to a bridge-like morphology of M1 (pheno A, Pheno A), accompanied by the congenital absence of the third molar (M3), or a single-root morphology of M1 (pheno B, Pheno B). F is the WT at 5 weeks of age. Edar + / - and Edar - / - A 3D reconstruction of mouse M1, with enamel shown in white, dentin in yellow, and pulp in red. The blue double-headed arrows indicate the pulp chamber height, i.e., the distance from the top to the bottom of the pulp chamber. G is the quantification of the parameters of M1 tooth at 5 weeks, which measures enamel volume, enamel thickness and total tooth volume (enamel + dentin + pulp). H is a micro-CT analysis of the mandibular incisors at 5 weeks, showing cross-sectional views (af) of the enamel at the early (a, c, e) and late (b, d, f) maturation stages. I represents the quantification of mandibular incisor (LI) tooth parameters at 5 weeks, measuring enamel volume and total tooth volume; J is a systemic absence. Edar A diagram illustrating the Mohr phenotype of mice expressing the gene. Edar The lack of τ desmosomes leads to the τ desmosome-like morphology of M1 (Pheno A) or single tooth (Pheno B); K is Edar - / - Enamel mineralization defects in the incisor; coronal section shows a blunt incisor shape and enlarged pulp chamber. ****p ＜0.0001 ,ns, not significant; each group n =5).

[0100] Picture 2 It is a systemic absence Edar The construction, breeding, and genotyping of mouse models of gene expression, including: A represents the breeding strategy for mice; B is obtained through PCR sequencing. Edar Genotyping of the knockout mice revealed that the 700 bp allele was the wild-type (WT) allele; the 200 bp allele was... Edar + / - Alleles; C is Edar - / - The embryonic lethality of mice is determined by the deviation of gene frequencies from Mendelian ratios, indicating that homozygotes are lethal.

[0101] Epithelial cell specificity Edar The absence of these features also leads to ectodermal dysplasia and tooth loss in mice. We will Edarfl / fl mice and K14 Cre Mice mating produced K14 Cre ;Edar fl / fl Mice, K14 Cre It is a kind of Cre Transgenic lines that activate only epithelial cells (e.g.) Picture 3 As shown in Figure A). Epithelial cell specificity was verified by Western blot analysis. Edar Conditional knockout mice showed that EDAR protein expression was almost completely absent in dental epithelial tissue (e.g., Picture 3 As shown in B). K14 Cre ;Edar fl / fl The mice exhibited severe ectodermal defects, similar to Edar - / - Mice and HED patients (e.g.) Picture 4 (As shown). Furthermore, in K14 Cre ;Edar fl / fl In mice, the mesial root and lingual root of the maxillary M1 are almost completely fused, although their complex origin can still be discerned from their surface morphology (e.g., Picture 3 C Picture 3 D、 Picture 3 E is shown. K14 Cre ;Edar fl / fl Mice also exhibited congenital M3 deletion at specific frequencies, with an incidence ranging from 30% to 70% in different regions, corresponding to phenotype A described earlier (e.g., Picture 3 (as shown in F). However, phenotype B was not observed, which means that M1 did not transform into a single-rooted tooth. K14 Cre ;Edar fl / fl The M1 mandibular region of mice exhibits a morphology similar to bovine dentition, characterized by increased pulp chamber height, significantly shortened distal roots, and reduced enamel and total tooth volume (e.g., ...). Picture 3 G, Picture 3 (As shown in H). Subsequently, pseudo-color imaging was displayed. K14 Cre ;Edar fl / fl The enamel mineralization of the mandibular incisors in mice was impaired. Statistical analysis showed that both the enamel and tooth volume of the incisors were reduced (e.g., Picture 3 I, Picture 3 (As shown in J). The schematic diagram illustrates the relationship with... Edar - / -Similar dental phenotypes observed in mice include M3 absence, reduced tooth size, bovine-like morphology, and rounded incisors (such as...). Picture 3 (as shown in K).

