WO2024103631A1 - Notch2nlc基因ggc重复扩增突变转基因小鼠及其构建方法和应用 - Google Patents

Notch2nlc基因ggc重复扩增突变转基因小鼠及其构建方法和应用 Download PDF

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WO2024103631A1
WO2024103631A1 PCT/CN2023/089834 CN2023089834W WO2024103631A1 WO 2024103631 A1 WO2024103631 A1 WO 2024103631A1 CN 2023089834 W CN2023089834 W CN 2023089834W WO 2024103631 A1 WO2024103631 A1 WO 2024103631A1
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mice
generation
mouse
gene
notch2nlc
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潘永诚
刘琼
唐北沙
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中南大学湘雅医院
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    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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    • C12N15/09—Recombinant DNA-technology
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    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C—CHEMISTRY; METALLURGY
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Definitions

  • the present invention belongs to the field of biomedical technology for constructing animal models, and in particular, relates to a NOTCH2NLC gene GGC repeat expansion mutant transgenic mouse and a construction method and application thereof.
  • NIID Neuronal intranuclear inclusion disease
  • the clinical manifestations of NIID are complex and diverse, mainly manifested as muscle weakness and dementia, in addition to Parkinson's-like symptoms, cerebellar ataxia, tremor, paroxysmal impaired consciousness, sensory disorders and movement disorders.
  • GGC repeat expansion of the NOTCH2NLC gene was reported as a pathogenic gene mutation for NIID, more and more NIID cases were diagnosed.
  • Animal models of human diseases are animal experimental subjects and materials established in biomedical scientific research that have similar manifestations to human diseases. They are an extremely important experimental model in modern biomedical research.
  • the research of experimental mice is the most widely used model animal in the field of modern biomedical research because of its easy breeding, high reproduction rate, high genetic purity, short generation cycle, small differences between individuals, metabolic type, physiology and pathology similar to humans, and advanced neural activity.
  • the main purpose of this application is to provide a transgenic mouse with a GGC repeat expansion mutation in the NOTCH2NLC gene and a construction method and application thereof, in order to solve the problem of constructing a stable animal model of neuronal nuclear inclusion disease.
  • This application has established the world's first transgenic mouse carrying the GGC repeat expansion mutation in the NOTCH2NLC gene through CRISPR/Cas9, embryonic stem cell targeting, microinjection and other technologies.
  • This transgenic mouse model can stably transmit the GGC repeat expansion mutation in the NOTCH2NLC gene from generation to generation, and can simulate various pathological characteristics and clinical phenotypes of patients.
  • This mouse model will be It provides an important animal model for the occurrence and development mechanism, drug screening and efficacy evaluation of diseases related to NOTCH2NLC gene GGC repeat expansion mutation.
  • the present application provides a method for constructing a transgenic mouse with a NOTCH2NLC gene GGC repeat expansion mutation, comprising the following steps:
  • mice with LoxP sequence and site-directed knock-in of NOTCH2NLC gene insert CAG-Loxp-Stop-Loxp-NOTCH2NLC-(GGC)n-3tag-WPRE-polyA expression frame into the Rosa26 gene locus in mice, breed and identify F0 generation positive mice; n is a natural number, and n ⁇ 60;
  • the F0 generation positive mice were mated and bred with wild-type mice to obtain F1 generation mice, and the positive F1 generation heterozygous conditional transgenic mice were identified;
  • Wild-type mice can be selected from C57BL/6 mice commonly used in the art.
  • n is 98.
  • step S1 CRISPR/Cas9 gene editing technology is used to insert the expression frame at a specific site.
  • step S1 the specific method of inserting the expression cassette at a fixed point using CRISPR/Cas9 gene editing technology is:
  • the gene sequence of the gRNA is: GGGGACACACTAAGGGAGCTTGG;
  • the obtained recombinant donor plasmid is transformed into STBL3 Escherichia coli competent cells for amplification. During amplification, the cells are cultured at 25-30° C. for 46-50 hours.
  • step S1 the F0 generation positive mice are identified by long-fragment PCR amplification and electrophoresis;
  • the primers used for the long fragment PCR amplification are:
  • Reverse primer II 5′-TGAGGGCAATCTGGGAAGGTT-3′.
  • Forward primer III 5′-GGGGGAGGGGAGTGTTGC-3′
  • Reverse primer IV 5′-TTCTTCCTGCCTGCCTTCTGTGAC-3′;
  • the corresponding mice to be tested are F0 generation positive mice.
  • step S2 positive F1 generation heterozygous conditional transgenic mice are identified by PCR amplification and sequencing, or by PCR amplification and electrophoresis;
  • the primers used in the PCR amplification are:
  • Identification primer II TCAGATTCTTTTATAGGGGACACA;
  • Identification primer IV AAAGTCCCGGAAAGGAGCTG;
  • mice After electrophoresis, when the 967bp and 604bp bands are generated at the same time, the corresponding tested mice are positive. Sex F1 generation heterozygous conditional transgenic mice.
  • mice to be tested are positive F1 generation heterozygous conditional transgenic mice.
  • the present application also provides a NOTCH2NLC gene GGC repeat expansion mutation transgenic mouse, which is constructed using the above method.
  • the present application also provides the use of the above-mentioned transgenic mice in preparing an animal model of neuronal intranuclear inclusion disease.
  • the present invention establishes a reliable and stable mouse model and constructs a stable animal model of neuronal intranuclear inclusion disease, which provides an important model for exploring the pathogenesis of diseases related to NOTCH2NLC gene GGC repeat expansion, and is an important tool for future drug development, screening, and exploration of effective treatment methods.
  • Figure 1 shows the conditional transgene of NLC obtained by CRISPR/Cas9 technology by site-directed knock-in of Rosa26 Schematic diagram of the principle of the mouse model.
  • FIG. 2 shows the recombinant Donor plasmid into which the NOTCH2NLC-(GGC)17or98-3tag sequence is inserted.
  • Figure 3 is the electrophoresis diagram of the enzyme digestion identification of the recombinant donor plasmid.
  • FIG. 4 is a schematic diagram showing the principle of the PCR identification method for F0 generation mice.
  • FIG. 5 is an electrophoresis diagram of PCR identification of homologous recombination-positive F0 generation mice.
  • FIG. 6 is a schematic diagram showing the principle of short-fragment PCR identification of conditional transgenic mice.
  • Figure 7 is the electrophoresis diagram of mouse tail gDNA PCR product identification.
  • FIG8 is a Sanger sequencing peak diagram of positive F3 generation mice.
  • FIG. 9 is an immunofluorescence image of brain tissue of model mice.
  • FIG. 10 is an immunofluorescence image of mouse muscle tissue.
  • FIG. 11 is a Nissl staining image of mouse brain tissue.
  • FIG. 12 is a HE staining image of mouse gastrocnemius muscle.
