A method for establishing an APP / PSEN1 double transgenic tree shrew model of Alzheimer's disease

Abstract

The present invention discloses a method for establishing an APP / PSEN1 double transgenic AD tree shrew model. For the first time, lentivirus carrying pathogenic genes APP and PSEN1 is used to infect the spermatogonial stem cells of tree shrews. The AD transgenic spermatogonial stem cells of tree shrews are injected into the testes of male tree shrews treated with busulfan. Then, the recipient male tree shrews are allowed to mate with wild-type female tree shrews. The obtained offspring AD transgenic tree shrews are identified to obtain a tree shrew model that produces overexpressed mutant APP and PSEN1 double transgenic Alzheimer's disease. The present invention is the first AD transgenic disease model of tree shrews in the world. A stable and heritable new tree shrew strain can be obtained through continuous hybridization of existing transgenic tree shrews; it can simulate the generation and metabolic process of the main pathogenic genes of AD pathology in vivo, reproduce the disease process, and prove through behavioral indicators that this model is significantly different from other AD models, and can be used for research on the pathogenic mechanism of AD, development of drug targets, and drug screening.

Description

Technical Field

[0001] The present invention relates to a biotechnology method, and particularly to a method for establishing an APP / PSEN1 double transgenic Alzheimer's disease animal model. Background Art

[0002] Alzheimer's disease (AD) is the only major disease with a continuously increasing prevalence rate. Establishing an effective AD animal model is of great significance for promoting the research on the pathogenesis of AD, disease prevention, early diagnosis, and drug development.

[0003] Currently, the AD animal models used in a large number of disease mechanism and drug studies are mainly rodents (mice, rats). However, due to the huge evolutionary status difference between rodents and humans, the AD mouse model cannot fully reproduce the pathological characteristics of AD, which greatly restricts the revelation of the pathogenesis of AD and the development of new drugs, making the AD mouse model have insurmountable limitations (Zahs and Ashe 2010; Webster et al. 2014). Non-human primates are the most similar to humans in evolution, and elderly non-human primates will show physiological or pathological characteristics similar to those of the elderly or AD patients. Non-human primates (such as Old World monkeys) can better simulate the AD phenotype (Souder et al. 2021). For example, the formation of Aβ plaques has been detected in the brain parenchyma and cerebral blood vessels of elderly cynomolgus monkeys. The recent paper "Synthetic amyloid-β oligomers drive early pathological progression of Alzheimer's Disease in nonhuman primates" discloses a large animal model of Alzheimer's disease in which typical AD neuropathological characteristics such as Aβ plaques, tau tangles, and neuroinflammation are induced in the brains of non-human primate cynomolgus monkeys. In this study, soluble AβOs were injected into the brain parenchyma of the bilateral white matter regions above the hippocampus of adult cynomolgus monkeys in batches. Due to the long life cycle of monkeys, in the existing related studies on establishing AD animal models based on non-human primates, pathological proteins are directly injected into specific brain regions to simulate the AD phenotype. However, this modeling method cannot simulate the natural onset process of AD, and the established animal model is not stably heritable, having certain limitations in application.

[0004] Compared with mice, tree shrews have a developed brain and a large brain-to-body weight ratio, making them an ideal animal model for studying nervous system diseases (including mental diseases such as depression and neurodegenerative diseases such as AD) (Yao 2017). The inventor team previously established a set of efficient and stable gene manipulation technology platforms for tree shrews through the spermatogonial stem cell approach, overcoming the bottleneck problems of tree shrew gene manipulation and generating the world's first transgenic tree shrew (Li et al. 2017). The APP and PSEN1 genes are pathogenic genes of familial AD and the main targets for establishing AD animal models. Through sequence alignment analysis, it was found that the APP and PSEN1 genes of tree shrews, as well as the entire AD pathogenic pathway, have a high homology with humans. The AD pathogenic mutation hotspot gene region is highly conserved between tree shrews and humans, and the brain gene expression pattern of tree shrews is very similar to that of primates (Fan et al. 2018). In contrast, mice and rats, which are currently widely used as AD animal models, are not conservative at many AD pathogenic mutation sites, and the brain gene expression pattern is very different from that of primates. Therefore, tree shrews have great advantages over rodents as AD animal models.