[0102] Despite being 5 weeks old K14 Cre ;Edar fl / fl M1 enamel thickness in mice and Edar fl / fl No significant differences were observed in mice, but a marked reduction was observed at 12 weeks of age (e.g. Picture 3 L, Picture 3 M). To investigate the cause of this change, scanning electron microscopy (SEM) was performed on the M1 molars of 5-week-old mice. The results showed that the surface of the mutant mice's molars was cracked, and the enamel prisms were clearly exposed, indicating a defect in enamel development (e.g., as shown in M). Picture 3 (as shown in N). These findings suggest that epithelial cells Edar The mice lacked enamel hardness and mineralization defects. In summary, epithelial cell specificity... Edar The absence was reproduced in the whole body Edar The phenotype observed in knockout mice and HED patients. Furthermore, this deficiency is associated with enamel defects and other developmental dental abnormalities.

[0103] Picture 3 It is a specific deficiency in epithelial cells. Edar The construction of a mouse model of gene expression and the changes in tooth morphology and mineralization, including: A is a specific deficiency in epithelial cells. Edar Strategies for constructing mouse models of gene expression; When B is P0 Edar fl / fl and K14 Cre ;Edar fl / fl Western blot analysis of EDAR expression in mouse dental epithelium; C is at 5 weeks of age Edar fl / fl and K14 Cre ;Edar fl / fl Representative sagittal and corresponding axial micro-CT images of the maxillary molars of mice; D is at 5 weeks of age Edar fl / fl and K14 Cre ;Edar fl / fl Views of the cheek and jaw of mouse maxilla M1; E is at 5 weeks of age K14Cre ;Edar fl / fl Top view of the mouse maxilla M1; F is at 5 weeks of age Edar fl / fl and K14 Cre ;Edar fl / fl Microscopic CT reconstruction of mouse mandibular teeth; G is at 5 weeks old Edar fl / fl and K14 Cre ;Edar fl / fl A 3D reconstruction of the mouse mandible M1, with enamel shown in white, dentin in orange, and pulp in red. The blue double-headed arrows indicate the pulp chamber height, i.e., the distance from the top to the bottom of the pulp chamber. H is a quantitative analysis of the parameters of the mandibular M1 tooth at 5 weeks of age, measuring enamel volume, tooth volume, pulp chamber height, and distal root length; I is a micro-CT reconstruction image of the mandibular incisor at 5 weeks of age. The cross sections (ad) show the enamel in the early (a, c) and late (b, d) maturation stages, respectively. J is a quantitative analysis of tooth parameters of the mandibular incisors (LI) at 5 weeks of age, measuring enamel and tooth volume; K is specifically absent in epithelial cells. Edar M1 mice with gene expression exhibit bovine tooth disease (similar to phenotype A), which causes enamel mineralization defects and rounded tooth morphology; L is at 24 weeks of age Edar fl / fl and K14 Cre ;Edar fl / fl Sagittal CT micro-image of mouse molars, with red arrows indicating areas of enamel wear; M is at 5 weeks or 24 weeks of age. Edar fl / fl and K14 Cre ;Edar fl / fl Quantitative analysis of enamel thickness in mouse M1 mice; N is at 5 weeks of age Edar fl / fl and K14 Cre ;Edar fl / fl Appearance and scanning electron microscopy (SEM) images of the mandibular molars of mice, with white arrows indicating clearly exposed enamel prisms on the surface of mutant mouse M1. *p<0.1; **p<0.01; ***p< 0.001；****p＜0.0001H, J, M: Each group n =6; N: n=3 per group).

[0104] Picture 4 yes K14 Cre ;Edar fl / fl A schematic diagram of ectodermal defect phenotypes in mice, including: complete hair loss of the tail and chest, curved tail tip, bald patches behind the ears, and underdeveloped eyelids.

[0105] Epithelial cells Edar The absence of these features impairs enamel mineralization and ameloblast function. Serial sections of tooth germs allow for more precise observation of morphological differences at 3.5 days (P3.5), 6.5 days (P6.5), 11.5 days (P11.5), and 35 days (P35) postnatal time. K14 Cre ;Edar fl / fl In mice, the number of cusps decreased, the roots shortened, and the pulp chamber height increased, although the eruption process appeared to proceed normally (e.g., Picture 5 (As shown in A). Furthermore, picric acid Sirius red (PSR) staining of M1 at P0 showed that, compared with... Edar fl / fl Compared to mice, mutant mice showed almost no positive signal at the enamel-dentin junction (e.g., Picture 5 (As shown in B). This finding indicates that the collagen network is incomplete, which may impair the mineralization process. (P11.5) K14 Cre ;Edar fl / fl Ameloblasts in mice are sparsely arranged, and the arrows indicate the gaps between them (e.g. Picture 5 (As shown in C). We also stained the mandibular incisors of P14 with hematoxylin and eosin (H&E), and the results showed that... Edar fl / fl compared to, K14 Cre ;Edar fl / fl Mice have only a small amount of enamel matrix formed (e.g.) Picture 5 (as shown in D).