  • FIG. 13 shows the results of the open-field experiment in mice.
  • FIG. 14 shows the results of a novel object recognition experiment in mice.
  • FIG. 15 shows the results of the mouse exercise ability test experiment.
  • FIG. 16 is a transcriptome analysis of brain tissue of transgenic mice.
  • mice Genetically engineered mice often use genetic techniques to knock out mouse homologous genes, insert foreign genes into mouse homologous genes, or insert foreign genes into other safe sites (the industry has confirmed that knocking in foreign genes at the rosa26 site is the most classic safe site, which will not affect the expression of other endogenous genes and is widely expressed in various cell types and developmental stages). Since the NOTCH2NLC gene is a human-specific gene and there is no homologous gene in the mouse genome, the only option is to insert the NOTCH2NLC gene GGC repeat expansion mutation at the Rosa26 site to achieve overexpression of the NOTCH2NLC gene GGC repeat expansion pathogenic mutation in mice.
  • Figure 1 is a schematic diagram of the principle of obtaining a conditional transgenic mouse model of NOTCH2NLC with site-directed knock-in of the Rosa26 locus using CRISPR/Cas9 technology.
  • gRNA Design guide RNA
  • Rosa26 locus Rosa26 locus
  • in vitro transcription kit Takara, 632635
  • the gene sequence of gRNA is: GGGGACACACTAAGGGAGCTTGG.
  • NOTCH2NLC-(GGC)17or98 sequence was cloned using cloning primers I and II, and connected with 3tag sequence synthesized by Gene Company through BamHI to obtain NOTCH2NLC-(GGC)17or98-3tag sequence.
  • FIG. 2 shows the recombinant Donor plasmid into which the NOTCH2NLC-(GGC)17or98-3tag sequence is inserted.
  • nucleotide sequence of NOTCH2NLC-(GGC)17-3tag (SEQ ID NO.1 in the sequence list) is:
  • the double-underlined part is the NOTCH2NLC gene
  • the black box marks the 17 GGC repeat sequences
  • the single-underlined part is the 3tag sequence.
  • nucleotide sequence of NOTCH2NLC-(GGC)98-3tag is:
  • the double underlined part is NOTCH2NLC gene, and the black box marks 98 GGC re-expressions.
  • the single underlined part is the 3tag sequence.
  • Figure 3 is an electrophoresis diagram of the restriction enzyme digestion identification of the recombinant donor plasmid.
  • Figure A shows the HindIII restriction enzyme digestion identification result of the homologous recombination plasmid carrying 17 GGC repeats, and the theoretical band sizes are 7677bp, 4224bp, 2445bp, 1525bp, 941bp, 389bp, 247bp, and 79bp (the product amount is small and the bands are not clear);
  • Figure B shows the BglII+XhoI restriction enzyme digestion identification result of the homologous recombination plasmid carrying 98 GGC repeats, and the theoretical band sizes are 7976bp, 5475bp, 1952bp, 1539bp, and 825bp.
  • the extraction method of the recombinant donor plasmid is as follows: the recombinant plasmid identified by enzyme digestion is transformed into STBL3 E. coli competent cells for amplification, and low-temperature long-term culture (such as 25-30°C for 48 hours) is used during amplification to improve the stability of the recombinant plasmid carrying 98 GGC repeats during amplification. Then the plasmid is extracted according to the steps of the commercial plasmid extraction kit.
  • FIG. 4 is a schematic diagram showing the principle of the PCR identification method for F0 generation mice.
  • Figure 5 is a PCR electrophoresis diagram of homologous recombination positive F0 generation mice.
  • Lane M is a marker
  • lane WT is a wild-type control
  • Figure A is a long-fragment PCR of the 5-end arm
  • the PCR band size of lanes 2, 4 and 5 is 5.1kb, which is consistent with WT, proving that the mice corresponding to lanes 2, 4 and 5 are wild-type
  • the PCR band sizes of lanes 1, 3 and 6 are 3.4kb and 5.1kb, proving that the corresponding mice 5-end arm targeting is successful and heterozygous
  • Figure B is a long-fragment PCR of the 3-end arm, and the PCR band sizes of lanes 1, 3 and 6 are 6.5kb and 3.6kb, proving that the corresponding mice 3-end arm targeting is successful and heterozygous.
  • the primer information for long fragment PCR identification is as follows:
  • Reverse primer II 5′-TGAGGGCAATCTGGGAAGGTT-3′.
  • Forward primer III 5′-GGGGGAGGGGAGTGTTGC-3′
  • Reverse Primer IV 5′-TTCTTCCTGCCTGCCTTCTGTGAC-3′.
  • the PCR reaction system and reaction conditions for the 5′ homology arm and the 3′ homology arm are the same.
  • the specific PCR reaction system is shown in Table 3.
  • the PrimeStar GXL kit (TaKaRa, Code No: R050A) was used.
  • the F0 generation mice are chimeras.
  • the cells do not necessarily carry exogenous genes, and need to be passaged to select F1 generation mice that can stably inherit the genes.
  • Figure 6 is a schematic diagram of the short fragment PCR identification principle of conditional transgenic mice.
  • the wild-type allele uses identification primers I and II, and the PCR product size should be 967bp; the transgenic allele uses identification primers III and IV, and the PCR product size should be 604bp.
  • Figure 7 is an electrophoresis diagram for identification of mouse tail gDNA PCR products.
  • Lane M is a marker
  • the theoretical PCR product band size of the wild-type mouse (WT) allele is 967bp
  • the theoretical PCR product band size of the transgenic mouse (Tg) allele is 604bp (regardless of 17 or 98 repeats). It can be seen that the mouse allele corresponding to lane 3 is one wt and one tg, so it is a heterozygous transgenic mouse; the mouse alleles corresponding to lanes 1, 2, 4, 5, 6, and 7 are both wt, so they are wild-type mice.
  • PCR primers are as follows:
  • Identification primer II TCAGATTCTTTTATAGGGGACACA;
  • Identification primer III GCGCAGGATCCTACCCATAC Identification primer IV: AAAGTCCCGGAAAGGAGCTG.
  • the PCR reaction system is shown in Table 5.
  • the 967bp product sequence (SEQ ID NO.3 in the sequence table) is:
  • the underlined part corresponds to the primer sequence.
  • the 604 bp product sequence (SEQ ID NO.4 in the sequence table) is:
  • the underlined part corresponds to the primer sequence.
  • the positive F1 generation mice need to be backcrossed with wild-type C57BL/6 mice for at least 2 generations before they can be hybridized with the cre tool mice.
  • the positive F1 generation mice were backcrossed with wild-type C57BL/6 mice for two generations to obtain positive F3 generation mice.
  • the genomic DNA of the obtained conditional transgenic mice was subjected to Sanger sequencing. The sequencing results are shown in FIG8 .
  • Figure 8 is a Sanger sequencing peak diagram of positive F3 generation mice.
  • Figure A shows the DNA sequencing peak diagram of the tail of NOTCH2NLC-(GGC)17 mice in the normal control group (hereinafter referred to as (GGC)17 mice), and
  • Figure B shows the DNA sequencing peak diagram of the tail of NOTCH2NLC-(GGC)98 mice in the mutant group (hereinafter referred to as (GGC)98 mice).
  • the underlined part shows the GGC repeat region of the NOTCH2NLC gene; it was found that the sequence inserted in the mouse was the same as the original design.
  • Figure 8 shows that the method of the present invention obtains a transgenic mouse with a GGC repeat expansion mutation in the NOTCH2NLC gene.
