A recombinant DNA construct, a non-human genetically engineered animal model and application thereof

Abstract

This invention provides a recombinant DNA construct, a non-human genetically engineered animal model, and their applications, relating to the fields of genetic engineering and biomedicine. The recombinant DNA construct operably connects, from its 5' to 3' end, the following: a promoter, a loxP-Stop-loxP sequence, a Notum gene coding sequence, a P2A self-cleaving polypeptide coding sequence, and an mBaojin fluorescent reporter gene coding sequence. This recombinant DNA construct utilizes the loxP-Stop-loxP sequence to achieve precise spatiotemporal regulation of gene expression, avoiding the lethal defects caused by continuous systemic expression. Simultaneously, the self-cleavage of the P2A sequence allows the Notum protein to be normally secreted extracellularly, while the highly sensitive fluorescent protein remains intracellular, effectively overcoming the technical challenge of inaccurate reverse localization in cells containing secretory proteins.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering and biomedicine, and more specifically, to a recombinant DNA construct, a non-human genetically engineered animal model, and their applications. Background Technology

[0002] The Wnt signaling pathway plays a crucial role in embryonic development, tissue regeneration, and the pathogenesis of various diseases. Notum, a highly conserved secretory enzyme, is a key negative regulator of this pathway. In-depth research into the expression patterns and biological functions of these secretory proteins at the in vivo level is of significant scientific importance for exploring related physiological and pathological mechanisms.

[0003] Currently, in vivo studies targeting specific gene functions primarily rely on existing conventional genetically engineered animal models. Common research methods in this field include in vitro validation at the cellular level or constructing conventional transgenic animals that continuously express the target gene systemically. Furthermore, when detecting and tracking the expression of specific proteins in tissues, current technologies mostly employ conventional molecular biology detection methods such as in situ hybridization and immunohistochemistry.

[0004] However, existing in vivo research tools and detection methods have significant limitations. On the one hand, for genes playing key regulatory roles, traditional systemic or constitutive sustained overexpression often leads to embryonic lethality or severe developmental abnormalities, making it impossible for researchers to achieve precise spatiotemporal regulation of gene expression in specific tissue microenvironments or developmental stages. On the other hand, because secreted proteins are released and diffuse into the extracellular matrix after translation, existing traditional detection techniques are cumbersome and have limited sensitivity, making it extremely difficult to accurately reverse-engineer which source cells synthesized these target proteins in complex living tissues.

[0005] In summary, there is an urgent need for a more advanced in vivo research tool in the current technology. Overcoming the bottleneck of ambiguous cell localization of secretory target proteins, while simultaneously addressing the lack of spatiotemporally specific induction control of target genes at the in vivo level, and achieving the organic unity of precise and controllable expression of target genes and highly sensitive in situ cell tracing, are pressing technical challenges that need to be addressed in this field.

[0006] In view of this, the present invention is hereby proposed. Summary of the Invention

[0007] The purpose of this invention is to provide a recombinant DNA construct, a non-human genetically engineered animal model, and its applications. The recombinant DNA construct achieves precise spatiotemporal induction of gene expression through the loxP-Stop-loxP sequence and utilizes the P2A sequence to specifically retain fluorescent proteins within the cell, thus effectively solving the technical problem of the inability to accurately track and locate source cells for secreted proteins in vivo.

[0008] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted: In a first aspect, the present invention provides a recombinant DNA construct in which the following are operably linked from the 5' end to the 3' end: a promoter, a loxP-Stop-loxP sequence, a Notum gene coding sequence, a P2A self-cleaving polypeptide coding sequence, and an mBaojin fluorescent reporter gene coding sequence.

[0009] In an optional implementation, the promoter is a systemic expression promoter or a tissue-specific promoter; Preferably, the promoter is a CAG promoter or a Col1a1 promoter.

[0010] Preferably, the promoter comprises a nucleotide sequence as shown in SEQ ID NO: 15.

[0011] In an optional embodiment, the loxP-Stop-loxP sequence includes a first loxP site, a Stop sequence, and a second loxP site sequentially from the 5' end to the 3' end; Preferably, the Stop sequence contains a transcription termination signal; Preferably, the Stop sequence comprises three tandem SV40 polyA sequences; Preferably, both the first loxP site and the second loxP site contain the nucleotide sequence shown in SEQ ID NO: 16; Preferably, the Stop sequence comprises a nucleotide sequence as shown in SEQ ID NO: 17.

[0012] In an optional embodiment, the 3' end of the mBaojin fluorescent reporter gene coding sequence is also operably linked with a WPRE sequence and a PolyA sequence in sequence; Preferably, the PolyA sequence is a bGH polyA sequence.

[0013] Preferably, the WPRE sequence comprises the nucleotide sequence shown in SEQ ID NO: 21; Preferably, the PolyA sequence comprises a nucleotide sequence as shown in SEQ ID NO: 22.

[0014] In an optional embodiment, the recombinant DNA construct is connected to two ends with a 5' homologous arm and a 3' homologous arm for site-specific integration at a genomic safe harbor site, respectively. Preferably, the safe harbor site is the H11 site; Preferably, the lengths of the 5' end homologous arm and the 3' end homologous arm are 1 kb; More preferably, the nucleotide sequence of the 5' homologous arm is shown in SEQ ID NO: 2; More preferably, the nucleotide sequence of the 3' homologous arm is shown in SEQ ID NO: 3.

[0015] In an optional embodiment, the Notum gene coding sequence comprises the nucleotide sequence shown in SEQ ID NO: 18; and / or, The P2A self-cleaving polypeptide coding sequence comprises the nucleotide sequence shown in SEQ ID NO: 19; and / or, The mBaojin fluorescent reporter gene coding sequence comprises the nucleotide sequence shown in SEQ ID NO: 20; and / or, The recombinant DNA construct contains a nucleotide sequence as shown in SEQ ID NO: 1.

[0016] In a second aspect, the present invention provides a recombinant expression vector comprising the recombinant DNA construct described in any of the foregoing embodiments.

[0017] Thirdly, the present invention provides a recombinant cell comprising the recombinant DNA construct described in any of the foregoing embodiments, or comprising the recombinant expression vector described in the foregoing embodiments; Preferably, the recombinant cells are non-human mammalian cells; Mouse cells are preferred.

[0018] Fourthly, this invention provides a method for constructing a non-human genetically engineered animal model of Cre-dependent Notum gene overexpression and in vivo tracing, comprising: The recombinant DNA construct or the recombinant expression vector described in any of the foregoing embodiments is introduced into a fertilized egg or embryonic stem cell of a non-human mammal. Preferably, the recombinant DNA construct is integrated into the H11 site of the genome of the fertilized egg or embryonic stem cell using CRISPR / Cas9 technology.

[0019] Fifthly, the present invention provides a primer set for detecting recombinant DNA constructs as described in any of the foregoing embodiments, or for identifying recombinant cells as described in the foregoing embodiments, or non-human genetically engineered animal models prepared as described in the foregoing embodiments, or for identifying non-human genetically engineered animal models as described in the foregoing embodiments; the primer set comprises at least one of the following primer combinations: A. First primer combination for amplifying the 5' integration region: the forward primer contains the sequence shown in SEQ ID NO: 4, and the reverse primer contains the sequence shown in SEQ ID NO: 5; B. A second primer combination for amplifying the 3' integration region: the forward primer contains the sequence shown in SEQ ID NO: 6, and the reverse primer contains the sequence shown in SEQ ID NO: 7; C. A third primer combination for distinguishing wild-type and transgenic alleles: comprising forward primer 1, forward primer 2 and reverse primer, wherein forward primer 1 contains the sequence shown in SEQ ID NO: 8, forward primer 2 contains the sequence shown in SEQ ID NO: 9, and reverse primer contains the sequence shown in SEQ ID NO: 10; D. A fourth primer combination for amplifying internal fragments of the recombinant DNA construct: the forward primer contains the sequence shown in SEQ ID NO: 11, and the reverse primer contains the sequence shown in SEQ ID NO: 12; E. A fifth primer combination for amplifying internal fragments of the recombinant DNA construct: the forward primer contains the sequence shown in SEQ ID NO: 13, and the reverse primer contains the sequence shown in SEQ ID NO: 14.

[0020] In a sixth aspect, the present invention provides a kit comprising the primer set described in the foregoing embodiments; Preferably, the kit further includes one or more of DNA extraction reagents and PCR amplification reagents.

