A bilateral cleft lip mouse model based on pitx2-cre conditional knockout of bmp4 and a construction method and application thereof

Abstract

The present application relates to the field of bioengineering, and particularly relates to a bilateral cleft lip mouse model based on Pitx2-Cre conditional knockout of Bmp4 as well as a construction method and application thereof. The purpose of the present application is to construct a stable bilateral cleft lip mouse model. It is found through experiments that the Bmp4 gene is expressed in the epithelium of the early frontonasal process and maxillary process of the lip as early as E9.25. The spatiotemporal expression pattern of the Pitx2 gene in the epithelium of these early primordiums 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, and found that this mouse model exhibits a 100% bilateral cleft lip phenotype. Compared with the reported Nestin-Cre;Bmp4 f / f model, the bilateral cleft lip model constructed by the present application is more stable, can better characterize human cleft lip genetic diseases, and has important research significance.

Description

Technical Field

[0001] This invention relates to the field of bioengineering, and in particular to a method based on... Pitx2-Cre Conditional knockout Bmp4 Bilateral cleft lip mouse model, its construction method and application. Background Technology

[0002] Orofacial clefts (OFC) are one of the most common congenital craniofacial structural defects in humans, and their pathogenesis is closely related to morphological developmental abnormalities in multiple craniofacial structures. This disease severely impairs a patient's appearance, speech, and mental well-being, usually requiring multidisciplinary care and treatment, and placing a significant burden on patients, their families, and society.

[0003] Orofacial clefts include 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 embryonic development, CL and CLP are often clinically grouped together as cleft lip with or without cleft palate (CL / P). Depending on whether other congenital malformations are present, CL / P can be further divided into non-syndromic cleft lip or palate (NSCL / P) and synthetic cleft lip or palate (SCL / P), with NSCL / P accounting for approximately 70%. Therefore, elucidating the pathogenesis of NSCL / P is of great clinical significance for the prevention and treatment of this disease, and this depends on understanding the molecular mechanisms related to lip and palate development. Because the development of the lips and palate is highly similar in humans and mice, mouse animal models have become important tools for studying their normal development and the causes of abnormalities. Currently, there are many reported cleft lip / palatal cleft (CL / P) mouse models, but very few CL mouse models exist, and the phenotypes of the few existing cleft lip models are unstable. Therefore, establishing stable cleft lip mouse models is extremely important for research on maxillofacial clefts.

[0004] Bmp4 The gene is a member of the TGF-β superfamily and is expressed in the three midfacial processes involved in lip development (lateral nasal process, medial nasal process, and maxillary process). This gene plays a crucial role in lip fusion during craniofacial development and is a candidate gene for cleft lip and palate. Studies of human NSCL / P using genetic methods such as linkage analysis, association studies, and whole-exome sequencing have revealed that... BMP4The genomic region 14q22-23 containing the gene is closely associated with susceptibility to NSCL / P. Among them, humans... BMP4 Gene polymorphisms RS762642, RS17563, and RS10130587 were associated with different forms of NSCL / P: the RS762642 locus is located at BMP4 Intron 1 of a gene, variants of which may alter DNA binding and transcriptional regulation.

[0005] Given Bmp4 Its key role in lip development and its correlation with human NSCL / P, constructing Bmp4 Conditional knockout mice are a feasible strategy for constructing cleft lip models. Liu et al. first used... Nestin-Cre Specifically knock out the entire BMP pathway activity (construction) Nestin-Cre;Bmpr1a null / flox (In mice), it was found that this mouse model resulted in bilateral cleft lip and palate. Further research was conducted using... Nestin-Cre Specific knockout Bmp4 Genetic analysis revealed that this mouse model also exhibited the bilateral cleft lip phenotype, but it 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 this invention is to provide a method based on Pitx2-Cre Conditional knockout Bmp4 This study aims to develop a bilateral cleft lip mouse model, its construction method, and its applications to address the problems existing in the aforementioned technologies.

[0007] To achieve the above objectives, the present invention provides the following solution: This invention provides a method based on Pitx2-Cre Conditional knockout Bmp4 Bilateral cleft lip mouse model ( Pitx2 Cre / + ; Bmp4 f / f The method for constructing a mouse includes the following steps: Will Pitx2-Cre tool mouse and Bmp4 f / f Mice mate and obtain Pitx2-Cre;Bmp4 f / + Mice; The Pitx2-Cre;Bmp4 f / + Mice and the Bmp4 f / f Mice were mated to obtain the bilateral cleft lip mouse model.