  • the transgenic mouse can pass the GGC repeat expansion mutation in the NOTCH2NLC gene to its offspring.
  • the mouse model established in the present application can stably transmit the mutation to the next generation.
  • positive F3 generation mice By further backcrossing the positive F3 generation mice with the wild-type C57BL/6 mice, positive F4 generation mice, positive F5 generation mice, positive F6 generation mice, and so on can be obtained.
  • transgenic mice expressing NOTCH2NLC gene in the whole body or in specific tissues or at specific developmental periods can be obtained.
  • the obtained positive F3 generation and above heterozygous positive mice are mated with each other, and the obtained homozygous mice are then hybridized with different cre tool mice, so that the proportion of positive mice in the offspring will be further increased.
  • conditional transgenic mice carrying LSL i.e., Loxp-Stop-Loxp
  • transgenic mice expressing NOTCH2NLC gene in the whole body or specific tissues or specific developmental periods can be obtained, thereby achieving the expression of NOTCH2NLC gene in the whole body or specific tissues or specific developmental periods.
  • LSL Loxp-Stop-Loxp
  • Cre-ERT2 mice are a class of mice that express a fusion protein containing a ligand binding region mutant (ERT) of the estrogen receptor and Cre recombinase.
  • Cre-ERT2 mice Only after Tamoxifen induction, Cre-ERT2 mice produce Cre recombinase activity. For example, conditional transgenic mice are hybridized with cardiac-specific Myh6-Cre-ERT2 tool mice, and positive offspring are given Tamoxifen induction at 2 months of age, which can achieve cardiac-specific expression of NOTCH2NLC gene. This is a commonly used method in the prior art and will not be repeated here.
  • Whether the animal model can simulate the clinical phenotype of the patient is a key indicator of whether the animal model is successfully established.
  • the main pathological phenotypes of NIID are widespread intranuclear inclusions and neuronal death, and the main clinical symptoms are muscle weakness and dementia.
  • the inventors of the present application hybridized the positive F3 conditional transgenic mice in Example 1 with the EIIa-cre tool mice that express the recombinase systemically, and obtained model mice (EIIa; (GGC) 17 and EIIa; (GGC) 98) that express the gene mutation systemically (Sanger sequencing shows that the NOTCH2NLC gene sequences of EIIa; (GGC) 17 and EIIa; (GGC) 98 mice are consistent with Figure 8), and detected the pathological and behavioral phenotypes of EIIa; (GGC) 17 and EIIa; (GGC) 98 mice.
  • EIIa; (GGC) 17 (or (GGC) 17 ) is a normal control, indicating a transgenic mouse with a GGC repeat expansion mutation of 17 times in the NOTCH2NLC gene; and EIIa; (GGC) 98 (or (GGC) 98 ) indicates a transgenic mouse with a GGC repeat expansion mutation of 98 times in the NOTCH2NLC gene.
  • polyG Intranuclear polyglycine
  • Figure 9 is an immunofluorescence image of the brain tissue of the model mouse, where green represents polyG protein aggregates and blue represents cell nuclei.
  • polyG protein aggregates were formed in the hippocampus, cerebellum, cortex, striatum and other brain regions of model mice around 50 days after birth, and most of them were located in the cell nucleus.
  • Figure 10 is an immunofluorescence image of mouse gastrocnemius muscle, wherein green represents polyG protein aggregates and blue represents cell nuclei.
  • polyG protein aggregates were also formed in the nuclei of gastrocnemius muscles of model mice around day 50 after birth.
  • FIG. 11 is a Nissl staining image of mouse brain tissue.
  • mice compared with the control group EIIa; (GGC) 17 mice, EIIa; (GGC) 98 mice had more pyknotic cells in the cortex and fewer Purkinje cells in the cerebellum, indicating neuronal death.
  • FIG. 12 is a HE staining image of mouse gastrocnemius muscle.
  • EIIa As shown in Figure 12, compared with the control group EIIa; (GGC) 17 mice, EIIa; (GGC) 98 mice showed pathological changes such as myocyte nuclear intranslocation, increased nuclei, and inflammatory infiltration, indicating that myocytes were damaged.
  • the behavioral phenotypes of transgenic mice were analyzed using multiple behavioral analysis platforms.
  • Figure 13 shows the results of the open field experiment of mice.
  • Figure A is the movement trajectory of mice in the open field box;
  • Figure B is a bar graph of the total movement time of mice in the open field box;
  • Figure C is a bar graph of the total movement distance of mice in the open field box.
  • Figure 14 shows the results of the novel object recognition experiment of mice.
  • Figure A is a schematic diagram of the experiment: the same objects were placed on both sides during the adaptation phase, and the objects on one side were replaced with new objects during the test phase;
  • Figure B is a bar graph of the preference ratios of the three types of mice for novel objects during the test phase.
  • the EIIa;(GGC) 98 transgenic mice were poorer at recognizing novel objects than the control group.
  • Rotarod test Place the mouse on a rotating rod and observe how long the mouse stays on the rod. The longer the time, the stronger its motor and coordination abilities.
  • Figure 15 shows the results of the mouse motor ability test experiment, where Figure A shows the time the mouse stayed on the rotarod, Figure B shows the time the mouse hung on the metal grid, and Figure C shows the number of times the mouse stood up and explored in the cylinder.
  • This example introduces the application of the transgenic mice of the present invention in studying the occurrence and development mechanism of NOTCH2NLC gene GGC repeat expansion-related diseases, drug screening and efficacy evaluation, including but not limited to the following examples:
  • conditional transgenic mice can be hybridized with tool mice that only express Cre enzymes in the above-mentioned tissues to achieve specific expression of NOTCH2NLC gene GGC repeat expansion in these tissues.
  • Cre-ERT2 mice are a type of mouse that expresses a fusion protein containing the estrogen receptor ligand binding region mutant (ERT) and Cre recombinase. Cre-ERT2 mice only produce Cre recombinase activity after tamoxifen induction.
  • conditional transgenic mice are hybridized with heart-specific Myh6-Cre-ERT2 tool mice, and positive offspring are given tamoxifen induction at 2 months of age, which can achieve specific expression of NOTCH2NLC gene at a specific age stage (2 months old) and a specific tissue organ (heart).
  • the above strategy can eliminate interference caused by abnormalities in other systems and conduct more targeted research on the pathogenesis of the disease and drug screening.
  • Figure 16 is a transcriptome analysis of brain tissue of transgenic mice.
  • Figure A shows the Venn diagram analysis results of genes down-regulated in the three brain regions of hippocampus, cortex and cerebellum
  • Figure B shows the Venn diagram analysis results of genes up-regulated in the three brain regions of hippocampus, cortex and cerebellum, among which 162 genes were down-regulated and 383 genes were up-regulated
  • Figure C shows the KEGG pathways enriched in genes down-regulated in the three brain regions
  • Figure D shows the KEGG pathways enriched in genes up-regulated in the three brain regions.