[0021] In a seventh aspect, the present invention provides an application of the recombinant DNA construct, the recombinant expression vector, or the recombinant cell as described in any of the foregoing embodiments, applicable to any of the following aspects: A. Application in the preparation of non-human genetically engineered animal models with Cre-dependent Notum gene overexpression and in vivo tracing; B. Application in the preparation of in vitro models for studying Wnt signaling pathways or tissue regeneration and repair; C. Application in the preparation of drug screening models for non-diagnostic therapeutic purposes, used to screen or evaluate drugs for intervention in metabolic diseases or cancer.

[0022] Compared with existing technologies, the beneficial effects of this invention are as follows: This recombinant DNA construct establishes a highly controllable inducible expression mechanism by operably linking the promoter, loxP-Stop-loxP sequence, and target gene sequence. Under natural conditions, the loxP-Stop-loxP sequence can effectively block the transcription of downstream genes, preventing systemic or constitutive sustained expression of the target gene, thereby effectively overcoming the defects of embryonic lethality or abnormal in vivo development caused by abnormal expression of key regulatory factors. When a specific recombinase is introduced into a specific microenvironment within the organism, the blocking sequence can be excised at specific sites and times, achieving precise induction of Notum gene expression in specific tissues or developmental stages.

[0023] Furthermore, this recombinant DNA construct creatively incorporates the P2A self-cleaving polypeptide coding sequence and the mBaojin fluorescent reporter gene coding sequence, placing them within the same open reading frame as the Notum gene coding sequence. Utilizing the unique self-cleaving property of the P2A sequence during translation, the Notum protein translated from the same messenger RNA is physically separated from the mBaojin fluorescent protein. This structure not only ensures that the Notum protein maintains its native secretory properties and is released into the extracellular matrix to perform its normal biological regulatory functions, but also promotes the stable retention of the highly sensitive mBaojin fluorescent protein within the original cell from which the protein was initially synthesized.

[0024] This unique structural design ingeniously overcomes the technical bottleneck of secreted factors being easily dispersed once released, making in-situ tracking extremely difficult using traditional detection methods. This construct successfully integrates in vivo spatiotemporally specific gene induction with highly sensitive source cell precise localization, providing an extremely sophisticated and intuitive tool for in-depth in vivo biological research. Attached Figure Description

[0025] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0026] Figure 1 This is a schematic diagram of the structural design of a recombinant DNA construct for Cre-dependent Notum gene overexpression and in vivo tracing provided in Example 1 of this application; Figure 2 This is a schematic diagram of a targeting strategy for precisely knocking the recombinant DNA construct into the mouse H11 safe harbor site, as provided in Embodiment 2 of this application. Figure 3Agarose gel electrophoresis results of PCR genotype identification of genetically engineered mice (F1 generation) provided in Example 3 of this application; Figure 4 This is a schematic diagram of the primer design and binding sites used for genotype identification and sequence integrity verification in Example 3 of this application; Figure 5 This is an agarose gel electrophoresis image of the PCR genotype identification of the offspring of the F1 generation positive mice and Dmp1-iCre mice in Example 4 of this application. Figure 6 This image shows the immunofluorescence staining results of a section of the mandibular bone tissue from the hybrid offspring mice in Example 4 of this application, which illustrates the difference in fluorescence expression in odontoblasts and osteoblasts between wild-type control mice and double-positive transgenic mice. Detailed Implementation

[0027] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0028] This application provides a recombinant DNA construct in which the following components are operably linked from the 5' end to the 3' end: a promoter, a loxP-Stop-loxP sequence, a Notum gene coding sequence, a P2A self-cleaving polypeptide coding sequence, and an mBaojin fluorescent reporter gene coding sequence.

[0029] The aforementioned 'operable ligation' means that the DNA fragments are not only physically connected in sequence, but more importantly, functionally connected. That is, the distances and open reading frames (ORFs) between them are correct, enabling the promoter to successfully drive the transcription of downstream sequences, and preventing frameshift mutations during translation of the downstream coding sequences.

[0030] For example, if the components are only physically connected in a conventional manner without being "operably connected", the Notum gene and P2A may be misread by ribosomes (i.e. frameshift mutations) due to base insertions or deletions that are not multiples of 3. This can lead to the downstream translation of a protein sequence with no biological function, and P2A will also lose its self-cleavage function.

[0031] The promoters mentioned above are regulatory elements that drive DNA transcription and are responsible for recruiting RNA polymerases to initiate the transcription process of the entire downstream gene.

[0032] The aforementioned loxP-Stop-loxP sequence (LSL) is an inducible transcriptional blocking element. Based on the Cre-loxP site-specific recombination system, under default conditions, the multiple transcriptional termination signals contained in the middle Stop sequence can effectively block the transcription of downstream genes. When a specific recombinase (Cre) is introduced, homologous recombination occurs between the two loxP sites, thereby cleaving the Stop sequence, thus releasing the transcriptional blockade and allowing the specific expression of downstream genes.

[0033] The Notum gene coding sequence mentioned above is the core target gene of this product, and its translation product is a secreted Wnt signaling pathway repressor protein.

[0034] The P2A self-cleaving polypeptide coding sequence described above is a functional element that plays a role in 'internal translational cleavage'. Its mechanism is 'ribosome jumping', that is, when translation passes through this sequence, the ribosome does not form peptide bonds, thereby directly separating the two proteins at the translational level without interrupting translation.

[0035] The above-mentioned mBaojin fluorescent reporter gene encoding sequence is a highly sensitive fluorescent protein gene. Its mechanism is to emit fluorescence under excitation at a specific wavelength, which is used to realize in situ detection and localization tracing of target cells in living tissues.

[0036] It is worth noting that this application, targeting the characteristics of Notum as a secretory protein, cleverly solves the problem of traditional gene expression tracking by combining a P2A self-cleavage sequence with a fluorescent reporter gene. Specifically, the Notum precursor protein itself usually contains a secretion signal peptide. After P2A-mediated intratranslational cleavage, the Notum protein can be normally secreted into the extracellular microenvironment along the endoplasmic reticulum-Golgi apparatus secretion pathway of the host cell to exert its physiological function of inhibiting the Wnt signaling pathway. Meanwhile, the mBaojin fluorescent protein, which is cleaved and separated, will be stably retained in the cytoplasm of the mother cell that translated the protein due to the lack of guidance from the secretion signal peptide. This design of "functional molecule exocytosis and tracer molecule retention" effectively avoids the bottleneck of secretory proteins easily diffusing in tissues after release, which makes it impossible to accurately reverse locate their source cells using conventional fluorescent fusion tags.

[0037] The aforementioned components, under an 'operably linked' structure, generate a highly innovative dual synergistic mechanism: On the one hand, the promoter, loxP-Stop-loxP sequence, and target gene work synergistically to effectively avoid systemic pan-expression of the Notum gene by utilizing the blocking effect of the LSL element. It is activated only at specific times and in specific cells when the specific Cre recombinase is introduced, completely overcoming the defect that continuous systemic overexpression can easily lead to lethality or developmental abnormalities in live embryos, and achieving precise spatiotemporal specific induction. On the other hand, the target gene, P2A sequence, and fluorescent reporter gene generate a targeted retention synergy of 'functional molecule exocytosis and tracer molecule retention'. With the help of P2A's unique intratranslational autocleavage mechanism, secretory Notum and non-secretory mBaojin are translated from the same mRNA, thus ensuring 1:1 absolute co-expression, and they rapidly separate after translation, allowing Notum to be normally secreted into the extracellular matrix to perform its biological function, while mBaojin remains firmly retained in the source cell where the protein was synthesized. This ingenious synergistic design, without affecting the natural secretion and activity of the target protein, significantly overcomes the traditional technical bottleneck of the inability to trace the source of secreted factors after diffusion. It allows researchers to precisely reverse locate and trace the source cells of the target gene in situ simply by detecting highly sensitive fluorescence within the cell.

[0038] In summary, the recombinant DNA construct provided in this application, through the organic tandem and functional synergy of the aforementioned specific components, completely overcomes the developmental abnormalities caused by the continuous systemic expression of key genes. At the same time, it breaks through the technical bottleneck of the diffusion and untraceability of secretory target proteins in living tissues, and successfully achieves a perfect unity between the spatiotemporal specific precise regulation of gene expression and highly sensitive in situ cell localization tracing.

[0039] In some embodiments, the promoter is a systemic expression promoter or a tissue-specific promoter; further, the promoter is a CAG promoter or a Col1a1 promoter. Preferably, the promoter comprises a nucleotide sequence as shown in SEQ ID NO: 15.