[0106] Given the crucial role of ameloblasts in enamel formation, we attempted to evaluate... Edar The effect on ameloblast mineralization function. To this end, we used specific shRNA to silence Edar in ALCs, an immortalized cell line derived from the mandibular molars of C57BL / 6J mice. Western blotting was then used to assess the knockdown efficiency (e.g., Edar). Picture 5As shown in E). Alizarin Red S (ARS) staining showed a significant reduction in induced shEdar-ALC calcified nodules and a marked decrease in staining intensity, indicating impaired mineralization capacity (e.g., Picture 5 (As shown in F). Subsequently, we performed quantitative polymerase chain reaction (qPCR) analysis on the two ameloblast lineages after mineralization induction. [The results showed that...] Edar And five key genes associated with enamel mineralization ( Amelx , Ambn , Enam , Amtn and Odam The results showed that knocking down cells... Edar The mRNA expression levels of all five genes were significantly downregulated (e.g. Picture 5 (As shown in G). These findings confirm the epithelial cells Edar It plays a key role in enamel development and ameloblast mineralization in mice.

[0107] Picture 5 It is a specific deficiency in epithelial cells. Edar The degree of enamel mineralization damage and ameloblast function in mice expressing the gene were assessed, including: A: At P3.5, P6.5, P11.5, and P35 Edar fl / fl and K14 Cre ;Edar fl / fl Images of the coronal surface of mouse mandibular M1 stained with hematoxylin and eosin (H&E); B: Picric acid Sirius red (PSR) staining at P0 showed that, K14 Cre ;Edar fl / fl The mutant showed reduced collagen deposition at the cusps, with the right box showing an image at a higher magnification. C: P11.5 days Edar fl / fl and K14 Cre Edar fl / fl E-cadherin immunofluorescence staining (IF) and H&E staining analysis were performed on the first mandibular molar of mice: the dashed line in the figure outlines the ameloblast layer, the red arrow indicates the gap between sparsely arranged ameloblasts, and Am represents ameloblasts. D: H&E staining results showed that, on day P14, compared with the control group, the formation of enamel matrix in the mandibular incisors of mutant mice was delayed. The right box shows the image at a higher magnification. The dashed line on the left indicates the position of the neck ring, and the dashed line on the right marks the site where enamel matrix deposition begins. The blue double arrows show the distance between the two locations. E: Western blot analysis confirmed that, compared to control cells, Edar The level of EDAR protein was decreased in ameloblast lineage cells knocked down (shEdar); F: Alizarin Red S (ARS) staining showed that the mineralization capacity of shEdar ameloblast lineage cells was reduced; G: mRNA expression of shEdar and shCtrl ameloblast lineages was detected by qPCR. Edar Extragenic enamel-specific genes (including) Amelx , Ambn , Enam , Amtn and Odam The expression of ); H: In Edar fl / fl and K14 Cre Edar fl / fl Immunofluorescence staining analysis of AMERX and AMBN was performed on the first mandibular molar of mice. *p<0.1, **p<0.01, ***p<0.001 Each group n =3) Single-cell RNA sequencing reveals epithelial cells Edar Knockout damages ameloblast differentiation To further investigate epithelial cell specificity Edar How the absence of P0 cells impairs ameloblast mineralization: an analysis of P0 cell regeneration Edar fl / fl (Control group, Ctrl) and K14 Cre ;Edar fl / fl Single-cell RNA sequencing (scRNA-seq) was performed on the mandibular M1 tooth germs of conditional knockout (cKO) mice. The experimental procedure is as follows: Picture 6 As shown in Figure A. After data integration, dimensionality reduction analysis identified seven distinct cell subpopulations (e.g., ...). Picture 6 B. Picture 9-10 (As shown). Pulp cells, dental follicle cells, and epithelial cells constituted the main cell types (82.13%). It was observed that... Eda It is highly expressed in both mesenchymal and epithelial cells, and its receptor... Edar The downstream adaptor protein EDARADD is primarily expressed in epithelial cells. This expression pattern supports the use of... K14 Cre ;Edar fl / fl Mouse achieves epithelial cell specificity Edar Reasons for the absence (e.g.) Picture 11(As shown).

