Bilateral cleft lip mouse model based on Pitx2-Cre conditional knockout of Bmp4 as well as construction method and application of bilateral cleft lip mouse model

Abstract

The invention relates to the field of bioengineering, in particular to a bilateral cleft lip mouse model based on Pitx2-Cre conditional knockout of Bmp4 as well as a construction method and application of the bilateral cleft lip mouse model. The invention aims to construct a stable bilateral cleft lip mouse model. Experiments find that the Bmp4 gene is expressed in the epithelium of the frontal nose process and the maxillary process at the early lip stage in E9.25. The spatio-temporal expression mode of the Pitx2 gene in the early primordium epithelium is highly consistent with the spatio-temporal expression mode of the Bmp4 gene. Based on this, the inventor constructs a Bmp4 gene knockout model driven by an endogenous Pitx2 gene promoter, and finds that the mouse model shows a 100% bilateral cleft lip phenotype. Compared with the reported Netin-Cre, the Nestin-Cre has the advantages that the Nestin The bilateral cleft lip model constructed by the method is more stable, can better represent genetic diseases of human cleft lip, and has important research significance.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering, and in particular to a bilateral cleft lip mouse model based on conditional knockout of Bmp4 by Pitx2-Cre, and a construction method and application thereof. Background Art

[0002] Orofacial clefts (OFC) are one of the most common congenital defects of the craniofacial structure in humans. Their pathogenesis is closely related to morphological abnormalities in the development of multiple craniofacial structures. According to the World Health Organization's latest global burden of disease statistics, the number of people suffering from OFC worldwide is as high as 4.6 million, with a prevalence of approximately 1 in 1,675. The incidence is relatively high in Asia, with the average incidence in China more than twice the global rate, particularly in the southwest region. The disease can severely impair the patient's appearance, speech, and psychology, often requiring multidisciplinary care and treatment, placing a significant burden on patients, their families, and society.

[0003] Orofacial clefts encompass various combinations of cleft lip and / or cleft palate. The most common types include cleft lip (CL), cleft lip and palate (CLP), and cleft palate (CP). Because the lip develops before the palate during embryogenesis, CL and CLP are often clinically combined into one group: cleft lip with or without cleft palate (CL / P). Depending on the presence of other congenital malformations, CL / P can be further divided into non-syndromic cleft lip or palate (NSCL / P) and syndromic cleft lip or palate (SCL / P), with NSCL / P accounting for approximately 70% of cases. Therefore, elucidating the pathogenesis of NSCL / P is of great clinical significance for the prevention and treatment of this condition, which depends on understanding the molecular mechanisms underlying lip and palate development. Because the development of the lip and palate is highly similar between humans and mice, mouse models have become an important tool for studying their normal development and the causes of abnormalities. While numerous CL / P mouse models have been reported, very few CL mouse models exist, and the few that do exist exhibit unstable phenotypes. Therefore, establishing a stable cleft lip mouse model is crucial for oral and maxillofacial cleft research.

[0004] The BMP4 gene, a member of the TGF-β superfamily, is expressed in three midfacial processes involved in lip development (the lateral nasal process, the medial nasal process, and the maxillary process). This gene plays a key role in lip fusion during craniofacial development and is a candidate gene for cleft lip and palate. Genetic studies of human NSCL / P using linkage analysis, association studies, and whole-exome sequencing have revealed that the genomic region 14q22-23, where the BMP4 gene is located, is closely associated with susceptibility to NSCL / P. Among these, the human BMP4 gene polymorphisms RS762642, RS17563, and RS10130587 have been associated with different forms of NSCL / P. The RS762642 locus is located in intron 1 of the BMP4 gene, and variants in this locus may alter DNA binding and transcriptional regulation. Studies have found that polymorphisms at this locus are significantly associated with both forms of NSCL / P (CP and CLP) in a southern Chinese population. The RS17563 locus, located in exon 4 of the BMP4 gene, has a T>C polymorphism that converts valine to alanine and is primarily associated with CL. This variant may increase the risk of NSCL / P in Asian and Caucasian populations but has been found to be protective in Brazilians. The intronic SNP RS10130587 polymorphism is a risk factor for CL / P in Asian populations.