[0040] In this embodiment, the promoter type in the recombinant DNA construct has been further optimized and configured. The promoter can be a systemic expression promoter or a tissue-specific promoter.

[0041] In a preferred embodiment, the systemic expression promoter is the CAG promoter. As a highly efficient, multi-functional, pan-expression promoter, the CAG promoter provides a strong transcriptional drive in most cell types of organisms. Combining the CAG promoter with a downstream loxP-Stop-loxP (LSL) element creates a synergistic mechanism at the construct level: the construct possesses pan-expression potential at the systemic level, but its actual transcription is strictly blocked by the LSL element. When applied to genetically engineered animal models, only a tool vector or tool animal carrying a tissue-specific Cre recombinase needs to be introduced to activate the Notum gene at any specified single organ or microenvironment. This combined design greatly enhances the versatility and flexibility of this recombinant DNA construct in multi-tissue, multi-organ biological research, and reduces model preparation costs.

[0042] In another preferred embodiment, the tissue-specific promoter is the Col1a1 promoter. The Col1a1 promoter is a type I collagen gene promoter with high osteoblast and connective tissue specificity. Binding the tissue-specific promoter to a downstream LSL element creates a "dual-lock" synergistic mechanism at the transcriptional level: successful transcription of the target gene requires not only specific Cre recombinase excision of the blocking sequence but also strict dependence on target cell-specific transcription factors to activate the promoter. This dual-lock mechanism significantly reduces potential basal leakage of exogenous genes in non-target tissues, providing extreme spatial precision for gene expression, making it particularly suitable for studies of the bone development or tissue regeneration microenvironment, which is highly sensitive to basal expression.

[0043] Furthermore, those skilled in the art will understand that the promoters are not limited to the specific examples described above. Based on the same spatiotemporally controllable regulation principle, the systemic expression promoters can also be replaced with promoters such as CMV, EF1α, or UBC; the tissue-specific promoters can also be replaced with liver-specific Albumin promoters, intestinal-specific Villin promoters, or neuron-specific Synapsin promoters, etc., depending on research needs. These equivalent substitutions all fall within the protection scope of the constructs in this application.

[0044] In some embodiments, the loxP-Stop-loxP sequence comprises a first loxP site, a Stop sequence, and a second loxP site sequentially from the 5' end to the 3' end; preferably, the Stop sequence contains a transcription termination signal; preferably, the Stop sequence comprises three tandem SV40 polyA sequences; preferably, both the first loxP site and the second loxP site contain the nucleotide sequence shown in SEQ ID NO: 16; preferably, the Stop sequence contains the nucleotide sequence shown in SEQ ID NO: 17.

[0045] In this embodiment, the internal fine structure of the gene "switch" element in the recombinant DNA construct, the loxP-Stop-loxP (LSL) sequence, has been further optimized and defined. The Stop sequence in the loxP-Stop-loxP sequence contains a transcription termination signal.

[0046] In studies of genetically engineered animal models, particularly those related to embryonic development, trace amounts of background leakage expression of the target gene in the uninduced state often lead to unexpected phenotypic interference or even lethal effects. To achieve absolute transcriptional silencing, in a preferred embodiment, the loxP-Stop-loxP sequence comprises, from the 5' end to the 3' end, a first loxP site, three tandem SV40 polyA sequences, and a second loxP site.

[0047] The first and second loxP sites are 34bp specific recombinase recognition target sequences aligned in the same direction; the three tandem SV40 (simian vacuolating virus 40) polyA sequences contained in the middle act as potent transcription termination elements.

[0048] This structure generates a highly robust blocking and induction synergistic mechanism within the system: under non-induction conditions (i.e., in the absence of Cre recombinase), when the upstream promoter-driven RNA polymerase crosses the first loxP site, it encounters a tandem transcription termination barrier composed of three strong SV40 polyA signals. While a single transcription termination signal often has a certain readthrough probability, the triple tandem structure employed in this construct forms a progressively layered transcriptional interception defense, effectively blocking the possibility of polymerase "readthrough" and ensuring that the downstream Notum gene and its subsequent sequences are in a state of strict transcriptional silencing before induction, thereby effectively avoiding developmental defects caused by abnormal expression of key regulatory genes.

[0049] Under induction conditions (i.e., when tissue-specific or time-specific Cre recombinases are introduced), the Cre enzyme specifically recognizes and binds to the homologous loxP sites at both ends of the sequence, efficiently catalyzing homologous recombination between the two sites. This results in the precise excision of the triple-tandem SV40 polyA blocking sequence between the two sites as a circular DNA molecule. After the blocking sequence is removed, only a loxP remnant remains at the original site, instantly opening the transcription pathway downstream of the promoter. This allows RNA polymerase to successfully enter and transcribe the subsequent Notum target gene. This design ensures an extreme silencing effect while controlling the length of the homologous recombination sequence within an optimal range, ensuring that the system can rapidly and efficiently switch to the expression state upon receiving the Cre induction signal, endowing gene expression with extremely high sensitivity and spatiotemporal controllability.

[0050] Furthermore, those skilled in the art will understand that, as an equivalent alternative, the transcription termination signal (Stop sequence) is not limited to the above-mentioned tandem SV40 polyA combination, but can also be replaced by transcriptional blocking elements known in the art, such as (including but not limited to) Neo gene termination box, rabbit β-globin polyA signal combination; the loxP site can also be replaced by orthogonal recombination site mutants such as lox2272 and lox511 according to the needs of multiple gene regulation. These alternative designs can all achieve the same inducible expression mechanism and fall within the protection scope of the construct of this application.

[0051] In some embodiments, the 3' end of the mBaojin fluorescent reporter gene coding sequence is operably linked with a WPRE sequence and a PolyA sequence in sequence; preferably, the PolyA sequence is a bGH polyA sequence. Preferably, the WPRE sequence comprises the nucleotide sequence shown in SEQ ID NO: 21; preferably, the PolyA sequence comprises the nucleotide sequence shown in SEQ ID NO: 22.

[0052] In this embodiment, to further ensure and significantly enhance the expression abundance of the target gene and the fluorescent reporter gene at the post-transcriptional level, the 3' end structure of the recombinant DNA construct was optimized. Specifically, the 3' end of the mBaojin fluorescent reporter gene coding sequence is operably linked with a WPRE sequence and a PolyA sequence in sequence.

[0053] The WPRE sequence is a post-transcriptional regulatory element of marmot hepatitis virus. During gene expression, the WPRE sequence can promote the formation of specific higher-order structures from the transcribed mRNA precursors. These structures can be efficiently recognized by the host cell's nuclear transport mechanisms, thereby significantly promoting the processing and maturation of the mRNA precursors and their export from the nucleus to the cytoplasm.

[0054] The downstream PolyA sequence works synergistically with the WPRE sequence. In a preferred embodiment, the PolyA sequence is the bGH polyA sequence (bovine growth hormone polyadenylation signal sequence). As a powerful transcription termination and tailing signal, bGH polyA guides the precise cleavage of newly synthesized mRNA at the 3' end and effectively adds a polyadenylated tail. This structure not only protects mRNA from rapid degradation by intracellular exonucleases, significantly prolonging the intracellular half-life of mRNA molecules, but also synergizes with translation initiation mechanisms, significantly improving the efficiency of ribosome-mediated translation initiation.

[0055] The WPRE and bGH polyA sequences at the ends of the recombinant DNA construct form a synergistic "post-transcriptional dual enhancement" mechanism. They are not only physically linked sequentially but also complement each other in biological function: WPRE accelerates the output of effective mRNA ("open source"), while bGH polyA enhances mRNA stability ("throttling"). This ingenious synergistic design allows the construct, upon activation by Cre recombinase, to maximize the conversion of limited transcripts into actual protein products. This not only ensures that the secretory Notum protein reaches a sufficient concentration to exert its inhibitory function on the Wnt signaling pathway, but more importantly, it promotes the accumulation of high abundance of mBaojin fluorescent protein in the source cells, ensuring strong fluorescence emission even in the complex microenvironment of living tissue. This design completely overcomes the technical bottlenecks often faced by in vivo fluorescence tracing, such as weak signals and easy quenching, greatly improving the sensitivity and reliability of this genetically engineered animal model in precise cell localization and tracing detection.