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Abstract

提供NOTCH2NLC基因GGC重复扩增突变转基因小鼠的构建方法，包括以下步骤：构建带有LoxP序列并定点敲入NOTCH2NLC基因的F0代阳性小鼠；将F0代阳性小鼠与野生型小鼠交配并繁殖，鉴定出阳性F1代杂合子条件性转基因小鼠；将阳性F1代杂合子条件性转基因小鼠与野生型小鼠回交m代，得到F (1+m)代杂合子条件性转基因小鼠；将F (1+m)代杂合子条件性转基因小鼠之间交配并繁殖，得到纯合子条件性转基因小鼠；将F (1+m)代杂合子条件性转基因小鼠或纯合子条件性转基因小鼠与cre工具小鼠杂交，获得转基因小鼠。所述方法解决了相关技术中的如何构建稳定的神经元核内包涵体病动物模型的技术问题。

Description

NOTCH2NLC基因GGC重复扩增突变转基因小鼠及其构建方法和应用 技术领域
本发明属于动物模型构建的生物医学技术领域，具体而言，涉及NOTCH2NLC基因GGC重复扩增突变转基因小鼠及其构建方法和应用。
背景技术
神经元核内包涵体病(Neuronal Intranuclear Inclusion Disease，NIID)是一种以广泛存在的神经元核内包涵体为特征的致死性神经退行性疾病。NIID的临床表现复杂多样，主要表现为肌无力和痴呆，此外还可见帕金森样症状、小脑共济失调、震颤、发作性的意识障碍、感觉障碍和运动障碍等症状。2019年NOTCH2NLC基因GGC重复扩增被报道为NIID致病基因突变后，越来越多的NIID病例得到确诊。随后，各国科学家陆续在亚洲人群原发性震颤、帕金森病、肌萎缩侧索硬化、阿尔茨海默病、额颞叶痴呆、脑白质病变和多系统萎缩等疾病部分患者中也检测到相同基因突变，而在欧洲人群相应疾病中没有或很少检测到该突变，这表明该基因突变可能在亚洲人群神经退行性疾病的发生中发挥重要作用。然而，NIID发病的具体分子机制尚无任何报道，亟待开展相关研究。
人类疾病的动物模型，是生物医学科学研究中所建立的具有人类疾病模似性表现的动物实验对象和材料，是现代生物医学研究中的一个极为重要的实验 对象和工具，有助于更方便、更有效地认识人类疾病的发生、发展规律和研究防治措施。实验小鼠由于容易饲养、繁殖率高、遗传上有较高的纯和度、世代周期短、个体间差异小、代谢类型、生理病理与人类接近、有高级神经活动等特点，成为现代生物医学研究领域中应用最广泛的模式动物。
2021年，法国科学家通过注射腺相关病毒过表达NOTCH2NLC基因GGC重复扩增，证明该模型能够部分模拟患者行为及病理表型；然而，通过注射腺相关病毒过表达方法建立的小鼠模型不能够稳定的向下一代传递该突变，从而导致该小鼠模型在探索疾病发病机制、开发治疗药物等方面的应用受到诸多限制。
针对相关技术中的如何构建稳定的神经元核内包涵体病动物模型的问题，目前尚未提出有效的解决方案。
发明内容
本申请的主要目的在于提供NOTCH2NLC基因GGC重复扩增突变转基因小鼠及其构建方法和应用，以解决构建稳定的神经元核内包涵体病动物模型的问题。本申请通过CRISPR/Cas9、胚胎干细胞打靶、显微注射等技术建立了世界上首个携带NOTCH2NLC基因GGC重复扩增突变的转基因小鼠，该转基因小鼠模型能够稳定的将NOTCH2NLC基因GGC重复扩增突变进行代际传递，能够模拟患者的各种病理特征及临床表型。该小鼠模型将为 NOTCH2NLC基因GGC重复扩增突变相关疾病的发生发展机制、药物筛选及疗效评价提供重要的动物模型。
为了实现上述目的，第一方面，本申请提供NOTCH2NLC基因GGC重复扩增突变转基因小鼠的构建方法，包括以下步骤：
S1，构建带有LoxP序列并定点敲入NOTCH2NLC基因的小鼠：在小鼠体内的Rosa26基因位点，定点插入CAG-Loxp-Stop-Loxp-NOTCH2NLC-(GGC)n-3tag-WPRE-polyA表达框，繁育并鉴定出F0代阳性小鼠；所述n为自然数，且n≥60；
S2，将F0代阳性小鼠与野生型小鼠交配并繁殖，获得F1代小鼠，鉴定出阳性F1代杂合子条件性转基因小鼠；
S3，将阳性F1代杂合子条件性转基因小鼠与野生型小鼠回交m代，其中，m为自然数，且m≥2，得到F(1+m)代杂合子条件性转基因小鼠；将F(1+m)代杂合子条件性转基因小鼠之间交配并繁殖，得到纯合子条件性转基因小鼠；
S4，将F(1+m)代杂合子条件性转基因小鼠或纯合子条件性转基因小鼠与cre工具小鼠杂交，获得转基因小鼠。
野生型小鼠可以选择本领域常用的C57BL/6小鼠等。
作为优选方案，步骤S1中，所述n为98。
作为优选方案，步骤S1中，采用CRISPR/Cas9基因编辑技术定点插入所述表达框。
作为优选方案，步骤S1中，采用CRISPR/Cas9基因编辑技术定点插入所述表达框的具体方法为：
1)、针对Rosa26位点设计gRNA靶序列，所述gRNA的基因序列为：GGGGACACACTAAGGGAGCTTGG；
2)、将外源NOTCH2NLC-(GGC)n-3tag通过AgeI和EcorV酶切，连接至donor质粒上，获得CAG-Loxp-Stop-Loxp-NOTCH2NLC-(GGC)n-3tag-WPRE-polyA重组donor质粒；
3)、将Cas9mRNA、gRNA及重组donor质粒进行小鼠受精卵注射，然后将注射后的小鼠受精卵移植到假孕母鼠中，孕育的小鼠为F0代小鼠。
作为优选方案，将获得的重组donor质粒转化入STBL3大肠杆菌感受态细胞中进行扩增，扩增时，在25-30℃培养46-50小时。
作为优选方案，步骤S1中，通过长片段PCR扩增和电泳的方法鉴定出F0代阳性小鼠；
所述长片段PCR扩增时的引物为：
长片段PCR 5′同源臂鉴定引物信息：
正向引物I：5′-GCCGGGCCTCGTCGTCTG-3′
反向引物II：5′-TGAGGGCAATCTGGGAAGGTT-3′。
长片段PCR 3′同源臂鉴定引物信息：
正向引物III：5′-GGGGGAGGGGAGTGTTGC-3′