[0005] Given the critical role of Bmp4 in lip development and its relevance to human NSCL / P, constructing Bmp4 conditional knockout mice is a feasible strategy for establishing a cleft lip model. Liu et al. first specifically knocked out the entire BMP pathway activity through Nestin-Cre (constructing Nestin-Cre; Bmpr1a null / flox The study, published in the journal Nature Communications, found that this mouse model developed bilateral cleft lip and palate. Further work using Nestin-Cre to specifically knock out the Bmp4 gene revealed that this mouse model also exhibited a bilateral cleft lip phenotype, but the model was unstable, with a 77.8% probability of bilateral cleft lip and a remaining 22.2% probability of unilateral cleft lip. Therefore, a more stable bilateral cleft lip model is urgently needed. Summary of the Invention

[0006] The purpose of the present invention is to provide a bilateral cleft lip mouse model based on Pitx2-Cre conditional knockout of Bmp4 and its construction method and application, so as to solve the problems existing in the above-mentioned prior art.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] The present invention provides a bilateral cleft lip mouse model based on Pitx2-Cre conditional knockout of Bmp4 (Pitx2 Cre / + ;Bmp4 f / f The construction method of the mouse comprises the following steps:

[0009] Pitx2-Cre tool mice and Bmp4 f / f Mice were mated to obtain Pitx2-Cre; Bmp4 f / + mice;

[0010] The Pitx2-Cre; Bmp4 f / + Mice and Bmp4 f / f The mice were mated to obtain the bilateral cleft lip mouse model.

[0011] The Pitx2-Cre tool mouse is a mouse in which the Cre coding region is expressed by the Pitx2 gene promoter.

[0012] Preferably, the Pitx2-Cre tool mouse is a mouse expressing the Cre coding region driven by the endogenous Pitx2 gene promoter, including inserting the Cre coding region sequence into the endogenous Pitx2 exon sequence, or connecting it after the Pitx2 stop codon through the ires element.

[0013] Preferably, the Pitx2-Cre tool mouse is a Pitx2 gene expressing Cre coding region driven by the endogenous Pitx2 gene promoter. Cre / + mice (the Cre coding region sequence was inserted into the endogenous Pitx2 exon sequence to ensure stable transcription and expression of Cre).

[0014] The present invention provides application of the above-mentioned bilateral cleft lip mouse model in studying the molecular mechanism of bilateral cleft lip.

[0015] The present invention provides use of a bilateral cleft lip mouse model constructed using the above-mentioned construction method in screening drugs for preventing and / or treating bilateral cleft lip.

[0016] The present invention provides an application of the bilateral cleft lip mouse model constructed by the above-mentioned construction method in the genetic research of bilateral cleft lip.

[0017] The present invention provides an application of a bilateral cleft lip mouse model constructed using the above-mentioned construction method in the study of the biological function of bilateral cleft lip.

[0018] The present invention provides the use of a bilateral cleft lip mouse model constructed using the above-mentioned construction method in the preparation of a bilateral cleft lip auxiliary diagnosis product.

[0019] The present invention provides an application of a bilateral cleft lip mouse model constructed using the above-mentioned construction method in the safety evaluation of drugs for treating bilateral cleft lip.

[0020] The present invention provides an application of a bilateral cleft lip mouse model constructed using the above-mentioned construction method in the study of lip development mechanisms.

[0021] The present invention discloses the following technical effects:

[0022] The purpose of the present invention is to construct a stable bilateral cleft lip mouse model and its construction method, study the molecular mechanism of bilateral cleft lip, explore the important role of Bmp4 gene in craniofacial lip development, and provide a stable mouse model and corresponding theoretical basis for case studies of CL / P, especially bilateral cleft lip.