[0056] Furthermore, those skilled in the art will understand that the post-transcriptional regulatory sequence can also be replaced by conventional equivalent substitutions. For example, the WPRE sequence can be replaced with a truncated and optimized WPRE mutant sequence (such as WPRE3) to accommodate stricter vector capacity limitations; the PolyA sequence can also be replaced with other types of eukaryotic tailing signals, such as SV40 polyA or rabbit β-globin polyA, depending on the actual requirements for target protein expression abundance. These equivalent substitution structures can all achieve similar post-transcriptional enhancement effects and fall within the protection scope of the claims of this application.

[0057] In some embodiments, the recombinant DNA construct is connected to two ends with a 5' homologous arm and a 3' homologous arm for site-specific integration at a genomic safe harbor site, respectively; preferably, the safe harbor site is an H11 site; preferably, the length of the 5' homologous arm and the 3' homologous arm is 1 kb; more preferably, the nucleotide sequence of the 5' homologous arm is as shown in SEQ ID NO: 2; more preferably, the nucleotide sequence of the 3' homologous arm is as shown in SEQ ID NO: 3.

[0058] In this embodiment of the application, in order to achieve stable inheritance and phenotypic consistency of the above-mentioned recombinant expression component in non-human mammals (such as mice), the sequence structures at both ends of the recombinant DNA construct were further designed and defined. Specifically, the two ends of the recombinant DNA construct are respectively connected to a 5' homologous arm and a 3' homologous arm for site-specific integration at genomic safe harbor sites.

[0059] The aforementioned "genomic safe harbor sites" are specific transcriptionally active regions on the host chromosome. This functional constraint requires that the insertion of large exogenous DNA fragments into these regions neither disrupt the normal physiological structure and function of the host's endogenous genes (i.e., avoiding developmental abnormalities or tumors caused by insertional mutations) nor result in the silencing of the inserted exogenous genes by epigenetic mechanisms such as host chromatin heterochromatinization, thus ensuring stable and efficient transcription of the target gene. Based on this mechanism, by introducing specific homologous arm sequences, cells can utilize their own homology-directed repair (HDR) pathway to precisely and seamlessly integrate the recombinant expression sequence located between two homologous arms into the host's target chromosome.

[0060] In a preferred embodiment, the safe harbor site is the H11 site. The aforementioned site-directed integration component (homologous arm sequence) and the aforementioned core expression elements (i.e., promoter, LSL sequence, and target gene sequence) together constitute a system-level synergistic mechanism that combines 'precise site-directed integration' and 'spatiotemporally controllable expression'. Specifically, if only the aforementioned controllable expression sequence is used and conventional random transgenic methods are employed, large exogenous DNA fragments are highly likely to insert into the host's lethal genes, or cause severe position effects and intergenerational gene silencing due to multiple random insertions; conversely, if only homologous arm knock-in is present without LSL blocking elements, the strong background open transcriptional activity at the H11 site will lead to uncontrolled widespread expression of the target gene.

[0061] This application organically combines the homologous knock-in mechanism at the H11 site with the internal LSL-induced expression mechanism, achieving a sophisticated synergy at the in vivo genome level: the homologous arm ensures that the entire recombinant DNA construct is safely and permanently anchored in the host chromosome as a single copy, endowing the model with extremely high intergenerational genetic stability; while expression elements such as LSL and CAG rely on the open chromatin environment of the H11 site to exert excellent inducible expression efficiency and phenotypic consistency. This systematic synergistic design effectively overcomes the dual bottlenecks of 'uncontrollable expression' and 'genetic instability' in traditional in vivo genetic engineering models.

[0062] In a further preferred embodiment, the length of the 5' and 3' homologous arms is 1 kb. In homologous recombination technology, the length of the homologous arms directly affects the efficiency of targeted knock-in. If the length is too short, the probability of homologous recognition and pairing is greatly reduced; if the length is too long, the overall molecular weight of the recombinant DNA construct will be too large, which not only increases the engineering difficulty of in vitro vector cloning, but also reduces the efficiency of delivering large DNA fragments into embryonic cells and increases cytotoxicity. The 1 kb length is the optimal balance parameter obtained through extensive experimental verification. It can ensure high homologous recombination efficiency mediated by gene editing tools (such as CRISPR / Cas9) while also taking into account the convenience of vector construction and subsequent delivery operations. More specifically, the nucleotide sequences of the 5' and 3' homologous arms are shown in SEQ ID NO: 2 and SEQ ID NO: 3, respectively, in the sequence listing of this application.

[0063] Furthermore, those skilled in the art will understand that the specific parameters of the safe harbor site and homologous arm can be adaptively adjusted according to the host species or editing strategy. For example, as an equivalent alternative, the safe harbor site can also be the Rosa26 site or the TIGAC site; the length of the homologous arm can be reasonably adjusted between 800bp and 1500bp according to the actual delivery vector capacity. Such adaptive changes based on the principle of homologous recombination can achieve equivalent targeted knock-in effects and fall within the protection scope of the embodiments of this application.

[0064] In some embodiments, the Notum gene coding sequence comprises the nucleotide sequence shown in SEQ ID NO: 18; and / or, the P2A self-cleaving polypeptide coding sequence comprises the nucleotide sequence shown in SEQ ID NO: 19; and / or, the mBaojin fluorescent reporter gene coding sequence comprises the nucleotide sequence shown in SEQ ID NO: 20; and / or, the recombinant DNA construct comprises the nucleotide sequence shown in SEQ ID NO: 1.

[0065] The nucleotide sequence shown in SEQ ID NO: 1 is the core preferred embodiment of the recombinant expression system of this application. It precisely achieves the "operable linkage" between the aforementioned promoter, loxP-Stop-loxP sequence, Notum target gene, P2A element, and mBaojin fluorescent reporter gene at the molecular level. In this specific sequence entity, the spacer sequences between each component, the translation initiation Kozak sequence, and the open reading frames (ORFs) between each coding region have undergone rigorous codon matching and structural optimization. This ensures that after the introduction of the specific Cre recombinase, homologous recombination can occur with the highest efficiency in the host cell, and the physically separated but strictly consistent secretory Notum protein and retained mBaojin fluorescent protein are precisely translated, thereby maximizing the synergistic effect of this construct in highly sensitive in vivo cell tracking and spatiotemporally controlled expression.

[0066] It should be noted that the terms "comprising" or "including" as used in this application are open-ended expressions. That is, in addition to strictly having the nucleotide sequence shown in SEQ ID NO: 1, the recombinant DNA construct may also have other nucleotide sequences that do not substantially change the function of the present invention attached to its 5' or 3' end, such as, but not limited to: restriction endonuclease recognition site sequences, protective base sequences, universal primer binding sequences for molecular cloning, and conventional plasmid backbone sequences (such as resistance selection genes, replication initiation sites, etc.) for in vitro amplification or viral packaging.

[0067] Furthermore, those skilled in the art should understand that, due to the degeneracy of the genetic code, synonymous mutations or codon optimization to adapt to host cell preferences in SEQ ID NO: 1 are conventional techniques in the field, provided that the final translated polypeptide amino acid sequence and the core secondary structure of each regulatory element are not altered. Therefore, variant nucleotide sequences that have at least 85%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 98% or 99% sequence identity with the nucleotide sequence shown in SEQ ID NO: 1, and still possess substantially the same Cre-dependent induced expression and P2A self-cleavage in vivo tracing function, are all considered equivalent to SEQ ID NO: 1 and equally fall within the protection scope of the embodiments of this application.

[0068] This application also provides a recombinant expression vector comprising the recombinant DNA construct described in any of the foregoing embodiments.

[0069] The recombinant expression vector is a complete nucleic acid molecular entity formed by recombining the core recombinant DNA construct of this application (i.e., a functional fragment containing a promoter, a loxP-Stop-loxP sequence, a Notum gene, a P2A sequence, an mBaojin sequence, and homologous arm sequences at both ends) into a vector backbone. Within this entity, an indispensable "amplification and delivery synergistic mechanism" is formed between the aforementioned core expression sequence and the vector backbone. Specifically, the core sequence endows the product with the biological functions of spatiotemporally controllable target gene induction and high-sensitivity in vivo fluorescence tracing; while the vector backbone, utilizing its own replication origin site and selection marker elements, endows the core sequence with the ability to achieve high-purity, exponential replication and amplification in in vitro hosts (such as *E. coli*), and effectively resists exonuclease degradation using its spatial conformation (such as a plasmid supercoiled structure).