反向引物IV：5′-TTCTTCCTGCCTGCCTTCTGTGAC-3′；
电泳后，5端臂产生泳道条带大小为3.4kb和5.1kb，且3端臂产生泳道条带大小为6.5kb和3.6kb时，对应的待测小鼠即为F0代阳性小鼠。
作为优选方案，步骤S2中，通过PCR扩增和测序的方法，或通过PCR扩增和电泳的方法鉴定出阳性F1代杂合子条件性转基因小鼠；
所述PCR扩增时的引物为：
鉴定引物I：TAAAGGCCACTCAATGCTCACTAA
鉴定引物II：TCAGATTCTTTTATAGGGGACACA；
鉴定引物III:GCGCAGGATCCTACCCATAC
鉴定引物IV:AAAGTCCCGGAAAGGAGCTG；
电泳后，同时产生967bp和604bp的泳道条带时，对应的待测小鼠即为阳 性F1代杂合子条件性转基因小鼠。
作为进一步优选方案，PCR扩增后经测序，若同时得到序列表中SEQ ID No.3和SEQ ID No.4的基因序列，则对应的待测小鼠为阳性F1代杂合子条件性转基因小鼠。
第二方面，本申请还提供了NOTCH2NLC基因GGC重复扩增突变转基因小鼠，是应用上述的方法构建得到的。
第三方面，本申请还提供了上述的转基因小鼠在制备神经元核内包涵体病动物模型中的应用。
本发明建立了一种可靠稳定的小鼠模型，构建了稳定的神经元核内包涵体病动物模型，为探索NOTCH2NLC基因GGC重复扩增相关疾病的发病机制提供了重要模型，更是未来进行药物开发、筛选，探索有效治疗方法的重要工具。
附图说明
构成本申请的一部分的附图用来提供对本申请的进一步理解，使得本申请的其它特征、目的和优点变得更明显。本申请的示意性实施例附图及其说明用于解释本申请，并不构成对本申请的不当限定。在附图中：
图1为CRISPR/Cas9技术获得Rosa26位点定点敲入NLC条件性转基 因小鼠模型的原理示意图。
图2为插入NOTCH2NLC-(GGC)17or98-3tag序列的重组Donor质粒。
图3为重组donor质粒酶切鉴定电泳图。
图4为F0代小鼠PCR鉴定方法原理图。
图5为同源重组阳性F0代小鼠的PCR鉴定电泳图。
图6为条件性转基因小鼠短片段PCR鉴定原理图。
图7为鼠尾gDNA PCR产物鉴定电泳图。
图8为阳性F3代小鼠桑格(sanger)测序峰图。
图9为模型小鼠的脑组织免疫荧光图。
图10为小鼠肌肉组织免疫荧光图。
图11为小鼠脑组织的尼氏(Nissl)染色图。
图12为小鼠腓肠肌HE染色图。
图13为小鼠的开场实验结果。
图14为小鼠的新物体识别实验结果。
图15为小鼠的运动能力检测实验结果。
图16为转基因小鼠脑组织转录组分析。
具体实施方式
为了使本技术领域的人员更好地理解本申请方案，下面将结合本申请实施 例中的附图，对本申请实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本申请一部分的实施例，而不是全部的实施例。基于本申请中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都应当属于本申请保护的范围。下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的试验材料，如无特殊说明，均购自常规生化试剂公司。
需要说明的是，在不冲突的情况下，本申请中的实施例及实施例中的特征可以相互组合。下面将参考附图并结合实施例来详细说明本申请。
实施例1 NOTCH2NLC基因GGC重复扩增突变转基因小鼠的建立
原理如下：基因工程小鼠常常通过遗传学技术在小鼠的基因组上敲除小鼠同源基因、在小鼠同源基因上插入外来基因或者在其他安全位点插入外来基因(业界已证实在rosa26位点敲入外源基因是最经典的安全位点，不会影响其他内源性基因的表达，且在各种细胞类型和发育阶段均有广泛表达)。由于NOTCH2NLC基因是人类特有的基因，在小鼠基因组中不存在同源基因，因此只能选择在Rosa26位点插入NOTCH2NLC基因GGC重复扩增突变来实现小鼠体内过表达NOTCH2NLC基因GGC重复扩增致病突变。
现有遗传学研究证实，NOTCH2NLC基因GGC重复次数40次以内不致病，超过60则可能致病。故GGC重复次数≥60理论上均可以用于构建本申 请的转基因小鼠，并可能具有类似的功能。基于对多核苷酸重复扩增疾病的研究经验，本申请选择17次重复作为正常对照，选择98次重复作为致病突变。考虑到CRISPR/Cas9技术周期短、效率高的优点，本申请采用CRISPR/Cas9技术，通过同源重组的方式，在Rosa26基因位点定点插入CAG-LSL-NOTCH2NLC-WPRE-polyA表达框。
图1为CRISPR/Cas9技术获得Rosa26位点定点敲入NOTCH2NLC条件性转基因小鼠模型的原理示意图。
NOTCH2NLC基因GGC重复扩增突变转基因小鼠的建立方法如下：
1)针对Rosa26位点设计guideRNA(gRNA)靶序列，按照体外转录试剂盒(Takara,632635)步骤进行体外转录获得gRNA。gRNA的基因序列为：GGGGACACACTAAGGGAGCTTGG。
以正常人及NIID患者基因组DNA为模板，使用克隆引物I和II克隆NOTCH2NLC-(GGC)17or98序列，并通过BamHI与基因公司合成的3tag序列连接，得到NOTCH2NLC-(GGC)17or98-3tag序列，当然上述序列也可以直接交由基因公司合成，再通过AgeI和EcorV酶切和连接的方式将NOTCH2NLC-(GGC)17or98-3tag序列插入到含有CAG-Loxp-Stop-Loxp-AgeI-EcorV-WPRE-polyA元件donor质粒上，获得CAG-Loxp-Stop-Loxp-NOTCH2NLC-(GGC)17or98-3tag-WPRE-polyA重组donor质粒(见图2)。 克隆引物I:克隆引物II：
表1 PCR反应体系
使用的是Q5高保真DNA酶(NEB，Code No：M0491)。
PCR反应条件见表2。
表2 PCR反应条件