[0023] The Nestin gene used to drive Cre gene expression is only expressed in the epithelium of the inner and outer nasal processes and maxillary processes of the lip structure from E10.0, while the Bmp4 gene is expressed in the epithelium of the early lip primordium (frontonasal process and maxillary process) as early as E9.25. Therefore, the reported Nestin-Cre; Bmp4 model can only knock out the Bmp4 gene from E10.0. In a specific embodiment, the present invention found through experiments that the Pitx2 gene is expressed earlier than the Nestin gene in the epithelium of the mouse facial primordium in the early stage of lip development, and the expression position of the Pitx2 gene in these early facial primordium epithelia is highly overlapped with the expression of the Bmp4 gene, that is, the present invention found that the spatiotemporal expression pattern of the Pitx2 gene in the early lip primordium is highly consistent with that of the Bmp4 gene. Based on this, the inventors constructed a Bmp4 gene knockout model driven by the endogenous Pitx2 gene promoter - Pitx2 Cre / + ;Bmp4 f / f Mice were found to exhibit 100% bilateral cleft lip phenotype. f / f The mouse cleft lip model showed an unstable bilateral cleft lip phenotype (77.8%). The Pitx2 constructed by the present invention Cre / + ;Bmp4 f / f All mice presented with bilateral cleft lip. Therefore, compared to the reported Nestin-Cre;Bmp4 model, the mouse model constructed in this invention is more stable and can better characterize human cleft lip genetic diseases, thus possessing important research significance. Furthermore, this invention has the following advantages:

[0024] 1. 100% bilateral cleft lip phenotype penetrance: Through Pitx2-Cre, the Bmp4 gene is precisely knocked out, resulting in 100% bilateral cleft lip in mutant embryos.

[0025] 2. High pathological consistency: The bilateral cleft lip phenotype matches the clinical characteristics of human non-syndromic cleft lip (NSCLP). BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0027] Figure 1 It is the technical roadmap of the present invention;

[0028] Figure 2 Figure 4 is the expression pattern of Nestin gene in the craniofacial region of mice at different developmental stages; whole-mount in situ hybridization was used to detect the expression of Nestin gene in the craniofacial region of E9.5, E10.0 and E10.5 mice; AF is the expression of Nestin gene in the craniofacial region of E9.5 mice, C is the magnification of the white box area in B, D is the section display, E and F are the magnifications of the red and black box areas in D; GL is the expression of Nestin gene in the craniofacial region of E10.0 mice, I is the magnification of the white box area in H, J is the section display, K and L are the red box areas in J and the black framed area are magnified. Note: at this time, the Nestin gene begins to be expressed in the epithelium of the frontonasal and maxillary processes; MQ is the expression of the Nestin gene in the craniofacial region of E10.5 mice, O is the magnification of the white framed area in H, P is a schematic diagram of the mouse section at E10.5, and Q is an in situ hybridization tissue section at E10.5; lnp is the lateral nasal process; mnp is the medial nasal process; mx is the maxillary process; mnd is the mandibular process; the scale bars of AC, GI, and MO are 200 μm, and the scale bars of DF, JK, and Q are 50 μm; the arrows indicate the Nestin gene expression areas;

[0029] Figure 3 The expression patterns of Pitx2 and Bmp4 genes in the mouse craniofacial region at different developmental stages; mnd represents the mandibular process; mx represents the maxillary process; scale bar is 200 μm;

[0030] Figure 4 For Pitx2 Cre / + ;R26R mTmG Expression patterns of the Cre gene in the craniofacial region of mice at different developmental stages; AB represents Pitx2 Cre / + ;R26R mTmG Cre gene activity in the frontonasal and maxillary processes of mice at E9.5; CD represents Pitx2 Cre / + ;R26R mTmG Cre gene activity in the epithelium at the fusion site of the medial and lateral nasal processes and the maxillary process epithelium of mice at E10.5; EF is Pitx2 Cre / + ;R26R mTmGCre gene activity in the fusion of the inner and outer nasal processes, the junction of the maxillary process and the outer nasal process, and the maxillary process epithelium of mice at E11.5; the upper lip development is complete at E12.5, Pitx2 Cre / + ;R26R mTmG Cre gene activity in the lip epithelium and maxillary epithelium of mice; fnp is the frontonasal process; mx is the maxillary process; lnp is the lateral nasal process; mnp is the medial nasal process; scale bars in A-H are 100 μm;