[0070] This collaborative design makes the recombinant expression vector an extremely stable and efficient tool for genetic engineering. In the process of creating non-human genetically engineered animal models, this recombinant expression vector can be directly delivered as a donor template to embryonic stem cells or fertilized eggs as a gene-editing system (such as CRISPR / Cas9). Its excellent physical and chemical stability ensures the maintenance of high concentrations of intact homologous arms and core chimeric sequences before intracellular homology-directed repair (HDR) occurs, thereby greatly improving the site-directed knock-in efficiency of the host genome's H11 site.

[0071] Those skilled in the art will understand that the term "vector" in this application includes, but is not limited to, various cloning vectors and expression vectors applicable to prokaryotic or eukaryotic cells. In one specific embodiment, the recombinant expression vector can be a non-viral plasmid vector, such as a plasmid constructed based on a conventional backbone like pUC, pBluescript, or pMD18-T, suitable for routine electroporation or microinjection of fertilized eggs; in other embodiments, depending on the actual need for efficient gene delivery to adult tissues or specific cell lines, the recombinant expression vector can also be integrated and packaged as a viral vector, such as a lentiviral vector, adeno-associated virus (AAV) vector, or adenovirus vector. As long as it carries and can successfully deliver the core recombinant DNA construct described in this application, it equally falls within the protection scope of the embodiments of this application.

[0072] This application also provides a recombinant cell comprising the recombinant DNA construct described in any of the foregoing embodiments, or comprising the recombinant expression vector described in the foregoing embodiments; preferably, the recombinant cell is a non-human mammalian cell; more preferably, a mouse cell.

[0073] The aforementioned recombinant cells are living entities with specific gene expression potential formed by introducing the artificially designed nucleic acid macromolecules into a living biological environment. The exogenous recombinant DNA sequence can exist in two states within these recombinant cells: it can be expressed transiently and freely in the cell nucleus as an independent recombinant expression vector; or, particularly when it carries a safe harbor homologous arm such as H11, the recombinant DNA construct can be stably integrated into the genome chromosome of the recombinant cell through the intracellular homology-directed repair (HDR) pathway, thereby achieving stable generational transmission with cell division.

[0074] In this system, the artificially recombinant DNA construct acts as the "blueprint" for functional instructions, while the recombinant cell acts as the "execution chassis" providing the transcription and translation machinery. The eukaryotic promoter, P2A autocleavage sequence, PolyA tailing signal, and secretion signal peptide of Notum protein contained in the construct are all highly dependent on the sophisticated ribosome translation system, endoplasmic reticulum and Golgi apparatus processing, and secretion pathways of eukaryotic cells (especially mammalian cells). Through this synergistic mechanism of "instruction and chassis execution," the recombinant cell, under the induction of a specific Cre recombinase, can precisely execute the extracellular secretion of the functional molecule (Notum) and the intracellular retention of the tracer molecule (mBaojin fluorescent protein), perfectly realizing the design concepts of spatiotemporal control and in-situ tracing at the real cell level.

[0075] Due to ethical and regulatory requirements and the needs of the aforementioned eukaryotic processing system, the recombinant cells are preferably non-human mammalian cells. In a further preferred embodiment, the recombinant cells are mouse cells, which achieve the most perfect functional match with the H11 homologous knock-in mechanism for mouse chromosome 11 that is preferably designed in this application.

[0076] Those skilled in the art will understand that the types of recombinant cells have broad applicability depending on the specific application scenario. On the one hand, when used to prepare live genetically engineered animal models, the recombinant cells can be totipotent or pluripotent cells in the early stages of development, such as fertilized eggs or embryonic stem cells from non-human mammals; on the other hand, when the system is directly used as an in vitro research tool, the recombinant cells can also be various somatic cells or immortalized cell lines, such as osteoblasts, fibroblasts, etc. These in vitro recombinant cell models can not only be used to verify the effectiveness of Cre recombination induction and P2A targeted isolation, but also serve as a high-throughput in vitro drug screening platform for rapidly evaluating interventional compounds or small molecule drugs targeting the Notum and Wnt signaling pathways, all of which fall within the protection scope of the embodiments of this application.

[0077] This application also provides a method for constructing a non-human genetically engineered animal model of Cre-dependent Notum gene overexpression and in vivo tracing, comprising: introducing the recombinant DNA construct or the recombinant expression vector described in any of the foregoing embodiments into a fertilized egg or embryonic stem cell of a non-human mammal; preferably, using CRISPR / Cas9 technology to precisely integrate the recombinant DNA construct into the H11 site of the genome of the fertilized egg or embryonic stem cell.

[0078] This application further provides a method for constructing a non-human genetically engineered animal model of Cre-dependent Notum gene overexpression and in vivo tracing. This method utilizes the aforementioned carefully designed recombinant DNA construct or recombinant expression vector as a donor template, combined with modern genome editing tools and embryo engineering techniques, to achieve precise knock-in and stable inheritance of exogenous genes at the in vivo level. Specifically, the construction method includes: introducing the recombinant DNA construct or recombinant expression vector described in any of the foregoing embodiments into fertilized eggs or embryonic stem cells of a non-human mammal.

[0079] Selecting fertilized eggs or embryonic stem cells as recipient cells is crucial to ensuring that recombinant sequences can achieve germline chimerism and be stably inherited by offspring. In practice, when the recipient is a fertilized egg, pronuclear microinjection is typically used to directly deliver the purified recombinant expression vector or linearized DNA construct into the pronucleus; when the recipient is an embryonic stem cell, techniques such as electroporation or liposome transfection can be used to mediate transmembrane delivery of nucleic acids.

[0080] In order to completely overcome the damage to endogenous genes (lethal mutations) and expression position effects (such as extremely unstable expression levels or intergenerational silencing) caused by the random insertion of exogenous DNA into the host genome in traditional random transgenic technology, in a preferred embodiment, this construction method further utilizes CRISPR / Cas9 technology to precisely integrate the recombinant DNA construct into the H11 site of the genome of the fertilized egg or embryonic stem cell.

[0081] The principle behind this knock-in mechanism is as follows: The mRNA (or protein) encoding the Cas9 nuclease, along with a guide RNA (sgRNA) specifically targeting the host H11 site, is co-injected into the fertilized egg along with the recombinant DNA construct of this application. Intracellularly, the sgRNA guides the Cas9 nuclease to precisely locate the H11 site, causing a DNA double-strand break (DSB). At this point, the cell's own DNA damage repair mechanism is activated. Because the recombinant DNA construct of this application carries homologous arms designed specifically for the H11 site at both ends, the cell preferentially initiates the homology-directed repair (HDR) pathway, using the recombinant DNA construct as a repair template to seamlessly and precisely integrate the internal "promoter-LSL-Notum-P2A-mBaojin" core functional sequence into the H11 safe harbor site.

[0082] This synergistic effect of "CRISPR-targeted cleavage + homologous arm-guided repair" greatly improves the efficiency and accuracy of site-specific integration, ensuring that the target sequence is precisely positioned as a single copy in the host genome. Subsequently, using conventional embryo transfer techniques, fertilized eggs or recombinant embryonic stem cells that have undergone the above microinjection and genome editing are injected into chimeric blastocysts and transplanted into pseudopregnant female recipient animals that have undergone estrus synchronization treatment, allowing them to develop to full term and give birth. After the offspring are born, genomic DNA is extracted from their tissue samples, and positive founder animals are screened by PCR identification. All somatic cells of the founder animal carry the Notum expression frame "sealed" by the blocked sequence at the H11 locus.

[0083] In practical in vivo biology research and drug screening applications, researchers only need to breed and mate this pioneering animal model with tool animals expressing tissue-specific Cre recombinases (such as Col1a1-Cre mice) to precisely induce the secretory expression of the Notum gene and intracellular mBaojin luminescent tracing in the specific tissue microenvironment of their double-positive offspring. This construction method is mature and reliable, fundamentally solving the technical barriers that have long limited in vivo research on secretory lethal regulators, and providing an extremely superior in vivo genetic engineering tool for subsequent large-scale tissue regeneration, signaling pathway regulation, and targeted drug development.

[0084] This application also provides a non-human genetically engineered animal model of Cre-dependent Notum gene overexpression and in vivo tracing, which is constructed using the construction method described in the foregoing embodiments.

[0085] The aforementioned macroscopic living model entity is constructed using the non-human genetically engineered animal model construction method described in the foregoing embodiments of this application, through targeted gene knock-in at the fertilized egg (or embryonic stem cell) level and subsequent bioengineering processes such as embryo transfer, breeding, and screening.