图2为插入NOTCH2NLC-(GGC)17or98-3tag序列的重组Donor质粒。
NOTCH2NLC-(GGC)17-3tag的核苷酸序列(序列表中SEQ ID NO.1)为：
其中，双下划线部分为NOTCH2NLC基因，黑框标记的是17次GGC重复序列，单下划线部分为3tag序列。
NOTCH2NLC-(GGC)98-3tag的核苷酸序列(序列表中SEQ ID NO.2)为：
其中，双下划线部分为NOTCH2NLC基因，黑框标记的是98次GGC重 复序列，单下划线部分为3tag序列。
将重组donor质粒通过酶切进行验证，结果见图3。
图3为重组donor质粒酶切鉴定电泳图。其中，A图为携带17次GGC重复同源重组质粒HindIII酶切鉴定结果，理论条带大小为7677bp、4224bp、2445bp、1525bp、941bp、389bp、247bp、79bp(产物量少看不清条带)；B图为携带98次GGC重复同源重组质粒BglII+XhoI酶切鉴定结果，理论条带大小为7976bp、5475bp、1952bp、1539bp、825bp。
两种重组donor质粒酶切后的电泳条带均符合预期。
重组donor质粒的提取方法为：将酶切鉴定正确的重组质粒，转化到STBL3大肠杆菌感受态细胞中进行扩增，扩增时采用低温长时培养(如25-30℃培养48小时)，以提高携带98次GGC重复重组质粒扩增时的稳定性。随后按照商业化质粒提取试剂盒步骤提取质粒。
2)将体外转录的100ng/μl Cas9 mRNA、50ng/μl gRNA及200ng/μl重组donor质粒采用显微注射的方式进行小鼠受精卵注射，将注射后的受精卵移植到假孕母鼠中，20天左右出生的小鼠为F0代小鼠；取小鼠尾尖约5mm按照鼠尾基因型快速鉴定试剂盒(碧云天D7283S)方法处理鼠尾，对打靶位点的5端臂和3端臂通过长片段PCR和电泳，对获得的F0代小鼠进行基因型鉴定，F0代小鼠PCR扩增原理见图4，鉴定结果见图5。
图4为F0代小鼠PCR鉴定方法原理图。
由图4可以看出：使用正向引物I和反向引物II，5端臂同源重组阳性基因组应扩增出3.4kb片段，阴性基因组应扩增出5.1kb片段；使用正向引物III和反向引物IV，3端臂同源重组阳性基因组应扩增出3.6kb片段，阴性基因组应扩增出6.5kb片段。
图5为同源重组阳性F0代小鼠的PCR鉴定电泳图。泳道M为Marker，泳道WT为野生型对照，图A是对5端臂进行长片段PCR，泳道2、4和5的PCR条带大小为5.1kb与WT一致，证明泳道2、4、5对应的小鼠为野生型；泳道1、3和6的PCR条带大小为3.4kb和5.1kb，证明对应的小鼠5端臂打靶成功，且为杂合子；B图是对3端臂进行长片段PCR，泳道1、3、6的PCR条带大小为6.5kb和3.6kb，证明对应的小鼠3端臂打靶成功，且为杂合子。
长片段PCR鉴定引物信息如下：
长片段PCR 5′同源臂鉴定引物信息：
正向引物I：5′-GCCGGGCCTCGTCGTCTG-3′
反向引物II：5′-TGAGGGCAATCTGGGAAGGTT-3′。
长片段PCR 3′同源臂鉴定引物信息：
正向引物III：5′-GGGGGAGGGGAGTGTTGC-3′
反向引物IV：5′-TTCTTCCTGCCTGCCTTCTGTGAC-3′。
5′同源臂和3′同源臂的PCR反应体系和反应条件均相同，具体的PCR反应体系见表3。
表3 PCR反应体系
使用的是PrimeStar GXL试剂盒(TaKaRa，Code No：R050A)。
PCR反应条件见表4。
表4 PCR反应条件
由于受精卵早期卵裂速度很快，因此得到的F0代小鼠为嵌合体，其生殖 细胞内不一定携带外源基因，需要进行传代以筛选可稳定遗传的F1代小鼠。
3)将所有阳性的F0代小鼠(雌鼠和雄鼠都可以)与野生型C57BL/6小鼠交配并繁殖，获得F1代小鼠，取小鼠尾尖约5mm按照鼠尾基因型快速鉴定试剂盒(碧云天D7283S)方法处理鼠尾，按照下述进行PCR和电泳鉴定，条件性转基因小鼠短片段PCR鉴定原理见图6，鉴定结果见图7。
图6为条件性转基因小鼠短片段PCR鉴定原理图。野生型等位基因使用鉴定引物I和II，应扩增出PCR产物大小为967bp；转基因等位基因使用鉴定引物III和IV，应扩增出PCR产物大小为604bp。
图7为鼠尾gDNA PCR产物鉴定电泳图。泳道M为Marker，野生型小鼠(WT)等位基因理论PCR产物条带大小为967bp，转基因小鼠(Tg)的等位基因理论PCR产物条带大小为604bp(不论17次重复还是98次重复)，可以看出泳道3对应的小鼠等位基因一条是wt一条是tg，所以是杂合子转基因小鼠；泳道1、2、4、5、6、7对应的小鼠两条等位基因均为wt，所以是野生型小鼠。
PCR引物如下：
鉴定引物I：TAAAGGCCACTCAATGCTCACTAA
鉴定引物II：TCAGATTCTTTTATAGGGGACACA；
鉴定引物III：GCGCAGGATCCTACCCATAC 鉴定引物IV：AAAGTCCCGGAAAGGAGCTG。PCR反应体系见表5。
表5 PCR反应体系
PCR反应条件见表6。
表6 PCR反应条件
967bp产物序列(序列表中SEQ ID NO.3)为：