[0008] The Pitx2-Cre tool mouse as a reasonPitx2 Gene promoter driven Cre Mice expressing the coding region.

[0009] Preferably, the aforementioned Pitx2-Cre Tool mice are from endogenous Pitx2 Gene promoter driven Cre Mice expressing the coding region, including those that... Cre Insertion of intrinsic source into coding region sequence Pitx2 In the exon sequence, or through ires Components connected in Pitx2 After the stop codon.

[0010] Preferably, the aforementioned Pitx2-Cre Tool mice are from endogenous Pitx2 Gene promoter driven Cre The encoding region represents Pitx2 Cre / + mice ( Cre Insertion of intrinsic source into coding region sequence Pitx2 In the exon sequence, to ensure Cre Stable transcription and expression.

[0011] This invention provides the application of the above-mentioned bilateral cleft lip mouse model in the study of the molecular mechanisms of bilateral cleft lip.

[0012] This invention provides the application of the bilateral cleft lip mouse model constructed using the above-described construction method in screening drugs for the prevention and / or treatment of bilateral cleft lip.

[0013] This invention provides the application of the bilateral cleft lip mouse model constructed using the above-described method in the genetic study of bilateral cleft lip.

[0014] This invention provides the application of the bilateral cleft lip mouse model constructed using the above-described construction method in the study of the biological function of bilateral cleft lip.

[0015] This invention provides the application of the bilateral cleft lip mouse model constructed using the above-described construction method in the preparation of auxiliary diagnostic products for bilateral cleft lip.

[0016] This invention provides the application of the bilateral cleft lip mouse model constructed using the above-described construction method in the safety evaluation of drugs for treating bilateral cleft lip.

[0017] This invention provides the application of the bilateral cleft lip mouse model constructed using the above-described construction method in the study of lip development mechanisms.

[0018] The present invention discloses the following technical effects: The purpose of this invention is to construct a stable mouse model of bilateral cleft lip and its construction method, to study the molecular mechanisms of bilateral cleft lip, and to explore...Bmp4 The important role of genes in craniofacial lip development provides a stable mouse model and corresponding theoretical basis for case studies of cleft lip / polylipidemia (CL / P), especially bilateral cleft lip.

[0019] Used for driving Cre Gene expression Nestin The gene only begins to be expressed in the epithelium of the nasal and maxillary processes on the inner and outer sides of the lip structure from E10.0 onwards. Bmp4 The gene was already expressed in the epithelium of the early lip primordia (frontonasal and maxillary processes) as early as E9.25, therefore it has been reported... Nestin-Cre;Bmp4 The model can only be knocked out starting from E10.0. Bmp4 Genes. In specific embodiments, this invention has discovered through experiments... Pitx2 Gene ratio Nestin The gene was expressed earlier in the facial primordium epithelium of mice during early lip development, and Pitx2 The expression location of genes in these early facial primordia epithels and Bmp4 Gene expression highly overlaps, as discovered in this invention. Pitx2 Spatiotemporal expression patterns of genes in early lip primordia and Bmp4 The genes are highly consistent. Based on this, the inventors constructed an endogenous... Pitx2 Gene promoter driven Bmp4 Gene knockout model — Pitx2 Cre / + ; Bmp4 f / f In mice, this mouse model was found to exhibit 100% bilateral cleft lip phenotype. Currently reported... Nestin-Cre;Bmp4 f / f The mouse cleft lip model exhibited an unstable bilateral cleft lip phenotype (77.8%). The method constructed in this invention... Pitx2 Cre / + ; Bmp4 f / f All mice exhibited bilateral cleft lip. Therefore, compared to previously reported... Nestin-Cre;Bmp4 The mouse model constructed in this invention is more stable and can better characterize human cleft lip genetic diseases, which is of significant research value. Furthermore, this invention also has the following advantages: 1. 100% penetrability of bilateral cleft lip phenotype: through Pitx2-Cre ,make sure Bmp4 The precise knockout of the gene resulted in 100% bilateral cleft lip in the mutant embryos.