[0031] Figure 5 For Bmp4 gene at E10.5 Pitx2 Cre / + ;Bmp4 f / f Knockout in mice; AB represents the expression of Bmp4 gene in the epithelium of the medial and lateral nasal process contact area and the epithelium of the maxillary process in E10.5 wild-type mice; CD represents the expression of Bmp4 gene in Pitx2 Cre / + ;Bmp4 f / f Knockout of the epithelium in the contact area of the inner and outer nasal processes and the epithelium of the maxillary process in mice; lnp is the lateral nasal process; mnp is the medial nasal process; mx is the maxillary process; mnd is the mandibular process; the yellow arrow indicates the area where the Bmp4 gene is knocked out in the mutant embryo; the scale bar is 200 μm;

[0032] Figure 6 For Pitx2 Cre / + ;Bmp4 f / f Craniofacial phenotypes of mice at birth at E18.5; A and E are the lip and palate phenotypes of wild-type mice; B and F are the lip and palate phenotypes of Pitx2 Cre / + ;Bmp4 f / f Mouse lip and palate phenotype; C and G are Pitx2 Cre / + ;Bmp4 f / f Mouse lip and palate phenotype; D and H are Pitx2 Cre / + ;Bmp4 f / f Mouse lip and palate phenotypes; scale bars in AD are 500 μm, and scale bars in EH are 1000 μm;

[0033] Figure 7 For Pitx2 Cre / + ;Bmp4 f / f Phenotypes of the hands and feet of mice born at E18.5; A: polydactyly; B: fused legs; scale bar: 500 μm;

[0034] Figure 8 For Pitx2 Cre / + ;Bmp4 f / fEarly craniofacial phenotypes of mouse development; AB represents the medial and lateral nasal processes of E10.5 wild-type mice; CD represents the E10.5 Pitx2 Cre / + ;Bmp4 f / f Phenotypes of the maxillary process and medial and lateral nasal processes of mouse embryos; the dotted line is the outline of the nasal fossa; the black arrow indicates the contact and fusion site of the medial and lateral nasal processes in wild-type embryos; * indicates that the medial and lateral nasal processes in mutant embryos are not in contact and fused; EH is E10.0 Pitx2 Cre / + ;Bmp4 f / f Comparison of the early craniofacial appearance of mouse embryos and wild-type mice; the dotted line represents the lateral outline of the nasal fossa before fusion; fnp is the frontonasal process; lnp is the lateral nasal process; mnp is the medial nasal process; mx is the maxillary process; mnd is the mandibular process; the scale bar is 200 μm. DETAILED DESCRIPTION

[0035] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0036] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0037] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0038] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0039] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0040] Unless otherwise specified, the materials used in the present invention are all purchased by those skilled in the art, and the methods used in the present invention are all methods well known to those skilled in the art.

[0041] Example 1 Pitx2 Cre / + ;Bmp4 f / f Mouse construction and phenotypic analysis

[0042] 1. Genetic tools and design

[0043] Cre tool mouse: Pitx2 Cre / + Mice were purchased from MMRRC (Cat#000126-UCD; RRID:MMRRC_000126-UCD), and the ires-Cre-PGKneomycin chromosome fragment was introduced into the PvuII and NruI sites of the fifth exon of Pitx2.

[0044] Target gene design: The target gene is: Bmp4 gene (NCBI Gene ID: 12159; Ensembl ID: ENSMUSG00000021835); Bmp4 f / f Mice were purchased from Jackson Laboratory, USA (Strain#: 016878; RRID: IMSR_JAX: 016878), and loxP sites were inserted on both sides of the fourth exon of the Bmp4 gene.

[0045] 2. Hybridization strategy: Pitx2 Cre / + Mice and Bmp4 f / f Mice were mated and the offspring were heterozygous for Pitx2 Cre / + ;Bmp4 f / + mice; the Pitx2 Cre / + ;Bmp4 f / + Mice and Bmp4 f / f Mice mating to obtain Pitx2 Cre / + ;Bmp4 f / f mice were designated as the experimental group; wild-type mice were designated as the control group.