[0108] Then, the epithelial cells were further classified, identifying neck ring cells (CL), early ameloblasts (eAM), epithelial cycle cells (epi-cycling), outer ameloblasts (OEE), pre-ameloblasts, secretory ameloblasts (sAM), intermediate layer (SI), and stellate reticulum (SR) (e.g.) Picture 6 (as shown in C). Picture 6 D shows the marker genes identified for CL, eAM, and sAM. It is worth noting that... Edar High expression in CL during this period (P0) is consistent with previous findings. Picture 6 E shows the proportion of each epithelial cell subset. Compared with the Ctrl group, the proportions of CL, eAM, and sAM populations decreased in the cKO group, while the proportion of pre-ameloblasts increased. Pseudo-temporal analysis revealed two distinct differentiation trajectories originating from CL cells. One branch leads sequentially to the OEE, SI, and SR populations, while the other branch, through pre-ameloblast intermediates, sequentially generates eAM and subsequently sAM lineages (e.g., ...). Picture 6 (as shown in F). These findings suggest that in the cKO group, the differentiation of pre-ameloblasts into eAM may be impaired, potentially interfering with the functional maturation of ameloblasts during enamel development.

[0109] In addition, differentially expressed genes (DEGs) were analyzed between the Ctrl group and the cKO group in the pre-ameloblast, sAM, and eAM populations (e.g., Picture 6 (As shown in GI). Gene ontology (GO) analysis of downregulated genes in the cKO group showed enrichment in biological processes related to tooth and bone development, key signaling pathways such as the Wnt signaling pathway, epithelial-mesenchymal interactions, and calcium ion transmembrane transport (e.g., GI). Picture 6 (As shown in JL). These genes are also related to basic cellular functions, including cell cycle, cell adhesion, energy metabolism, and protein transport. Histological analysis using H&E staining further confirmed this. K14 Cre ;Edar fl / fl The number of eAM and sAM is reduced in mice (e.g. Picture 6 (As shown in M). Finally, based on the Venn diagram of downregulated DEGs shared by the pre-ameloblast, sAM, and eAM populations, six genes persistently downregulated in ameloblasts were identified, including Dlx3 , Shh , MSX2 , Bambi , Adgrf4 and IRX5 (like Picture 6(N is shown). The violin diagram illustrates the expression levels of these genes across epithelial subgroups in the Ctrl and cKO groups (e.g., ...). Picture 12 (As shown). Among these genes, Dlx3 It has attracted particular attention due to its recognized role in tooth development and enamel formation. In summary, the focus is on... Edar fl / fl and K 14 Cre ;Edar fl / fl Differences in cell composition were observed between the epithelial cells, and the epithelial cells were reported. Edar Defects can lead to abnormal differentiation of pre-ameloblasts, ultimately resulting in enamel formation defects. Among candidate genes, Dlx3 It is most likely caused by epithelial cells Edar Downstream targets for signal modulation.