[0020] 2. High pathological consistency: The bilateral cleft lip phenotype matched the clinical characteristics of human nonsyndromic cleft lip (NSCLP). Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 This is a technical roadmap of the present invention; Figure 2 for Nestin Gene expression patterns in the craniofacial region of mice at different developmental stages; detection using whole-cell in situ hybridization. Nestin Gene expression in the craniofacial region of E9.5, E10.0, and E10.5 mice; among which, AF was... Nestin Gene expression in the craniofacial region of E9.5 mice; C is a magnified view of the white-framed area in B; D is a slice view; E and F are magnified views of the areas outlined in red and black in D; GL is... Nestin Gene expression in the craniofacial region of E10.0 mice. I is a magnified view of the white box area in H, J is a slice view, and K and L are magnified views of the areas outlined in red and black boxes in J. Note: At this time... Nestin The gene begins to be expressed in the epithelium of the frontonasal and maxillary processes; MQ is Nestin Gene expression in the craniofacial region of E10.5 mice; O is an enlarged view of the white box area in H; P is a schematic diagram of a section of E10.5 mice; Q is a section of in situ hybridization tissue from E10.5 mice; 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 for AC, GI, and MO are 200 μm, and the scale bars for DF, JK, and Q are 50 μm; arrows indicate... Nestin Gene expression regions; Figure 3 for Pitx2 and Bmp4 Gene expression patterns in the craniofacial region of mice at different developmental stages; where mnd represents the mandibular process; mx represents the maxillary process; scale bar is 200 μm; Figure 4 for Pitx2 Cre / + ;R26R mTmG In the craniofacial region of mice at different developmental stages Cre Gene expression patterns; where AB represents Pitx2 Cre / + ;R26R mTmG In mice, the frontonasal process and maxillary process of E9.5 Cre Gene activity status; CD is Pitx2 Cre / + ;R26RmTmG In mice, the epithelium at the fusion of the medial and lateral nasal processes of E10.5 and the epithelium of the maxillary process... Cre Gene activity status; EF is Pitx2 Cre / + ;R26R mTmG In mice, at the fusion of the medial and lateral nasal processes of E11.5, at the junction of the maxillary process and the lateral nasal process, and in the epithelium of the maxillary process... Cre Gene activity status; upper lip development is complete when GH is E12.5. Pitx2 Cre / + ;R26R mTmG In mice, the epithelium of the lip and maxillary process Cre Gene activity status; fnp represents the frontonasal process; mx represents the maxillary process; lnp represents the lateral nasal process; mnp represents the medial nasal process; the scale bar of AH is 100μm; Figure 5 for Bmp4 The gene is at E10.5 Pitx2 Cre / + ;Bmp4 f / f Knockout in mice; where AB represents Bmp4 Gene expression in the epithelium of the medial and lateral nasal process contact area and the maxillary process of E10.5 wild-type mice; CD is Bmp4 Genes in Pitx2 Cre / + ;Bmp4 f / f Knockout of the epithelium in the contact area between the lateral and medial nasal processes and the maxillary process in mice; lnp represents the lateral nasal process; mnp represents the medial nasal process; mx represents the maxillary process; mnd represents the mandibular process; yellow arrows indicate mutant embryos. Bmp4 The region where the gene was knocked out; scale bar is 200 μm. Figure 6 for Pitx2 Cre / + ;Bmp4 f / f Craniofacial phenotype of mice E18.5 at birth; where A and E represent the lip and palate phenotype of wild-type mice; B and F represent... 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 / fMouse lip and palate phenotypes; the scale bar for AD was 500 μm, and the scale bar for EH was 1000 μm; Figure 7 for Pitx2 Cre / + ; Bmp4 f / f Phenotypes of hands and feet at birth in mice E18.5; where A represents polydactyly; B represents fused legs; scale bar is 500 μm; Figure 8 for Pitx2 Cre / + ;Bmp4 f / f Early craniofacial phenotypes in mice; where AB represents the medial and lateral nasal processes of E10.5 wild-type mice; CD represents E10.5... Pitx2 Cre / + ;Bmp4 f / f Phenotype of the maxillary process and medial and lateral nasal processes in mouse embryos; dashed lines represent the outline of the nasal fossa; black arrows indicate the fusion point of the medial and lateral nasal processes in wild-type embryos; * indicates that the medial and lateral nasal processes in mutant embryos are not fused; EH is E10.0. Pitx2 Cre / + ;Bmp4 f / f Comparison of early craniofacial morphology between mouse embryos and wild-type mice; dashed lines represent the nasal cavity outline before fusion on the side; fnp represents the frontonasal process; lnp represents the lateral nasal process; mnp represents the medial nasal process; mx represents the maxillary process; mnd represents the mandibular process; scale bar is 200 μm. Detailed Implementation

[0023] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0024] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0025] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0026] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0027] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0028] Unless otherwise specified, all materials used in this invention are commonly purchased by those skilled in the art, and all methods used in this invention are well known to those skilled in the art.