[0005] References

[0006] Fan Y, Luo R, Su LY, Xiang Q, Yu D, Xu L, Chen JQ, Bi R, Wu DD, Zheng P et al. 2018. Does the Genetic Feature of the Chinese Tree Shrew (Tupaia belangeri chinensis) Support Its Potential as a Viable Model for Alzheimer's Disease Research?J Alzheimers Dis 61:1015 - 1028.

[0007] Li CH, Yan LZ, Ban WZ, Tu Q, Wu Y, Wang L, Bi R, Ji S, Ma YH, Nie WH et al. 2017.

[0008] Long - term propagation of tree shrew spermatogonial stem cells in culture and successful generation of transgenic offspring. Cell Res 27:241 - 252.

[0009] Souder DC, Dreischmeier IA, Smith AB, Wright S, Martin SA, Sagar MAK, Eliceiri KW, Salamat SM, Bendlin BB, Colman RJ et al. 2021. Rhesus monkeys as a translational model for late-onset Alzheimer's disease. Aging Cell 20:e13374.

[0010] Webster SJ, Bachstetter AD, Nelson PT, Schmitt FA, Van Eldik LJ. 2014. Using mice to model Alzheimer's dementia: an overview of the clinical disease and the preclinical behavioral changes in 10 mouse models. Front Genet 5:88.

[0011] Yao YG. 2017. Creating animal models, why not use the Chinese tree shrew (Tupaia belangeri chinensis)? Zool Res 38:118 - 126.

[0012] Zahs KR, Ashe KH. 2010. 'Too much good news' - are Alzheimer mouse models trying to tell us how to prevent, not cure, Alzheimer's disease? Trends Neurosci 33:381 - 389. Summary of the Invention

[0013] The object of the present invention is to provide a method for establishing a heritable, easily repeatable, stable, and simple and convenient-to-operate tree shrew Alzheimer's disease animal model in view of the deficiencies of existing AD animal models.

[0014] The present invention achieves its object in the following manner:

[0015] A method for establishing an APP / PSEN1 double transgenic Alzheimer's disease tree shrew model, in which lentivirus carrying the pathogenic genes APP and PSEN1 is used to infect tree shrew spermatogonial stem cells to obtain AD transgenic tree shrew spermatogonial stem cells. The AD transgenic tree shrew spermatogonial stem cells are injected into the testes of male tree shrews treated with busulfan, and then the recipient male tree shrews are mated with wild-type female tree shrews. The obtained offspring AD transgenic tree shrews are identified to obtain a double transgenic Alzheimer's disease tree shrew model that produces overexpressed mutant APP and PSEN1.

[0016] Preferably, the obtaining of the AD transgenic tree shrew spermatogonial stem cells includes the following steps:

[0017] S1 Lentivirus preparation: The most important pathogenic genes APP and PSEN1 of familial Alzheimer's disease with pathogenic mutations are inserted behind the EF1α promoter through XbaI and NotI restriction enzyme sites, and the CDS region of APP is inserted. The CDS region of PSEN1 is inserted behind the CMV promoter to establish a lentiviral vector for the APP and PSEN1 genes. The constructed lentiviral vector, packaging plasmid psPAX, and pMD2.G are co-transfected into HEK293T cells, and the virus supernatant is collected at 48 h and 72 h respectively, and concentrated by ultracentrifugation to obtain lentivirus; K670N / M671L CDS region, and the CDS region of PSEN1 is inserted behind the CMV promoter M139V / M146L / H163R CDS region, establish a lentiviral vector for the APP and PSEN1 genes, co-transfect the constructed lentiviral vector and packaging plasmids psPAX and pMD2.G into HEK293T cells, and collect the virus supernatant at 48 h and 72 h respectively, and concentrate by ultracentrifugation to obtain lentivirus;