[0110] Picture 6 When it is P0 Edar fl / fl (Control group; Ctrl) and K14 Cre ;Edar fl / fl Single-cell transcriptome analysis of the M1 tooth germ in the mandibular region of mice (conditional knockout; cKO), including: A is a single-cell suspension prepared from tooth germs for scRNA-seq; B is a dimensionality-reduced visualization of the M1 single-cell transcriptome data at P0 in Ctrl and cKO mice, with cells divided into 7 types; C is a UMAP visualization of odontogenic epithelial cells, further divided into eight subgroups with different transcriptional characteristics; D represents the expression level of representative marker genes in CL (cervical ring), eAM (early ameloblasts), and sAM (secretory ameloblasts); E is a proportion diagram of dental epithelial cell types, showing the contribution of molar tooth germs in Ctrl and cKO mice at P0. The proportion of ameloblasts was higher in the cKO group (16.8% vs 16.1%), while the proportions of eAM (7.2% vs 9.4%) and sAM cells (7.3% vs 8.9%) were lower. F is a pseudo-temporal trajectory analysis, which reveals a lineage analysis originating from the OEE / SR / SI cell population, with one branch differentiating into ameloblasts (pre-ameloblasts, eAM, and sAM) and the other branch differentiating into cervical ring cells (CL). G to I are volcano plots showing differentially expressed genes in pre-ameloblasts, eAM, and sAM at P0. Blue represents genes downregulated in cKO, and red represents genes upregulated in cKO. J to L: Gene enrichment analysis of differentially expressed genes in pre-ameloblasts, eAM, and sAM to investigate their functions; M: Diagram of mandibular incisor development at P14 and Edar fl / fl and K14 Cre Eda rfl / fl H&E staining results of eAM and sAM in mouse mandibular incisors; cell count on the right shows a reduction in the number of eAM and sAM in mutants. N: Venn diagram showing genes downregulated in pre-ameloblasts, eAM, and sAM (*p<0.1; n=3 per group).

[0111] Picture 7 This represents the transcriptional characteristics of epithelial subpopulations in developing mouse mandibular molar germs. The feature map illustrates the expression patterns of representative marker genes in each epithelial subpopulation.

[0112] Picture 8 yes Edar fl / fl and K14 Cre Eda rfl / fl H&E staining images of the mandibular first molar (M1) of mice at 7.5 days (P7.5) and 11.5 days after birth. Yellow dashed circles indicate epithelial root sheath cells.

[0113] Picture 9 yes Edar fl / fl (Ctrl) and K14 Cre ;Edar fl / fl Quality control (QC) metrics for the (cKO) molar tooth germ scRNA-seq dataset: violin plots and scatter plots show the distribution of three QC metrics (nFeature_RNA, nCounts_RNA, and percent.mt) in P0 mandibular first molar tooth germ Ctrl and cKO cells. Correlation analysis of UMI counts with gene count and mitochondrial gene percentage was performed to assess data quality and identify potential outliers.

[0114] Picture 10 This is a transcriptional feature of different cell types in the mandibular molar tooth germ of P0 mice. The feature map shows the expression level of marker genes for each cell type.

[0115] Picture 11 yes Eda , Edar and Edaradd Expression in mesenchymal cells, including: A: Bubble chart display Eda , Edar and Edaradd Expression in all cell clusters; B: UMAP visualization of mesenchymal cell populations; C: Mesenchymal cells in the Ctrl and cKO groups Eda Comparison of expressions; D: Violin diagram comparing dental mesenchymal cells in the Ctrl group and cKO group mice. Eda The level of expression. ****p< 0.0001 ).

[0116] Picture 12 It is different epithelial cell subsets Dlx3 , Shh , MSX2 , IRX5 , Adgrf4 and Bambi Comparison of expression levels between the conditional knockout group (green) and the control group (blue).

[0117] Edar Through regulation Dlx3 Regulation of ameloblast function Therefore, DLX3 immunofluorescence staining was performed on P0 mouse tooth germ sections, and the results were observed. K14 Cre Edar fl / fl The DLX3 signal intensity was significantly reduced in mice (e.g. Picture 13 As shown in Figure A), this is consistent with the scRNA-seq results. In shEdar-ALCs, qPCR and Western blot analysis further confirmed this. Dlx3 Decreased expression at both mRNA and protein levels (e.g.) Picture 13 (As shown in BC). To investigate Dlx3 To investigate whether it is regulated by NF-κB, a ChIP-qPCR experiment was performed. The results showed that p65 can bind to... Dlx3 boot subregion (such as Picture 13 (as shown in D), and this binding was reduced to about one-third in shEdar ALCs. Consistently, shEdar ALCs exhibited elevated cytoplasmic IκB-α levels and reduced nuclear translocation of p65 and phosphorylated p65, indicating weakened NF-κB pathway activation (e.g., as shown in D). Picture 13 (As shown in E). These data support the regulation of Edar through the NF-κB pathway. Dlx3 The perspective of transcription.