[0046] 3. Build Pitx2 Cre / + ;R26R mTmG Mouse: Pitx2 Cre / + Mice and R26R mTmG Mice were obtained by mating.

[0047] 4. The technical route of this embodiment is as follows Figure 1 Specifically, the whole-body in situ hybridization and sectioning of Bmp4, Pitx2 and Nestin genes in wild-type mice were performed to compare the expression differences of Pitx2 and Nestin genes, as well as the expression overlap with Bmp4 gene; Cre / + ;R26R mTmG The mice were observed by fluorescence and sectioned to further confirm the location of Cre gene expression driven by Pitx2 gene promoter; Cre / + ;Bmp4 f / f Whole-mount in situ hybridization was performed on mice to verify the knockout efficiency of Bmp4 gene; Cre / + ;Bmp4 f / f Phenotyping of craniofacial defects in mice was performed.

[0048] 5. Verification experiments and results

[0049] (1) Whole-mount in situ hybridization was used to detect the expression of Nestin gene in the craniofacial region of wild-type mice at E9.5, E10.0 and E10.5. Figure 2 As shown. The results showed that the Nestin gene of E9.5 wild-type mice was expressed in the mesenchyme of the frontonasal process, but not in the epithelium of the frontonasal process and maxillary process where the Bmp4 gene was expressed. The Nestin gene of E10.5 wild-type mice was expressed in the epithelium of the fusion of the medial and lateral nasal processes and the maxillary process. The Nestin gene began to be expressed in the epithelium of the frontonasal process and maxillary process at E10.0. The expression of Pitx2 and Bmp4 genes in the craniofacial region of wild-type mice at different stages from E9.25 to E10.5 was also detected by whole-mount in situ hybridization. The results are shown as follows. Figure 3 The results showed that the expression patterns of Pitx2 and Bmp4 genes were highly overlapping, and both were expressed in the epithelium of the frontonasal and maxillary primordia at the early stage of lip development as early as E9.25, much earlier than the expression time of Nestin gene. Cre / + ;R26R mTmG The expression pattern of Cre driven by the Pitx2 gene promoter was observed by immunofluorescence in mice. Figure 4 The results show that Pitx2 Cre / + ;R26R mTmG The mice showed significant Cre gene activity in the frontonasal and maxillary processes at E9.5 ( Figure 4 A and B in Pitx2 Cre / + ;R26R mTmG Cre gene activity was observed in the epithelium at the fusion site of the medial and lateral nasal processes and the epithelium of the maxillary process at E10.5 ( Figure 4 C and D in (EF): Pitx2Cre / + ;R26R mTmG Cre gene activity was observed in the fusion of the medial and lateral nasal processes, the junction of the maxillary process and the lateral nasal process, and the epithelium of the maxillary process at E11.5. Figure 4 E and F); GH: upper lip development is complete at E12.5, Pitx2 Cre / + ;R26R mTmG The Cre gene is continuously active in the lip epithelium and maxillary epithelium of mice ( Figure 4 G and H). This shows that E9.5-E12.5 Pitx2 Cre / + ;R26R mTmG The Cre gene activity of mice is consistent with the expression of the Pitx2 gene in wild-type mice. It is also active in the frontonasal and maxillary epithelial processes of the early lip development primordium before E9.5. These results indicate that the Bmp4 gene begins to regulate lip development as early as E9.25. f / f The mouse model using Nestin-Cre mice cannot knock out the Bmp4 gene before E10.0, which may be the reason for the unstable phenotype of the model; however, the use of Pitx2-Cre mice can knock out the Bmp4 gene throughout the entire period of lip development, which may result in a more stable bilateral cleft lip model.