[0018] S2 Virus infection to obtain AD transgenic tree shrew spermatogonial stem cells: The prepared virus solution is used to infect tree shrew spermatogonial stem cells. 24 hours after virus infection, through fluorescence microscopy observation, the spermatogonial stem cells successfully integrated with the APP K670N / M671L gene express red fluorescent protein, and the spermatogonial stem cells successfully integrated with the PSEN1 M139V / M146L / H163R gene express green fluorescent protein.

[0019] Preferably, the male tree shrews are obtained by intraperitoneal administration of busulfan (35 mg / kg) to remove endogenous spermatogenic cells of the tree shrews.

[0020] Furthermore, the transgenic spermatogonial stem cells are resuspended in PBS and injected into the seminiferous tubules of recipient tree shrews. Two months later, the recipient male tree shrews are mated with wild-type female tree shrews to breed F1 generation AD transgenic tree shrews.

[0021] Furthermore, the F1 generation AD transgenic tree shrews are used as male and female parents for mating to obtain F2 generation AD transgenic tree shrews. The obtained F2 generation AD transgenic tree shrews are used as male and female parents for mating to obtain F3 generation AD transgenic tree shrews. The above steps of mating are continued 5 - 10 times to obtain Fn generation AD transgenic tree shrews.

[0022] Preferably, the obtained F1 to Fn generation AD transgenic tree shrews are identified, including the molecular, pathological, and behavioral characteristics of AD.

[0023] The present invention also protects the above-obtained F1-Fn generation transgenic tree shrews for use in studying the pathogenic mechanism of AD, drug research and development, and drug screening.

[0024] Based on tree shrews, a type of primate, and combined with spermatogonial stem cell transgenic operation technology, the present invention establishes a transgenic tree shrew model for AD, which is also the first transgenic disease model of tree shrews, providing an important basis and animal model for the mechanism research and drug development of AD.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] 1. The present invention provides a method for establishing a tree shrew Alzheimer's disease model using lentivirus in tree shrews.

[0027] 2. The present invention provides a method for quickly, efficiently, and hereditarily establishing a tree shrew Alzheimer's disease model.

[0028] 3. The APP and PSEN1 genes are overexpressed in the constructed lentiviral vector, and these genes are the most important pathogenic genes of familial Alzheimer's disease. Therefore, the established tree shrew Alzheimer's disease model can well simulate the genetic mechanism of human Alzheimer's disease, and thus this model has great application potential in the basic research of Alzheimer's disease and the testing of drugs.

[0029] 4. The number of transgenic tree shrews produced by the present invention is sufficient to fully meet the needs of subsequent research on the pathogenesis of human Alzheimer's disease, exploring new therapeutic targets, and developing therapeutic drugs.

[0030] 5. Based on the characteristics that tree shrews are close to primates in the evolutionary position and the AD pathogenic pathway is very conservative, the established Alzheimer's disease tree shrew model of the present invention has more advantages than traditional rodent models and is also the first transgenic tree shrew Alzheimer's disease model at present. Brief Description of the Drawings

[0031] Figure 1 Schematic diagram of the lentiviral overexpression vector structure of APP and PSEN1 genes;

[0032] Figure 2 Spermatogonial stem cell diagram of the transgenic system expressing APP and PSEN1, where Figure 2 A is the spermatogonial stem cell diagram of the transgenic system expressing APP, Figure 2 B is the spermatogonial stem cell diagram of the transgenic system expressing APP with fluorescence, Figure 2 C is the spermatogonial stem cell diagram of the transgenic system expressing PSEN1;Figure 2 Figure D shows spermatogonial stem cells of the PSEN1 transgenic system with fluorescence expression;