[0086] Specifically, in this animal model, a single copy of the exogenous recombinant expression sequence “promoter-LSL-Notum-P2A-mBaojin” is stably integrated into specific safe harbor sites (such as the mouse H11 site) in the genome of all somatic cells and germ cells. This site-specific genome modification in early development ensures that this artificially designed complex functional module can be transmitted in a highly stable and consistent germline manner across generations of animal reproduction.

[0087] In terms of macroscopic physiological phenotype, this animal model possesses a highly unique "Cre-dependent" and "resting-excitation synergistic mechanism." Due to the potent transcriptional blocking effect of the loxP-Stop-loxP (LSL) sequence, under natural conditions (i.e., without the introduction of Cre recombinase), the exogenous Notum gene in all cells of this model animal is strictly silenced. This characteristic allows the model to completely avoid embryonic lethality or severe developmental malformations caused by abnormal overexpression of key Wnt pathway regulators, ensuring that the genetically engineered animal can survive, develop, and reproduce normally like wild-type animals, making it a "universal resting chassis model" with extremely high application potential.

[0088] When placed in actual in vivo research settings, this "resting chassis" exhibits a high degree of spatiotemporal controllability and tracking accuracy. Researchers can introduce induction signals using extremely simple techniques (e.g., mating the chassis animal with a tool animal expressing a tissue-specific Cre recombinase, or locally injecting a viral vector encoding Cre recombinase into a specific organ). In the presence of Cre recombinase, cells in the target microenvironment within the model animal undergo precise genomic recombination, excising transcriptional blocking sequences. Subsequently, relying on the intratranslational autocleavage mechanism of the P2A sequence, the target cells simultaneously produce functional secretory Notum proteins to regulate the local Wnt signaling network and generate high-intensity non-secretory mBaojin fluorescent protein that remains firmly embedded intracellularly.

[0089] This series of in vivo biochemical reactions allows researchers to precisely locate and trace the source of Notum protein secretion directly in vivo or in tissue sections using extremely bright fluorescence signals. This genetically engineered animal model perfectly overcomes the developmental lethality bottleneck of traditional systemic overexpression models and breaks through the technical barrier that secreted target proteins cannot be traced in situ at the cell level after diffusion in complex living tissues. It provides a highly standardized, sensitive, and precise in vivo research platform for elucidating the pathogenesis of metabolic diseases, exploring the tissue regeneration microenvironment, and evaluating the in vivo pharmacodynamics of anti-tumor targeted drugs. Those skilled in the art will understand that the non-human genetically engineered animal model is preferably a rodent (such as a mouse or rat), but it can also be reasonably extended to other non-human experimental mammal strains according to specific experimental needs and adaptive adjustments to homologous sequences. The animal entities carrying the relevant characteristics bred based on the core nucleic acid construct of this application all fall within the protection scope of the embodiments of this application.

[0090] This application embodiment also provides a primer set, which is used to detect the recombinant DNA construct described in any of the foregoing embodiments, or to identify the recombinant cells described in the foregoing embodiments, or the non-human genetically engineered animal models prepared in the foregoing embodiments, or to identify the non-human genetically engineered animal models as described in the foregoing embodiments; the primer set includes at least one of the following primer combinations (specifically shown in Table 1): Table 1. Primers and their corresponding sequences in the primer set

[0091] In Table 1, NO. represents the “SEQ ID NO:” number.

[0092] A. First primer combination for amplifying the 5' integration region: the forward primer contains the sequence shown in SEQ ID NO: 4, and the reverse primer contains the sequence shown in SEQ ID NO: 5.

[0093] B. A second primer combination for amplifying the 3' integration region: the forward primer contains the sequence shown in SEQ ID NO: 6, and the reverse primer contains the sequence shown in SEQ ID NO: 7.

[0094] The first and second primer combinations described above employ a specific "cross-boundary amplification" principle. One primer is anchored to the outer side of the homologous arm of a safe harbor target site (such as the H11 site) in the host genome, while the other primer is anchored inside the exogenous recombination sequence. This design establishes rigorous validation in the spatial dimension: the target band of the expected size can only be amplified when the exogenous recombinant DNA construct is precisely and seamlessly integrated into the host target locus through homology-directed repair (HDR). This completely eliminates false positive results caused by random off-target insertion or unintegrated free residues of the exogenous gene.

[0095] C. A third primer combination for distinguishing wild-type and transgenic alleles: comprising forward primer 1, forward primer 2 and reverse primer, wherein forward primer 1 contains the sequence shown in SEQ ID NO: 8, forward primer 2 contains the sequence shown in SEQ ID NO: 9 and reverse primer contains the sequence shown in SEQ ID NO: 10.

[0096] This combination employs a three-primer multiplex PCR reaction system mechanism. It competitively amplifies the host's original wild-type alleles and transgenic alleles with site-directed knock-in of foreign sequences. By utilizing the molecular weight differences of different amplification products, it can accurately determine the genetic purity (i.e., wild-type, heterozygous, or homozygous) of the target individual in a one-step process during live animal breeding.

[0097] D. A fourth primer combination for amplifying internal fragments of the recombinant DNA construct: the forward primer contains the sequence shown in SEQ ID NO: 11, and the reverse primer contains the sequence shown in SEQ ID NO: 12; E. A fifth primer combination for amplifying internal fragments of the recombinant DNA construct: the forward primer contains the sequence shown in SEQ ID NO: 13, and the reverse primer contains the sequence shown in SEQ ID NO: 14.

[0098] Both the fourth and fifth primer pairs are anchored to the core functional regions of the recombinant macromolecule (e.g., spanning the promoter, LSL, Notum, and P2A). This aims to perform cross-validation from a sequence continuity perspective, ensuring that no internal sequence breaks, inversions, or large-scale deletions of functional domains occur during the complex intracellular delivery and homologous recombination modification of large exogenous DNA fragments, thereby guaranteeing the structural integrity of the core payload.

[0099] In summary, the five primer combinations provided in this application synergistically constitute a multi-dimensional cross-validation system for preventing false positives, which includes "boundary localization + internal integrity confirmation + allele purity determination." This provides a rigorous and efficient gold standard for identifying the genetically engineered live models in this application.

[0100] Furthermore, those skilled in the art will understand that the term "comprising" here indicates that the primer sequence does not exclude the additional non-specific accessory sequences attached to the 5' or 3' end of SEQ ID NO: 4-14. For example, to adapt to specific molecular diagnostic platforms, adding restriction endonuclease recognition sites, protecting bases, or covalently modifying various fluorescent reporter groups (such as FAM, VIC, Cy5, etc.) to the ends of the above primers for quantitative real-time PCR detection does not change their core function of specifically binding to the target sequence. Simultaneously, synonymous additions, deletions, or degenerate substitutions of individual bases in the primer sequence without affecting annealing specificity are also considered equivalent technical features and equally fall within the scope of protection of the claims of this application.

[0101] This application also provides a kit containing the primer set described in the foregoing embodiments; preferably, the kit further includes one or more of DNA extraction reagents and PCR amplification reagents.

[0102] This application also provides an in vitro detection or identification method for purposes other than disease diagnosis and treatment, which uses the primer set or kit described in the foregoing embodiments to perform PCR amplification of the nucleic acid of the sample to be tested; preferably, the sample to be tested is a tissue or cell sample of a non-human mammal.

[0103] The above methods are mainly used to accurately identify the genotypes of recombinant cells cultured in vitro or non-human genetically engineered animal models at the molecular level, so as to verify the accuracy of targeted integration and the purity of genetic lines.

[0104] The detection or identification method includes: extracting genomic nucleic acid from the sample to be tested (preferably a tail or auricle tissue sample from a non-human mammal or a cell sample cultured in vitro) as a template; subsequently, using the specific primer set provided in the foregoing embodiments of this application or an identification kit containing the primer set, the nucleic acid template is amplified by polymerase chain reaction (PCR) under the catalysis of a suitable buffer system and DNA polymerase; finally, the amplified products are analyzed by conventional nucleic acid electrophoresis or quantitative fluorescence signal acquisition technology. This in vitro detection method, because it excludes direct clinical purposes of disease diagnosis and treatment, is fully applicable to basic life science research, quality control and genotyping in animal model breeding, and can confirm the site-directed knock-in status and integrity of exogenous recombinant sequences at the genomic level with extremely high specificity and sensitivity, thereby significantly reducing the breeding and trial-and-error costs of ineffective models.