其中下划线部分对应的是引物序列。
604bp产物序列(序列表中SEQ ID NO.4)为：
其中下划线部分对应的是引物序列。
因前两代条件性转基因小鼠的基因背景不纯净，故需要将阳性F1代小鼠再与野生型C57BL/6小鼠至少回交2代以后，才能与cre工具小鼠杂交。
将阳性F1代小鼠与野生型C57BL/6小鼠回交2代，得到阳性F3代小鼠，对得到的条件性转基因小鼠基因组DNA进行桑格(sanger)测序，测序结果见图8。
图8为阳性F3代小鼠桑格(sanger)测序峰图。其中图A显示正常对照组NOTCH2NLC-(GGC)17小鼠(以下简称(GGC)17小鼠)鼠尾DNA测序峰图，图B显示突变组NOTCH2NLC-(GGC)98小鼠(以下简称(GGC)98小鼠)鼠尾DNA测序峰图。下划线部分显示为NOTCH2NLC基因GGC重复区域；发现小鼠插入的序列与最初的设计相同。
图8表明，本发明方法获得了NOTCH2NLC基因GGC重复扩增突变转基因小鼠。该转基因小鼠能够将NOTCH2NLC基因GGC重复扩增突变传递给子代。
本申请中建立的小鼠模型能够稳定的向下一代传递该突变，将阳性F3代小鼠继续与野生型C57BL/6小鼠回交，可得阳性F4代小鼠、阳性F5代小鼠、阳性F6代小鼠等等。
将阳性F3代及以上的条件性转基因小鼠与不同的cre工具小鼠杂交，就可以获得全身或者特定组织或特定发育时期表达NOTCH2NLC基因的转基因 小鼠。
或者，将获得的阳性F3代及以上的杂合子阳性小鼠之间交配，得到的纯合子小鼠再与不同的cre工具小鼠杂交，这样后代中阳性小鼠的比例会进一步提高。
由于本申请建立的是携带LSL(即Loxp-Stop-Loxp)元件的条件性转基因小鼠，将条件性转基因小鼠与不同的cre工具小鼠杂交，就可以获得全身或者特定组织或特定发育时期表达NOTCH2NLC基因的转基因小鼠，从而实现全身或者特定组织或特定发育时期表达NOTCH2NLC基因。例如，与只在神经系统表达cre重组酶的Nestin-cre小鼠杂交，可以实现只在神经系统表达NOTCH2NLC基因。Cre-ERT2小鼠是一类含有雌激素受体的配体结合区突变体(ERT)与Cre重组酶的融合蛋白表达的小鼠，只有当Tamoxifen诱导后，Cre-ERT2小鼠才产生Cre重组酶活性。如将条件性转基因小鼠与心脏特异性的Myh6-Cre-ERT2工具鼠杂交，阳性后代在出生2月龄时，给予Tamoxifen诱导，可以实现心脏特异性表达NOTCH2NLC基因。这是现有技术中常用的方法，在此不再赘述。
实施例2 NOTCH2NLC基因GGC重复扩增突变转基因小鼠的病理和行为表型分析
动物模型能否模拟患者的临床表型是动物模型是否成功建立的关键指标。 NIID的主要病理表型是广泛存在的核内包涵体及神经元死亡，其主要临床症状为肌无力和痴呆。因此，本申请发明人将实施例1中的阳性F3代条件性转基因小鼠与全身表达重组酶的EIIa-cre工具鼠杂交，得到了全身表达该基因突变的模型小鼠(EIIa；(GGC)17和EIIa；(GGC)98)(sanger测序显示EIIa；(GGC)17和EIIa；(GGC)98小鼠NOTCH2NLC基因序列与图8一致)，并检测了EIIa；(GGC)17和EIIa；(GGC)98小鼠的病理和行为表型。
下述中，EIIa；(GGC)17(或(GGC)17)为正常对照，表示NOTCH2NLC基因17次GGC重复扩增突变转基因小鼠；EIIa；(GGC)98(或(GGC)98)表示NOTCH2NLC基因98次GGC重复扩增突变转基因小鼠。
1.该模型小鼠不同组织器官内形成核内多聚甘氨酸(polyG)聚集物。
图9为模型小鼠的脑组织免疫荧光图。其中，绿色表示polyG蛋白聚集物，蓝色表示细胞核。
如图9所示，在出生后50天左右的模型小鼠的小鼠海马、小脑、皮层、纹状体等脑区形成polyG蛋白聚集物，且多数位于细胞核内。
图10为小鼠腓肠肌免疫荧光图。其中，绿色表示polyG蛋白聚集物，蓝色表示细胞核。
如图10所示，在出生后50天左右的模型小鼠的腓肠肌细胞核内也形成polyG蛋白聚集物。
2.这些形成聚集物的组织出现神经元死亡或肌纤维病变等。
图11为小鼠脑组织的尼氏(Nissl)染色图。
如图11所示，与对照组EIIa；(GGC)17小鼠相比，EIIa；(GGC)98小鼠在皮层(cortex)出现更多的固缩的细胞，小脑(cerebellum)浦肯野细胞数量更少，提示神经元死亡。
图12为小鼠腓肠肌HE染色图。
如图12所示，与对照组EIIa；(GGC)17小鼠相比，EIIa；(GGC)98小鼠出现肌细胞核内移、细胞核增多、炎性浸润等病理改变，提示肌细胞受损。
3.通过多种行为学分析平台对转基因小鼠的行为表型进行了分析。
3.1开场实验可以检测模型小鼠在自由活动状态下的运动能力。实验结果见图13。
图13为小鼠的开场实验结果。其中，A图为小鼠在开场箱中的运动轨迹图；B图为小鼠在开场箱中的总运动时间柱状图；C图为小鼠在开场箱中的总运动距离柱状图。
如图13所示，与对照组野生型(WT)小鼠和EIIa；(GGC)17小鼠相比，EIIa；(GGC)98转基因小鼠的总运动时间和运动距离减少。
3.2新物体识别实验，可以检测小鼠对新旧物体的识别，从而判断是否存在认知缺陷，实验结果见图14。
图14为小鼠的新物体识别实验结果。其中，A图为实验示意图：适应环节两侧放置相同物品，测试环节将其中一侧物品更换为新物品；B图为测试环节三种小鼠对新物体的偏好比例柱状图。
如图14所示，与对照组野生型(WT)小鼠和EIIa；(GGC)17小鼠相比，EIIa；(GGC)98转基因小鼠对新物体的识别较对照组差。
3.3运动能力检测实验。
转棒实验：将小鼠放置在转动的转棒上，观察小鼠在转棒上停留的时间，时间越长代表运动和协调能力越强。
悬挂实验：将小鼠放置在铁丝网后倒置，观察小鼠抓住铁丝网的时间，时间越长代表运动和协调能力越强。
站立实验：将小鼠放置在透明圆柱体内，观察小鼠站立并用前肢触碰圆柱体壁的次数，次数越多代表小鼠运动能力越强。
实验结果见图15。
图15为小鼠的运动能力检测实验结果。其中，A图为小鼠停留在转棒仪上的时间；B图为小鼠悬挂在金属网格上的时间；C图为小鼠在圆柱体中站立探索的次数。
如图15所示，在运动能力专项检测中，我们发现EIIa；(GGC)98转基因小鼠在转棒仪上停留的时间明显减少，悬挂在金属网格上的时间减少，站立探索 的次数减少，均提示EIIa；(GGC)98转基因小鼠存在运动障碍。
上述多种实验结果表明，EIIa；(GGC)98转基因小鼠能够模拟患者常见的肌无力、痴呆等表型。
实施例3 NOTCH2NLC基因GGC重复扩增突变转基因小鼠的应用
本实施例介绍了本发明的转基因小鼠在研究NOTCH2NLC基因GGC重复扩增相关疾病中发生发展机制、药物筛选及疗效评价等方面的应用，包括但不限于下列示例：
(1)NIID患者除了神经系统受到损伤外，在心脏、肾脏等多种系统存在病理改变。故可以将条件性转基因小鼠与仅在上述组织表达Cre酶的工具小鼠杂交，实现在这些组织中特异性表达NOTCH2NLC基因GGC重复扩增，例如，Cre-ERT2小鼠是一类含有雌激素受体的配体结合区突变体(ERT)与Cre重组酶的融合蛋白表达的小鼠，只有当Tamoxifen诱导后，Cre-ERT2小鼠才产生Cre重组酶活性。如将条件性转基因小鼠与心脏特异性的Myh6-Cre-ERT2工具鼠杂交，阳性后代在出生2月龄时，给予Tamoxifen诱导，可以实现特定年龄阶段(2月龄)、特定组织器官(心脏)特异性表达NOTCH2NLC基因。上述策略可以排除其他系统异常带来的干扰，对疾病的发病机制和药物筛选进行更针对性的研究。
(2)本申请发明人利用深度RNA测序技术，对NOTCH2NLC基因GGC 重复扩增的小鼠中与疾病表型相关的皮层、海马、小脑三个脑区进行了转录组分析，结果见图16。
图16为转基因小鼠脑组织转录组分析。其中，A图为海马(hippocampus)、皮层(cortex)和小脑(cerebellum)三个脑区下调表达的基因维恩图分析结果，B图为海马(hippocampus)、皮层(cortex)和小脑(cerebellum)三个脑区上调表达基因维恩图分析结果，其中共同下调162个基因，共同上调基因383个；C图为三个脑区共同下调基因富集的KEGG通路；D图为三个脑区共同上调基因富集的KEGG通路。
由图16可知，通过比较三者之间的异同，本申请发明人发现：1)三个脑区中小脑差异表达基因数量最多，皮层第二，海马最少，提示小脑可能是三个脑区中转录组受影响最严重的脑区，这提示我们小脑可能在NIID的发病中发挥重要贡献；2)三个脑区之间共同上调或者共同下调的差异表达基因占比较高，对这些共同改变的差异表达基因进行KEGG功能富集发现，共同上调的通路与免疫反应有关，而共同下调的通路与钙离子、cAMP信号通路有关。这些共同改变的通路可能是参与NIID发病的重要通路，为未来的机制研究和药物开发提供了重要线索。
以上所述仅为本申请的优选实施例而已，并不用于限制本申请，对于本领域的技术人员来说，本申请可以有各种更改和变化。凡在本申请的精神和原则 之内，所作的任何修改、等同替换、改进等，均应包含在本申请的保护范围之内。