[0033] Figure 3 This is the PCR and sequencing identification of transgenic positive tree shrews, Figure 3 A shows the PCR determination results and sequencing identification of transgenic positive tree shrews, Figure 3 B shows the sequencing results of the APP gene of transgenic positive tree shrews, Figure 3 C shows the sequencing results of the PSEN1 gene of transgenic positive tree shrews;

[0034] Figure 4 Figure shows the identification results of the PSEN1 gene of F2 generation tree shrews;

[0035] Figure 5 Figure shows the results of significant overexpression of APP and PSEN1 genes at the RNA level in primary cells of transgenic AD positive tree shrews;

[0036] Figure 6 Figure shows the results of overexpression of APP and PSEN1 proteins in the brains of transgenic AD positive tree shrews; among them Figure 6 A and Figure 6 B are immunoblotting and statistical charts of overexpression of APP and PSEN1 proteins; Figure 6 C is a figure showing the overexpression of Flag-tagged protein in tree shrew brain slices; Figure 6 D is a figure showing the immunoblotting results of Flag-tagged protein in tree shrew brain tissue;

[0037] Figure 7 Figure shows the comparison of the exploration frequencies of transgenic AD positive tree shrews and the control group in the hole board experiment;

[0038] Figure 8 Figure shows the comparison of the exploration frequencies of transgenic AD positive tree shrews and the control group in the novel object recognition experiment;

[0039] Figure 9 Figure shows the comparison of the levels of AD pathological molecule Aβ in the brains of transgenic AD positive tree shrews and the control group; among them, Figure 9 A and Figure 9 B are immunoblotting and quantitative charts of different forms of Aβ, Figure 9 C is a figure showing the immunohistochemical results of Aβ molecules in brain slices of control tree shrews and AD transgenic tree shrews; Figure 9 D is a figure showing the immunofluorescence results of Aβ molecules in brain slices of control tree shrews and AD transgenic tree shrews;

[0040] Figure 10 Figure shows the comparison of the levels of AD pathological molecule phosphorylated tau protein in the brains of transgenic AD positive tree shrews and the control group; among them, Figure 10 A and Figure 10Figure B shows the immunoblot and quantification of different phosphorylated Tau proteins. Figure 10 Figure C shows the immunohistochemical results of phosphorylated Tau protein molecules in brain sections of control tree shrews and AD transgenic tree shrews.

[0041] Figure 11 Figure shows the activation of glial cells in the brains of AD transgenic positive tree shrews and the control group. Among them, Figure 11 Figure A and Figure 11 Figure B show the immunoblot and statistical results of molecular markers of astrocytes and microglia. Figure 11 Figure C shows the immunohistochemical results of the astrocyte marker molecule GFAP in brain sections of control tree shrews and AD transgenic tree shrews.

[0042] Figure 11 Figure D shows the immunohistochemical results of the microglia marker molecule IBA1 in brain sections of control tree shrews and AD transgenic tree shrews. Detailed implementation methods

[0043] To make the present invention easier to understand, the following further elaborates the present invention in combination with specific examples, drawings and tables. Of course, these examples are only used to illustrate the present invention and not to limit the scope of the present invention.

[0044] Example 1: A method for establishing a tree shrew model of Alzheimer's disease

[0045] 1. Construction of lentiviral vector

[0046] The most important pathogenic genes APP and PSEN1 of familial Alzheimer's disease containing pathogenic mutations were inserted behind the EF1α promoter through XbaI and NotI restriction enzyme sites, and the CDS region of APP was inserted. K670N / M671L The CDS region of PSEN1 was inserted behind the CMV promoter to establish lentiviral vectors for APP and PSEN1 genes ( M139V / M146L / H163R ). Figure 1 )

[0047] 2. Virus packaging

[0048] The constructed lentiviral vector, packaging plasmid psPAX, and pMD2.G were co-transfected into HEK293T cells. After 6 - 8 hours of transfection, the medium was changed, and the virus supernatant was collected at 48h and 72h respectively. The virus was concentrated by centrifugation at 25000rpm and 4°C for 2.5h, and the virus was resuspended in PBS.