[0105] This application also provides an application of the recombinant DNA construct, the recombinant expression vector, or the recombinant cell as described in any of the foregoing embodiments, applicable to any of the following aspects: A. Application in preparing a non-human genetically engineered animal model for Cre-dependent Notum gene overexpression and in vivo tracing; B. Application in preparing an in vitro model for studying the Wnt signaling pathway or tissue regeneration and repair; C. Application in preparing a drug screening model for non-diagnostic and therapeutic purposes for screening or evaluating drugs for intervening in metabolic diseases or cancer.

[0106] This application further clarifies the diverse downstream application scenarios of the aforementioned recombinant DNA construct, recombinant expression vector, and recombinant cells. Based on the synergistic advantages of this recombinant system in the fields of induced expression and tracing, it can be specifically applied to any of the following important aspects: Firstly, as a core donor material, it plays an irreplaceable role in the preparation of non-human genetically engineered animal models for Cre-dependent Notum gene overexpression and in vivo tracing. It fundamentally provides the upstream material basis for stable inheritance and targeted editing.

[0107] Secondly, it has applications in preparing in vitro models for studying the Wnt signaling pathway or tissue regeneration and repair. Notum, a highly conserved secretory hydrolase, is a key negative regulator of the Wnt signaling pathway, which is deeply involved in regulating the maintenance of stemness and regeneration and repair of adult tissues. Using the recombinant cells provided in this application to construct in vitro models, the in vitro targeted secretion of Notum protein can be achieved after introducing Cre recombinase. Researchers can then directly and precisely analyze the molecular cascade reaction of Notum inhibiting the Wnt pathway and its pathological mechanism in tissue regeneration disorders at the cellular or tissue microenvironment level.

[0108] Thirdly, it possesses extremely high translational medicine value and can be applied to the preparation of drug screening models for non-diagnostic and therapeutic purposes, used to screen or evaluate drugs for intervention in metabolic diseases or cancer. It is known that abnormal activity of the Notum and Wnt signaling pathways is closely related to metabolic diseases such as osteoporosis and various malignant tumors. Utilizing the mechanism of "strict co-translation of Notum functional molecules and mBaojin tracer molecules" designed in this application, those skilled in the art can develop this recombinant cell or animal model into a high-throughput drug screening platform. When screening specific compound libraries or small molecule drugs in vitro or in vivo, researchers do not need to rely on cumbersome target protein immunoassays; they only need to directly and quantitatively read the signal intensity changes of the mBaojin fluorescent reporter gene using high-throughput fluorescence detection equipment to accurately and rapidly reflect the intervention efficacy of candidate drugs on Notum expression. This application provides a highly sensitive and standardized engineered detection platform for the development of targeted new drugs against the Wnt / Notum pathway (such as preliminary screening and efficacy evaluation of anti-osteoporosis drugs or anti-tumor drugs).

[0109] The present invention will be further illustrated below with specific embodiments. However, it should be understood that these embodiments are merely for the purpose of more detailed illustration and should not be construed as limiting the present invention in any way.

[0110] Example 1: Design and preparation of recombinant DNA constructs (site-targeting vectors) 1. Purpose of the study: In this embodiment, a Cre-dependent Notum overexpression and fluorescent labeling recombinant DNA construct (targeting vector) is provided, which aims to provide the core material basis for the subsequent construction of site-directed knock-in genetically engineered mice.

[0111] 2. Experimental methods: Using conventional molecular cloning techniques, functional elements were sequentially assembled and operatively linked in physical order from the 5' end to the 3' end to construct a recombinant expression vector. The specific linked element sequences are as follows: (1) CAG promoter (systemic high-efficiency expression promoter); (2) loxP-Stop-loxP sequence (LSL blocking element, containing three tandem SV40 polyA transcription termination signals, with loxP sites at both ends). (3) Mouse Notum gene coding sequence; (4) P2A self-cleaving polypeptide coding sequence; (5) The coding sequence of the mBaojin fluorescent reporter gene; (6) WPRE (posttranscriptional regulatory element) and bGH polyA (tailing signal).

[0112] Subsequently, a 5' homologous arm (5' HR) and a 3' homologous arm (3' HR) of approximately 1 kb in length from the mouse H11 locus were inserted at the 5' and 3' ends of the aforementioned core expression sequence, respectively, to form a complete homologous recombination targeting vector.

[0113] 3. Experimental Results and Analysis: This embodiment successfully designed and constructed a complete recombinant DNA construct. (Reference) Figure 1 This structural design not only ensures that the target gene is in a tightly transcribed state before being induced by Cre enzyme, but also guarantees that Notum and mBaojin can be co-transcribed via the same mRNA after Cre enzyme removes the Stop sequence, and achieve 1:1 post-translational physical separation using the P2A ribosomal skipping mechanism. The homologous arms at both ends provide the structural basis for subsequent precise knock-in.

[0114] Example 2: Construction of a genetically engineered animal model (mouse) 1. Purpose of the investigation: In this embodiment, a Cre-dependent Notum gene overexpression and in vivo tracing non-human genetically engineered animal model was prepared to investigate the feasibility of the knock-in of the recombinant DNA construct described in Example 1 in live fertilized eggs.

[0115] 2. Experimental methods: (1) Targeted strategy design: Using CRISPR / Cas9 genome editing technology, a specific guide RNA (sgRNA) was designed for the safe harbor site (H11 site) on mouse chromosome 11 to guide the Cas9 nuclease to generate DNA double-strand breaks at this site.

[0116] (2) Microinjection and embryo transfer: The recombinant DNA construct prepared in Example 1 was extracted and purified as a donor template for homology-directed repair (HDR). Cas9 mRNA (or protein), H11-specific sgRNA, and the recombinant DNA construct were mixed in a predetermined ratio and introduced into the fertilized eggs of wild-type C57BL / 6J mice using prokaryotic microinjection technology.

[0117] (3) Reproduction and propagation: The surviving fertilized eggs after injection were transplanted into the oviducts of pseudopregnant female mice for development. When the mice were full-term, the F0 generation founder mice were obtained. Positive F0 generation mice were selected and mated with wild-type mice to obtain F1 generation heterozygous mice.

[0118] 3. Experimental Results and Analysis: refer to Figure 2The experimental results showed that, under the precise cutting and guidance of the long homologous arm (about 1kb) of the CRISPR / Cas9 system, the cells spontaneously initiated the homology-directed repair mechanism, successfully inserting the Notum-P2A-mBaojin core sequence controlled by the LSL element precisely and in a single copy into the mouse H11 safe harbor site, obtaining healthy F1 generation genetically engineered mice, and verifying the efficiency and reliability of the modeling process.

[0119] Example 3: Genotyping and Integrity Verification of Genetically Engineered Mice 1. Purpose of the study: In this example, PCR genotyping was performed on the F1 generation mice bred in Example 2, and wild-type mice were used as a negative control. The aim was to confirm whether the exogenous recombination sequence was accurately knocked into the target site and to identify its allele status.

[0120] 2. Experimental methods: Genomic DNA was extracted from F1 generation mice and wild-type control mice as PCR amplification templates. Based on the recombinant construct of this application and the sequence characteristics of the mouse H11 site, a series of identification primers were synthesized (i.e., SEQ ID NO: 4 to SEQ ID NO: 14 as described in the aforementioned embodiments, as shown in Table 1).

[0121] Using the primer set described above, multiplex amplification was performed in a standard PCR reaction system for verification, and the banding of the amplification products was then detected by agarose gel electrophoresis.

[0122] 3. Experimental Results and Analysis: Reference Figure 3 and Figure 4 .

[0123] (1) Validation of the accuracy of the target boundary: combined with Figure 4 The primer design diagram shown uses a first primer combination (PCR1) that crosses the 5' boundary between the H11 genome and the exogenous vector, and a second primer combination (PCR2) that crosses the 3' boundary for amplification. Figure 4 The expected size of the specific cross-boundary amplification fragment is indicated (e.g., PCR1 expected to be 1674 bp, PCR2 expected to be 1604 bp). Figure 3 Electrophoresis results showed no abnormal amplification in the wild-type control group (WT), while the target mouse sample from the F1 generation successfully amplified the variant. Figure 4 The expected target bands were of uniform size. This confirms that the large exogenous recombination sequence has been precisely anchored and knocked into the H11 site.