[0029] Example 1 Pitx2 Cre / + ;Bmp4 f / f Mouse construction and phenotypic analysis 1. Genetic Tools and Design Cre tools mouse: Pitx2 Cre / + Mice were purchased from MMRRC, Inc., USA (Cat# 000126-UCD; RRID: MMRRC_000126-UCD). ires-Cre-PGKneomycin Chromosomal fragment introduction Pitx2 The PvuII and NruI sites in exon 5.

[0030] 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), with loxP insertion sites. Bmp4 Flanking the fourth exon of the gene.

[0031] 2. Hybridization strategy:Pitx2 Cre / + mice and Bmp4 f / f Mice were mated to produce offspring with a heterozygous genotype. Pitx2 Cre / + ;Bmp4 f / + Mice; the aforementioned Pitx2 Cre / + ;Bmp4 f / + mice and Bmp4 f / f Mice mate to obtain Pitx2 Cre / + ; Bmp4 f / f Mice were designated as the experimental group; wild-type mice served as the control group.

[0032] 3. Construction Pitx2 Cre / + ;R26R mTmG Mice: by Pitx2 Cre / + mice and R26R mTmG Obtained by mating mice.

[0033] 4. The technical approach of this embodiment is as follows: Figure 1 As shown, specifically: firstly, wild-type mice... Bmp4 , Pitx2 and Nestin Genes were subjected to whole-body in situ hybridization and sectioning for comparison. Pitx2 and Nestin Differences in gene expression, and with Bmp4 Gene expression overlap; for Pitx2 Cre / + ;R26R mTmG The mice were subjected to fluorescence observation and sectioning to further confirm the results. Pitx2 Gene promoter driven Cre Location of gene expression; Pitx2 Cre / + ;Bmp4 f / f Whole-body in situ hybridization was performed in mice to verify Bmp4 Gene knockout efficiency; Pitx2 Cre / + ;Bmp4 f / f The craniofacial defect phenotypes of mice were analyzed.

[0034] 5. Verification experiment and results (1) Detection of wild-type mice by whole-body in situ hybridization technique NestinGene expression in the craniofacial region at E9.5, E10.0, and E10.5 was as follows: Figure 2 As shown. The results indicate that E9.5 wild-type mice... Nestin The gene is expressed in the frontonasal mesenchyme, while in the expression Bmp4 The gene is not expressed in the epithelium of the frontonasal and maxillary processes of E10.5 wild-type mice. Nestin The gene is expressed in the epithelium of the fusion site of the inner and outer nasal processes and the maxillary process. Nestin Gene expression only begins in the epithelium of the frontonasal and maxillary processes at E10.0. This was also detected in wild-type mice via whole-cell in situ hybridization. Pitx2 and Bmp4 The expression of the gene in the craniofacial region at different time points from E9.25 to E10.5 is shown in the following results. Figure 3 As shown. The results indicate that, Pitx2 Genes and Bmp4 The gene expression patterns highly overlap, and both genes were already expressed in the epithelium of the frontonasal and maxillary process primordia in early lip development as early as E9.25, which is more advanced than... Nestin Gene expression occurs much earlier. This invention further constructs... Pitx2 Cre / + ;R26R mTmG Mice, observed by immunofluorescence Pitx2 Gene promoter driven Cre The expression pattern, the result is as follows Figure 4 As shown. The results show that, Pitx2 Cre / + ;R26R mTmG Mice showed obvious abnormalities in the E9.5 frontonasal process and maxillary process. Cre Gene activity ( Figure 4 (A and B in the text) Pitx2 Cre / + ;R26R mTmG Mice have epithelial cells at the fusion site of the medial and lateral nasal processes and the epithelium of the maxillary process at E10.5. Cre Gene activity ( Figure 4 (C and D in the middle); EF: Pitx2 Cre / + ;R26R mTmG Mice showed [symptoms] at the fusion of the medial and lateral nasal processes of E11.5, at the junction of the maxillary process and the lateral nasal process, and in the epithelium of the maxillary process. Cre Gene activity ( Figure 4 (E and F in the text); GH: Upper lip development is complete at E12.5. Pitx2 Cre / + ;R26R mTmG In mice, the epithelium of the lip and maxillary process CreGenes have continuous activity ( Figure 4 (G and H in the text). Therefore, E9.5-E12.5 Pitx2 Cre / + ;R26R mTmG mouse Cre Gene activity and wild-type mice Pitx2 Gene expression was consistent, and activity was also observed in the epithelium of the frontonasal and maxillary processes of early lip development primordia as early as E9.5. These results indicate that... Bmp4 Genes began regulating lip development as early as E9.25, while existing reports... Nestin-Cre;Bmp4 f / f Mouse model using Nestin-Cre Mouse knockout before E10.0 was not possible. Bmp4 Genes, which may be the reason for the phenotypic instability of this model; and utilizing Pitx2-Cre Mice can be knocked out throughout the entire lip development process. Bmp4 Genetic analysis may yield a more stable bilateral cleft lip model.