[0049] 3. Lentiviral infection of tree shrew spermatogonial stem cells

[0050] Prepare a total cell number of 3x10 6 -5x10 6The spermatogonial stem cells of tree shrews were resuspended with the prepared virus incubation solution (1 ml of spermatogonial stem cell medium + 1 μl of Polybrene (5 mg / ml) + 100 μl of concentrated virus), placed in an incubator for suspension infection for 24 hours, centrifuged at 1000 rpm for 5 minutes, the virus incubation solution was discarded, and the cells were resuspended with fresh spermatogonial stem cell medium and seeded onto the feeder cells prepared in advance. 24 hours after virus infection, by observing under a fluorescence microscope, the spermatogonial stem cells that successfully integrated the APP K670N / M671L gene expressed red fluorescent protein, and the spermatogonial stem cells that successfully integrated the PSEN1 M139V / M146L / H163R gene expressed green fluorescent protein ( Figure 2 ).

[0051] 4. Injection of transgenic spermatogonial stem cells and reproduction of tree shrews

[0052] One-year-old male tree shrews were prepared and given busulfan intraperitoneally (35 mg / kg) to remove endogenous spermatogenic cells of the tree shrews. The cultured transgenic spermatogonial stem cells were resuspended with PBS at a concentration of approximately 1×10 4 cells / μL. 100 μl of cell suspension was prepared for each recipient tree shrew, and the transgenic spermatogonial stem cells were injected into the seminiferous tubules of the recipient tree shrews through the efferent ducts. Two months later, the recipient tree shrews could be mated with female tree shrews to obtain offspring transgenic tree shrews.

[0053] 5. Genotype identification of offspring

[0054] The ear tissues of the offspring tree shrews were collected, and the genomic DNA was extracted using the AXYGEN genomic DNA extraction kit. Genotype detection primers were designed according to the lentiviral vector sequence and the target gene sequence (Appendix 1), and PCR was used to detect whether the exogenous mutant APP and PSEN1 genes were integrated into the genome of the tree shrews. The detection results showed that there were transgenic positive tree shrews among the offspring tree shrews. Sequencing verification of the PCR products found that the transgenic AD gene-positive tree shrews indeed carried the familial AD pathogenic variants ( Figure 3 ). It was shown that transgenic AD gene tree shrews had been successfully obtained and the model was successfully established. After gene identification, among the current 84 F1-generation tree shrews, there were 19 transgenic positive tree shrews, and the transgenic positive acquisition rate was 22%. Further, the positive F1-generation transgenic tree shrews were mated with each other. Currently, 19 offspring F2-generation tree shrews were obtained, among which 16 were transgenic positive, and the positive rate was 84% ( Figure 4 ).

[0055] In the future, the above operations will be repeated. The obtained F2-generation AD transgenic tree shrews will be used as male and female parents for mating to obtain F3-generation AD transgenic tree shrews, and the above steps will be continued for mating 5 - 10 times to obtain Fn-generation AD transgenic tree shrews.

[0056] Example 2: Successful expression of foreign genes in AD gene-transferred tree shrews

[0057] Ear tissues of AD gene-transferred positive tree shrews were collected to establish ear fibroblasts. Detection by real-time fluorescence quantitative PCR primers (Appendix 1) found that the APP and PSEN1 genes were significantly overexpressed in the primary cells of AD gene-transferred positive tree shrews ( Figure 5 ). Brains of AD gene-transferred tree shrews (F1 generation) were collected. Western blot analysis showed that the expression levels of the APP gene and the PSEN1 gene in the cerebral cortex of AD gene-transferred tree shrews were significantly higher than those of control tree shrews ( Figure 6 A-B). Moreover, the overexpressed APP protein could be detected by the exogenous tag Flag ( Figure 6 C-D), indicating that the exogenous APP gene was successfully overexpressed in the brains of transgenic tree shrews.