[0124] (2) Genotype and internal integrity verification: combined with Figure 4Multiplex PCR amplification was performed using the third primer combination (PCR3: F3-1 / F3-2 / R3). F3-1 / R3 amplified the wild-type allele (expected 447 bp), while F3-2 / R3 amplified the transgenic allele (expected 548 bp). Figure 3 PCR3 results showed that F1 generation mice exhibited heterozygous genotype characteristics representing targeted knock-in (with two bands simultaneously). Furthermore, amplification results for the fourth (PCR5, expected 1072 bp) and fifth primer combinations (PCR6, expected 2510 bp) targeting the core functional region within the large fragment showed that all internal core sequences could be amplified with complete specific bands, without any large fragment internal deletions or breaks.

[0125] 4. Conclusion: The molecular-level identification results above demonstrate that this application successfully constructed and obtained F1 generation positive genetically engineered mice carrying the complete "promoter-LSL-Notum-P2A-mBaojin" expression cassette. This model achieved the designed microstructure at the genomic level, laying a reliable physical foundation for subsequent Cre-dependent in vivo gene-induced expression and biological function studies.

[0126] Example 4: Crossing with tissue-specific Cre mice and verification of Cre-dependent expression 1. Purpose of the study: In this embodiment, the F1 generation mice obtained in Example 2 were crossed with Dmp1-iCre mice to construct transgenic mice that specifically overexpress Notum in odontoblasts and osteoblasts and are accompanied by fluorescent tracing. The aim is to verify the Cre-dependent induction expression efficacy of the recombinant DNA construct of this application in vivo.

[0127] 2. Experimental Methods: F1 generation positive mice (carrying the LSL-Notum-P2A-mBaojin sequence) identified in Example 2 were crossbred with Dmp1-iCre mice (specifically expressing Cre recombinase in odontoblasts and osteoblasts). Genomic DNA was extracted from the offspring mouse tissues for PCR genotyping. Double-transgenic mice identified as positive (Dmp1-iCre; Notum flox / +) and single-transgenic negative control mice (Notum flox / +) were euthanized at one week of age. Mandibular bone tissue was isolated, fixed, decalcified, and frozen sectioned. Immunofluorescence staining was then performed, and the expression of green fluorescence in the sections was observed using a fluorescence microscope.

[0128] 3. Experimental Results and Analysis: (1) Genotyping: such as Figure 5As shown, samples that simultaneously contain a Dmp1-iCre specific amplification band and a Notum transgenic allele specific amplification band (548bp) in the electrophoresis results are identified as successfully constructed double-positive transgenic hybrid mice (Dmp1-iCre; Notum flox / +).

[0129] (2) Immunofluorescence staining and tracing verification: such as Figure 6 As shown, no green fluorescence signal was observed in mandibular bone sections from negative control mice lacking Cre recombinase (Notum flox / +), demonstrating that the LSL blocking sequence exerts a strict transcriptional silencing effect under natural conditions. However, in double-positive transgenic mice (Dmp1-iCre; Notum flox / +), odontoblasts and surrounding osteoblast regions of the mandible exhibited strong, specific green fluorescence signals. This indicates that the Cre recombinase successfully excised the LSL sequence, triggering efficient co-expression of the downstream Notum and mBaojin genes, and that the mBaojin fluorescent protein remained stably within the source cells, enabling precise tracking.

[0130] 4. Conclusion: The above in vivo validation results demonstrate that this application successfully constructed a Cre-dependent in vivo induction expression model of the Notum gene. This model achieves tight background control and highly specific spatial activation, laying a reliable in vivo tool foundation for in-depth research on the biological functions of the Notum and Wnt signaling pathways in specific tissue microenvironments.

[0131] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A recombinant DNA construct, characterized in that, The recombinant DNA construct is operably linked from the 5' end to the 3' end with: a promoter, a loxP-Stop-loxP sequence, a Notum gene coding sequence, a P2A self-cleaving polypeptide coding sequence, and an mBaojin fluorescent reporter gene coding sequence.

2. The recombinant DNA construct as described in claim 1, characterized in that, The recombinant DNA construct has at least one of the following characteristics: A. The promoter is a systemic expression promoter or a tissue-specific promoter; B. The loxP-Stop-loxP sequence includes a first loxP site, a Stop sequence, and a second loxP site sequentially from the 5' end to the 3' end. C. The 3' end of the mBaojin fluorescent reporter gene coding sequence is also operably linked with a WPRE sequence and a PolyA sequence; D. The recombinant DNA construct is connected to two ends of a 5' homologous arm and a 3' homologous arm for site-specific integration at the genome safe harbor site, respectively. E. The Notum gene coding sequence comprises the nucleotide sequence shown in SEQ ID NO: 18; F. The P2A self-cleaving polypeptide coding sequence comprises the nucleotide sequence shown in SEQ ID NO: 19; G. The encoding sequence of the mBaojin fluorescent reporter gene contains the nucleotide sequence shown in SEQ ID NO: 20; H. The recombinant DNA construct contains the nucleotide sequence shown in SEQ ID NO:

1.

3. The recombinant DNA construct as described in claim 2, characterized in that, The promoter is a CAG promoter or a Col1a1 promoter; and / or, the promoter comprises a nucleotide sequence as shown in SEQ ID NO: 15; and / or, The Stop sequence contains a transcription termination signal; and / or, the Stop sequence comprises three tandem SV40 polyA sequences; and / or, both the first loxP site and the second loxP site contain the nucleotide sequence shown in SEQ ID NO: 16; and / or, the Stop sequence contains the nucleotide sequence shown in SEQ ID NO: 17; and / or, The PolyA sequence is a bGH polyA sequence; and / or, the WPRE sequence comprises the nucleotide sequence shown in SEQ ID NO: 21; and / or, the PolyA sequence comprises the nucleotide sequence shown in SEQ ID NO: 22; and / or, The safe harbor site is the H11 site; and / or, the length of the 5' homologous arm and the 3' homologous arm is 1 kb; and / or, the nucleotide sequence of the 5' homologous arm is as shown in SEQ ID NO: 2; and / or, the nucleotide sequence of the 3' homologous arm is as shown in SEQ ID NO:

3.

4. A recombinant expression vector, characterized in that, It includes the recombinant DNA construct according to any one of claims 1-3; The recombinant expression vector contains a nucleotide sequence as shown in SEQ ID NO:

23.

5. A recombinant cell, characterized in that, It comprises the recombinant DNA construct according to any one of claims 1-3, or the recombinant expression vector according to claim 3.

6. A method for constructing a non-human genetically engineered animal model of Cre-dependent Notum gene overexpression and in vivo tracing, characterized in that, include: The recombinant DNA construct according to any one of claims 1-3, or the recombinant expression vector according to claim 4, is introduced into fertilized eggs or embryonic stem cells of non-human mammals.

7. A primer set, characterized in that, The primer set is used to detect the recombinant DNA construct according to any one of claims 1-3, or to identify the recombinant cells according to claim 5, or the non-human genetically engineered animal model prepared according to claim 6; the primer set comprises at least one of the following primer combinations: A. First primer combination for amplifying the 5' integration region: the forward primer contains the sequence shown in SEQ ID NO: 4, and the reverse primer contains the sequence shown in SEQ ID NO: 5; B. A second primer combination for amplifying the 3' integration region: the forward primer contains the sequence shown in SEQ ID NO: 6, and the reverse primer contains the sequence shown in SEQ ID NO: 7; C. A third primer combination for distinguishing wild-type and transgenic alleles: comprising forward primer 1, forward primer 2 and reverse primer, wherein forward primer 1 contains the sequence shown in SEQ ID NO: 8, forward primer 2 contains the sequence shown in SEQ ID NO: 9, and reverse primer contains the sequence shown in SEQ ID NO: 10; D. The fourth primer combination for amplifying the internal fragments of the recombinant DNA construct: the forward primer contains the sequence shown in SEQ ID NO: 11, and the reverse primer contains the sequence shown in SEQ ID NO: 12; E. A fifth primer combination for amplifying internal fragments of the recombinant DNA construct: the forward primer contains the sequence shown in SEQ ID NO: 13, and the reverse primer contains the sequence shown in SEQ ID NO:

14.

8. A reagent kit, characterized in that, It includes the primer set as described in claim 7.

9. The application of a recombinant DNA construct as described in any one of claims 1-3, a recombinant expression vector as described in claim 4, or a recombinant cell as described in claim 5, characterized in that, Applicable to any of the following aspects: A. Application in the preparation of non-human genetically engineered animal models with Cre-dependent Notum gene overexpression and in vivo tracing; B. Application in the preparation of in vitro models for studying Wnt signaling pathways or tissue regeneration and repair; C. Application in the preparation of drug screening models for non-diagnostic therapeutic purposes, used to screen or evaluate drugs for intervention in metabolic diseases or cancer.

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