[0035] (2) To Pitx2 Cre / + ;Bmp4 f / f In mice Bmp4 The gene expression level was verified, and the results were as follows: Figure 5 As shown. In situ hybridization results show: at the fusion of the medial and lateral nasal processes and in the maxillary process... Bmp4 mRNA expression levels were significantly downregulated, i.e. Bmp4 Genes in Pitx2 Cre / + ;Bmp4 f / f It was successfully knocked out in mice.

[0036] (3) To 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 As shown. The results showed that the lip and palate of wild-type mice were normally developed ( Figure 6 (A, E in the text) Pitx2 Cre / + ;Bmp4 f / f The mice presented with bilateral cleft lip and complete cleft palate. Figure 6 (B, F in the middle) Pitx2 Cre / + ;Bmp4 f / f The mice presented with bilateral cleft lip and cleft palate. Figure 6 (C, G) Pitx2 Cre / + ;Bmp4 f / f The mice presented with bilateral cleft lip and cleft soft palate. Figure 6 (D, H); E12.5 and later Pitx2 Cre / + ;Bmp4 f / f All mouse embryos showed a phenotype of bilateral cleft lip, with or without cleft palate, i.e., 100% bilateral cleft lip, with various types of cleft palate; some mutant mice also showed phenotypes such as polydactyly and syndactyly (Table 1 and 1). Figure 7 The bilateral cleft lip phenotype first appeared in E10.5. Compared with the wild type, the medial and lateral nasal processes are not fused, and the medial nasal process and maxillary process are smaller in size. This size reduction is abnormal and can be observed as early as E10.0.

[0037] Table 1 Pitx2 Cre / + ;Bmp4 f / f Mouse phenotypic statistics (4) Pitx2 Cre / + ;Bmp4 f / f The craniofacial phenotype of mice in early development was observed, and the results were as follows: Figure 8 As shown. The results showed that the medial and lateral nasal processes of E10.5 wild-type mice began to contact and fuse; E10.5 Pitx2 Cre / + ; Bmp4 f / f The maxillary process and medial and lateral nasal processes of mouse embryos are relatively small, and the medial and lateral nasal processes are not fused; E10.0 Pitx2 Cre / + ;Bmp4 f / f Compared to wild-type mice, mouse embryos have flatter nasal basements on both the inner and outer sides and smaller maxillary processes.

[0038] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method based on Pitx2-Cre Conditional knockout Bmp4 The method for constructing a bilateral cleft lip mouse model is characterized by, Includes the following steps: Will Pitx2-Cre tool mouse and Bmp4 f / f Mice mate and obtain Pitx2-Cre;Bmp4 f / + Mice; The Pitx2-Cre;Bmp4 f / + mice and Bmp4 f / f Mice were mated to obtain the bilateral cleft lip mouse model; The Pitx2-Cre tool mouse as a reason Pitx2 Gene promoter driven Cre Mice expressing genes.

2. The application of a bilateral cleft lip mouse model constructed using the method described in claim 1 in the study of the molecular mechanisms of bilateral cleft lip.

3. The application of a bilateral cleft lip mouse model constructed using the construction method described in claim 1 in screening drugs for the prevention and / or treatment of bilateral cleft lip.

4. The application of a bilateral cleft lip mouse model constructed using the method described in claim 1 in the genetic study of bilateral cleft lip.

5. The application of a bilateral cleft lip mouse model constructed using the method described in claim 1 in the study of the biological function of bilateral cleft lip.

6. The application of a bilateral cleft lip mouse model constructed using the construction method described in claim 1 in the preparation of a bilateral cleft lip auxiliary diagnostic product.

7. The application of a bilateral cleft lip mouse model constructed using the method described in claim 1 in the safety evaluation of drugs for treating bilateral cleft lip.

8. The application of a bilateral cleft lip mouse model constructed using the construction method described in claim 1 in the study of lip development mechanism.

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