[0118] Functionally, forced expression in shEdar ALCs Dlx3 DLX3 protein levels were restored (e.g.) Picture 13(as shown in FG), and saved the previous cause Edar Knockdown and downregulation of the mRNA expression of multiple enamel-related genes (such as...) Picture 13 (As shown in H). Finally, after mineralization induction and ARS staining, Edar Knockdown of cells revealed impaired mineralization capacity, while Dlx3 Overexpression can salvage this defect (e.g.) Picture 13 As shown in Figure I).

[0119] Picture 13 yes Edar Knock down Dlx3 This phenotype expresses and impairs ALC mineralization, and can be transmitted through... Dlx3 Overexpression rescue, including: A: Immunofluorescence analysis of DLX3 showed that at P0... K14 Cre ;Edar fl / fl Mice prefer the lower jaw M1 Dlx3 Reduced expression; B: qPCR analysis results showed that, compared with shCtrl ameloblastic lineage cells, shEdar ALCs... Dlx3 Decreased mRNA expression; C: Western blot analysis confirmed a decrease in DLX3 protein levels in shEdar cells. The heatmap below shows the phase nucleus-paired DLX3 protein levels normalized with Vinculin as an internal control, indicating a 41.2% decrease compared to the control group. D: Chromatin immunoprecipitation assay was performed in shCtrl and shEdar ameloblast lineage cells to detect p65 in... Dlx3 Occupancy status on the promoter; E: Western blot analysis of proteins related to the EDAR / NF-κB signaling pathway in ameloblast lineage cells; F: qPCR analysis showed that, Dlx3 Expression is reduced in shEdar ameloblast lineage cells, while exogenous expression is reduced. Dlx3 Overexpression can restore its expression level; G: Western blot confirmed that Dlx3-HA was overexpressed in both shCtrl and shEdar ameloblast lineages (cells transfected with empty vector were used as controls). H: Overexpression in shEdar ameloblast lineage cells, as determined by qPCR. Dlx3 It can upregulate specific enamel genes (including Amelx , Ambn , Enam , Amtn and Odam The expression of ); I: Alizarin Red S staining shows Dlx3 After overexpression, the mineralization capacity of shEdar ameloblast lineages was restored (** p < 0.01, ***p < 0.001 Each group n =3).

[0120] The preferred embodiments of the present invention have been described in detail above with reference to the accompanying drawings; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention. This includes combining various technical features in any other suitable manner; these simple modifications and combinations should also be considered as part of the content disclosed in this invention and are all within the protection scope of this invention.

Claims

1. A method for constructing a mouse model, characterized in that, The mouse model has at least epithelial cells present. Edar A gene frameshift mutation, and the frameshift mutation leads to Edar The functional expression of the gene-encoded protein is lost; The method includes obtaining the mouse model by conditional deletion or systemic knockout.

2. The method according to claim 1, wherein, The mouse model is characterized by a specific deficiency in epithelial cells. Edar Mouse models of gene expression or systemic loss Edar Mouse models of gene expression; And / or, the mouse model Edar Deletion of exon 4 and / or exon 5 of the gene.

3. The method according to claim 2, wherein, The specific absence in the epithelial cells Edar Methods for constructing mouse models of gene expression include: (1) Edar Genes carry LoxP site Edar fl / fl Mouse and epithelial cell-specific expression Cre Mice were hybridized with the gene and screened to obtain the genotype of [gene name missing]. Cre; Edar fl / + Offspring mice; (2) Cre; Edar fl / + mice and Edar fl / fl Mouse backcrossing resulted in the acquisition of specific deletions in epithelial cells. Edar Mouse models of gene expression; Preferably, the LoxP The site is located Edar Flanking exon 4 and / or exon 5 of the gene; Preferably, the epithelial cells specifically express Cre Genes are driven by either the keratin 14 promoter or the keratin 5 promoter. Cre It is achieved through genes.