[0050] (2) Pitx2 Cre / + ;Bmp4 f / f The expression of Bmp4 gene in mice was verified, and the results were as follows Figure 5 In situ hybridization results showed that the expression level of Bmp4 mRNA in the fusion of the medial and lateral nasal processes and the maxillary process was significantly downregulated, indicating that the Bmp4 gene was expressed in Pitx2 Cre / + ;Bmp4 f / f was successfully knocked out in mice.

[0051] (3) Pitx2 Cre / + ;Bmp4 f / f The embryonic phenotype of mice was observed, and the results are shown in Table 1. Figure 6 and Figure 7 The results showed that the lip and palate of wild-type mice developed normally ( Figure 6 A, E in Pitx2 Cre / + ;Bmp4 f / f Mice presented with bilateral cleft lip and complete cleft palate ( Figure 6 B, F in Pitx2 Cre / + ;Bmp4 f / f Mice presented with bilateral cleft lip and cleft hard palate ( Figure 6 C, G in Pitx2 Cre / + ;Bmp4 f / fMice presented with bilateral cleft lip and cleft soft palate ( Figure 6 D, H); Pitx2 after E12.5 Cre / + ;Bmp4 f / f All mouse embryos showed bilateral cleft lip, with or without cleft palate, i.e. 100% of them had bilateral cleft lip, and cleft palate was divided into different cases; some mutant mice also had phenotypes such as polydactyly and clasped legs (Table 1 and Figure 7 The bilateral cleft lip phenotype first appeared at E10.5. Compared with the wild type, the medial and lateral nasal processes were not fused, and the medial nasal process and maxillary process were smaller in size. The abnormal reduction in size was observed as early as E10.0.

[0052] Table 1 Pitx2 Cre / + ;Bmp4 f / f Mouse phenotypic statistics

[0053] Gestational age total Bilateral cleft lip cleft palate Polydactyl Legs together E14.5 9 9 8 8 1 E18.5 20 20 12 14 5 total 29 29(100％) 20 22 6

[0054] (4) Pitx2 Cre / + ;Bmp4 f / f The craniofacial phenotypes of mice were observed during early development. Figure 8 The results showed that the medial and lateral nasal processes of wild-type mice began to contact and fuse at E10.5; Cre / + ;Bmp4 f / f The maxillary process and the medial and lateral nasal processes of mouse embryos are small in size, and the medial and lateral nasal processes are not in contact and fused; E10.0Pitx2 Cre / + ;Bmp4 f / f Compared with wild-type mice, the medial and lateral nasal placodes of mouse embryos are flatter and the maxillary processes are smaller.

[0055] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for constructing a bilateral cleft lip mouse model based on conditional knockout of Bmp4 by Pitx2-Cre, characterized in that: The following steps are involved: Pitx2-Cre tool mice and Bmp4 f / f Mice were mated to obtain Pitx2-Cre; Bmp4 f / + mice; The Pitx2-Cre; Bmp4 f / + Mice and Bmp4 f / f The mice were mated to obtain the bilateral cleft lip mouse model.

2. The construction method according to claim 1, characterized in that The Cre tool mouse is a mouse in which the Cre gene is expressed by the Pitx2 gene promoter.

3. Use of a bilateral cleft lip mouse model constructed using the construction method according to claim 1 or 2 in studying the molecular mechanism of bilateral cleft lip.

4. Use of a bilateral cleft lip mouse model constructed using the construction method according to claim 1 or 2 in screening drugs for preventing and / or treating bilateral cleft lip.

5. Use of a bilateral cleft lip mouse model constructed using the construction method according to claim 1 or 2 in genetic research on bilateral cleft lip.

6. Use of a bilateral cleft lip mouse model constructed using the construction method according to claim 1 or 2 in the study of the biological functions of bilateral cleft lip.

7. Use of the bilateral cleft lip mouse model constructed using the construction method according to claim 1 or 2 in preparing a bilateral cleft lip auxiliary diagnosis product.

8. Use of a bilateral cleft lip mouse model constructed using the construction method according to claim 1 or 2 in the safety evaluation of drugs for treating bilateral cleft lip.

9. Use of a bilateral cleft lip mouse model constructed using the construction method according to claim 1 or 2 in the study of lip development mechanisms.

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