Claims (10)

  1. NOTCH2NLC基因GGC重复扩增突变转基因小鼠的构建方法，其特征在于，包括以下步骤：
    S1，构建带有LoxP序列并定点敲入NOTCH2NLC基因的小鼠：在小鼠体内的Rosa26基因位点，定点插入CAG-Loxp-Stop-Loxp-NOTCH2NLC-(GGC)n-3tag-WPRE-polyA表达框，繁育并鉴定出F0代阳性小鼠；所述n为自然数，且n≥60；
    S2，将F0代阳性小鼠与野生型小鼠交配并繁殖，获得F1代小鼠，鉴定出阳性F1代杂合子条件性转基因小鼠；
    S3，将阳性F1代杂合子条件性转基因小鼠与野生型小鼠回交m代，其中，m为自然数，且m≥2，得到F(1+m)代杂合子条件性转基因小鼠；将F(1+m)代杂合子条件性转基因小鼠之间交配并繁殖，得到纯合子条件性转基因小鼠；
    S4，将F(1+m)代杂合子条件性转基因小鼠或纯合子条件性转基因小鼠与cre工具小鼠杂交，获得转基因小鼠。
  2. 如权利要求1所述的构建方法，其特征在于，步骤S1中，所述n为98。
  3. 如权利要求1所述的构建方法，其特征在于，步骤S1中，采用CRISPR/Cas9基因编辑技术定点插入所述表达框。
  4. 如权利要求3所述的构建方法，其特征在于，步骤S1中，采用CRISPR/Cas9基因编辑技术定点插入所述表达框的具体方法为：
    1)、针对Rosa26位点设计gRNA靶序列，所述gRNA的基因序列为：GGGGACACACTAAGGGAGCTTGG；
    2)、将外源NOTCH2NLC-(GGC)n-3tag通过AgeI和EcorV酶切，连接至donor质粒上，获得CAG-Loxp-Stop-Loxp-NOTCH2NLC-(GGC)n-3tag-WPRE-polyA重组donor质粒；
    3)、将Cas9 mRNA、gRNA及重组donor质粒进行小鼠受精卵注射，然后将注射后的小鼠受精卵移植到假孕母鼠中，孕育的小鼠为F0代小鼠。
  5. 如权利要求4所述的构建方法，其特征在于，将获得的重组donor质粒转化入STBL3大肠杆菌感受态细胞中进行扩增，扩增时，在25-30℃培养46-50小时。
  6. 如权利要求1所述的构建方法，其特征在于，步骤S1中，通过长片段PCR扩增和电泳的方法鉴定出F0代阳性小鼠；
    所述长片段PCR扩增时的引物为：
    长片段PCR 5′同源臂鉴定引物信息：
    正向引物I：5′-GCCGGGCCTCGTCGTCTG-3′
    反向引物II：5′-TGAGGGCAATCTGGGAAGGTT-3′；
    长片段PCR 3′同源臂鉴定引物信息：
    正向引物III：5′-GGGGGAGGGGAGTGTTGC-3′
    反向引物IV：5′-TTCTTCCTGCCTGCCTTCTGTGAC-3′；
    电泳后，5端臂产生泳道条带大小为3.4kb和5.1kb，且3端臂产 生泳道条带大小为6.5kb和3.6kb时，对应的待测小鼠即为F0代阳性小鼠。
  7. 如权利要求1所述的构建方法，其特征在于，步骤S2中，通过PCR扩增和测序的方法，或通过PCR扩增和电泳的方法鉴定出阳性F1代杂合子条件性转基因小鼠；
    所述PCR扩增时的引物为：
    鉴定引物I：TAAAGGCCACTCAATGCTCACTAA
    鉴定引物II：TCAGATTCTTTTATAGGGGACACA；
    鉴定引物III:GCGCAGGATCCTACCCATAC
    鉴定引物IV:AAAGTCCCGGAAAGGAGCTG；
    电泳后，同时产生967bp和604bp的泳道条带时，对应的待测小鼠即为阳性F1代杂合子条件性转基因小鼠。
  8. 如权利要求7所述的构建方法，其特征在于，PCR扩增后经测序，若同时得到序列表中SEQ ID No.3和SEQ ID No.4的基因序列，则对应的待测小鼠为阳性F1代杂合子条件性转基因小鼠。
  9. NOTCH2NLC基因GGC重复扩增突变转基因小鼠，其特征在于，是应用权利要求1-8任一项所述的方法构建得到的。
  10. 权利要求9所述的转基因小鼠在制备神经元核内包涵体病动物模型中的应用。
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Publication number Priority date Publication date Assignee Title
CN115851833B (zh) * 2022-11-15 2023-09-15 中南大学湘雅医院 Notch2nlc基因ggc重复扩增突变转基因小鼠及其构建方法和应用

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112094893A (zh) * 2019-06-17 2020-12-18 杭州方夏生物科技有限公司 神经元核内包涵体病notch2nlc基因ggc重复扩增检测试剂盒
CN112608939A (zh) * 2020-11-27 2021-04-06 复旦大学附属肿瘤医院 一种条件性mTERT过表达的小鼠模型的构建方法及其应用
JP2021108552A (ja) * 2020-01-08 2021-08-02 公立大学法人横浜市立大学 神経核内封入体病患者の検出方法
US20220025460A1 (en) * 2020-07-21 2022-01-27 The University Of Tokyo Method and kit for determining neuromuscular disease in subject
CN115851833A (zh) * 2022-11-15 2023-03-28 中南大学湘雅医院 Notch2nlc基因ggc重复扩增突变转基因小鼠及其构建方法和应用

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112553299A (zh) * 2019-09-10 2021-03-26 北京大学第一医院 Notch2nlc基因ggc重复序列扩增的方法
CN112029765A (zh) * 2020-07-30 2020-12-04 南京医科大学附属逸夫医院 一种Metrnl基因条件性敲除小鼠模型制作方法
CN112852875B (zh) * 2021-02-26 2022-10-21 福建省立医院 示踪肿瘤T淋巴细胞浸润的CD3e转基因小鼠模型的构建方法

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112094893A (zh) * 2019-06-17 2020-12-18 杭州方夏生物科技有限公司 神经元核内包涵体病notch2nlc基因ggc重复扩增检测试剂盒
JP2021108552A (ja) * 2020-01-08 2021-08-02 公立大学法人横浜市立大学 神経核内封入体病患者の検出方法
US20220025460A1 (en) * 2020-07-21 2022-01-27 The University Of Tokyo Method and kit for determining neuromuscular disease in subject
CN112608939A (zh) * 2020-11-27 2021-04-06 复旦大学附属肿瘤医院 一种条件性mTERT过表达的小鼠模型的构建方法及其应用
CN115851833A (zh) * 2022-11-15 2023-03-28 中南大学湘雅医院 Notch2nlc基因ggc重复扩增突变转基因小鼠及其构建方法和应用

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
A'NAN CHEN, QIAN HAIRONG: "Neurodegeneration-related mechanism and clinical research progress of neuronal intranuclear inclusion body disease", CHINESE JOURNAL OF DIFFICULT AND COMPLICATED CASES, vol. 21, no. 4, 18 April 2022 (2022-04-18), pages 423 - 427, XP093170516 *
BOIVIN MANON; DENG JIANWEN; PFISTER VéRONIQUE; GRANDGIRARD ERWAN; OULAD-ABDELGHANI MUSTAPHA; MORLET BASTIEN; RUFFENACH FRANK;: "Translation of GGC repeat expansions into a toxic polyglycine protein in NIID defines a novel class of human genetic disorders: The polyG diseases", NEURON, ELSEVIER, AMSTERDAM, NL, vol. 109, no. 11, 21 April 2021 (2021-04-21), AMSTERDAM, NL, pages 1825, XP086588353, ISSN: 0896-6273, DOI: 10.1016/j.neuron.2021.03.038 *
PAN YONGCHENG, JIANG YING; WAN JUAN; HU ZHENGMAO; JIANG HONG; SHEN LU; TANG BEISHA; TIAN YUN; LIU QIONG: "Expression of expanded GGC repeats within NOTCH2NLC causes cardiac dysfunction in mouse models", CELL & BIOSCIENCE, BIOMED CENTRAL LTD, LONDON, UK, vol. 13, no. 1, 29 August 2023 (2023-08-29), London, UK , pages 157, XP093170512, ISSN: 2045-3701, DOI: 10.1186/s13578-023-01111-6 *
YU JIAX, TONGLING LIUFU, YILEI ZHENG, JIN XU, LINGCHAO MENG, WEI ZHANG, YUN YUAN, DAOJUN HONG, NICOLAS CHARLET-BERGUERAND, ZHAOXI: "CGG repeat expansion in NOTCH2NLC causes mitochondrial dysfunction and progressive neurodegeneration in drosophila model", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, NATIONAL ACADEMY OF SCIENCES, vol. 119, no. 41, 3 October 2022 (2022-10-03), pages e2208649119, XP093170515, ISSN: 0027-8424 *

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