4. The method according to claim 2, wherein, The whole body missing Edar Methods for constructing mouse models of gene expression include: introducing gene expression into the mouse genome using a gene editing system. Edar Loss-of-function mutations in gene pairs result in systemic deficiency. Edar Mouse models of gene expression; Preferably, the whole-body absence Edar Methods for constructing mouse models of gene expression include: (1) For mice Edar Guide RNAs are designed from exons 4 and / or 5 of the gene and adjacent non-coding regions. (2) The guide RNA and Cas9 protein were introduced into mouse zygotes and gene editing was performed using the CRISPR / Cas9 system to obtain edited zygotes; (3) The edited fertilized eggs were transplanted into pseudopregnant female mice to obtain F0 generation mice. Edar Genetically modified mice; (4) Screening through genotype identification Edar Allele heterozygous mutation ( Edar + / - ) mice; (5) Edar + / - Mice mate with each other to acquire systemic [viruses / conditions]. Edar Homozygous gene knockout Edar - / - A mouse model of ).

5. The method according to any one of claims 1-4, wherein, The mouse model exhibited a developmental abnormality phenotype associated with hypohidrotic ectodermal dysplasia; Preferably, the developmental abnormality phenotype includes dental developmental abnormalities and / or other ectodermal organ developmental abnormalities; More preferably, the abnormal tooth development phenotype includes at least one of the following: missing third molars, reduced number of teeth, reduced tooth size, root fusion, reduced number of cusps, molars exhibiting a bovine tooth-like morphology, enamel hypoplasia, or mineralization defects. More preferably, the other ectodermal organ developmental abnormalities include at least one of the following: sparse or absent hair, reduced number or dysfunction of sweat glands, abnormal development of the skin and its appendages, or corneal epithelial abnormalities. More preferably, the abnormal development of the skin and its appendages includes at least one of the structural abnormalities of hair follicles, sebaceous glands, apocrine sweat glands, and toenails.

6. A kind Edar Cell lines with gene functional loss are characterized by, The cell line Edar The gene contains a frameshift mutation, and the frameshift mutation leads to Edar The functional expression of the gene-encoded protein is lost.

7. The cell line according to claim 6, characterized in that, The cell line is derived from a mouse model constructed by the method described in any one of claims 1-5.

8. The application of the mouse model constructed by the method according to any one of claims 1-5 or the cell line according to claim 6 or 7, characterized in that, The application includes at least one of the following: (1) Screening or evaluation for prevention, relief or treatment Edar Candidate drugs, gene therapy methods, cell therapy, or other biological agents that target gene expression defects are preferred for screening purposes. Edar Candidate drugs for gene expression defects; (2) Research Edar The role of signaling pathways in the development, differentiation, or homeostasis of ectodermal organs; Preferably, the ectodermal organs include at least teeth, hair, sweat glands, skin and their appendages, or cornea.

9. The application according to claim 8, wherein, The screening is used for treatment. Edar Approaches for candidate drugs with gene expression defects include: (1) Treating a mouse model constructed by the method of any one of claims 1-5 or a cell line of claim 6 or 7 with the candidate drug; (2) Compare the changes in mouse models or cell lines before and after treatment to determine whether the candidate drugs have an improving effect; (3) Identify candidate drugs that have an improving effect as potential therapeutic agents; Preferably, the changes in the mouse model include at least one of the following: changes in tooth phenotype, changes in the phenotype of other ectodermal organs, and changes in the expression levels of marker genes related to enamel mineralization or cell proliferation; Preferably, the changes in the cell line include changes in the expression levels of marker genes related to enamel mineralization or cell proliferation in the cells; More preferably, the tooth phenotype includes at least one of the following: number of teeth, tooth size, root morphology, number of cusps, enamel mineralization density, and thickness. More preferably, the other ectodermal organ phenotypes include at least one of hair density and morphology, number and function of sweat glands, structure of skin and its appendages, and corneal integrity; More preferably, the biomarker gene includes Dlx3 , Amelx , Ambn , Enam , Amtn , Odam At least one of them.

10. The application according to claim 9, wherein, The improvement effect includes at least one of the following: (1) Improvement of tooth phenotype in mouse models; (2) Improvement of phenotypes of other ectodermal organs in mouse models; (3) Reverse or alleviate the abnormal expression of the marker genes; (4) The differentiation capacity and function of the ectoderm-derived cells or tissues with impaired function are improved or restored.

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