[0058] Example 3: Behavioral abnormalities in AD gene-transferred tree shrews

[0059] The hole-board experiment found that AD gene-transferred tree shrews were reluctant to be trained by experimenters, and the frequency of exploring the hole board was significantly lower than that of the control group. Even a large proportion showed no behavior of exploring the hole board and could not complete the learning and memory tasks ( Figure 7 ). Similarly, in the novel object recognition test, the frequency of AD gene-transferred tree shrews exploring objects was significantly lower than that of control tree shrews ( Figure 8 ), and these behavioral phenotypes indicated that AD gene-transferred tree shrews had behavioral abnormalities. Some studies have found that behavioral abnormalities occur in the early stage of AD, including lack of interest in external things, senile depression, etc. This shows that the AD gene-transferred tree shrews of the present invention are similar to AD patients in these aspects and exhibit behavioral phenotypes closer to humans than mouse AD models.

[0060] Example 4: Appearance of early AD pathological features in the brains of AD gene-transferred tree shrews

[0061] The core pathological symptoms of AD include three parts: 1) accumulation of β-amyloid (Aβ); 2) hyperphosphorylation of Tau protein; 3) gliosis and neuronal loss. To explore whether AD molecular pathological phenotypes appeared in the early stage of AD gene-transferred tree shrews, we conducted AD molecular pathological analysis on 18-month-old transgenic positive tree shrews. The results showed that compared with normal control tree shrews, the level of Aβ in the brains of transgenic tree shrews increased ( Figure 9 ), the level of phosphorylated Tau increased ( Figure 10 ), and gliocyte activation occurred ( Figure 11)。These features are consistent with the early pathological state of AD. The lifespan of the tree shrews we raised is generally 8 - 10 years. The 18 - month - old AD transgenic tree shrews established and detected in this invention are in the prime of life. Some pathological features of late - stage AD, such as Aβ plaques, neurofibrillary tangles (NFT), and severe brain atrophy, need to be continuously tracked and observed. The AD transgenic tree shrews established in this invention have successfully integrated the exogenous APP and PSEN1 genes with pathogenic mutations into the tree shrew genome and can correctly express the exogenous genes, and the animals show AD - like behaviors and pathological phenotypes. This invention provides a new animal model for the mechanism research and drug development of AD.

[0062] Appendix 1. Primers used for DNA identification and gene expression identification in this invention

[0063]

Claims

1. A method for establishing an APP / PSEN1 double transgenic Alzheimer's disease tree shrew model, characterized in that: Using lentivirus to carry pathogenic genes APP and PSEN1 infecting the spermatogonial stem cells of tree shrews to obtain AD gene - transfected spermatogonial stem cells of tree shrews, injecting the AD gene - transfected spermatogonial stem cells of tree shrews into the testes of male tree shrews treated with busulfan, then mating the recipient male tree shrews with wild - type female tree shrews, and identifying the obtained offspring AD transgenic tree shrews to obtain over - expressed mutant APP and PSEN1 double - transgenic Alzheimer's disease tree shrew model; The acquisition of the AD gene - transfected tree shrew spermatogonial stem cells includes the following steps: S1 Lentivirus preparation: Insert the most important pathogenic gene of familial Alzheimer's disease containing pathogenic variants APP and PSEN1 through the XbaI and NotI restriction enzyme sites, insert the APP K670N / M671L CDS region behind the EF1α promoter, and insert the PSEN1 M139V / M146L / H163R CDS region behind the CMV promoter to establish APP and PSEN1 lentiviral vectors of the genes. Co-transfect the constructed lentiviral vectors and packaging plasmids psPAX and pMD2.G into HEK293T cells, collect the viral supernatants at 48 h and 72 h respectively, and obtain lentiviruses by ultracentrifugation and concentration. S2 virus-infected transgenic AD gene tree shrew spermatogonial stem cells. The prepared virus solution was used to infect the spermatogonial stem cells of tree shrews. 24 hours after virus infection, it was observed under a fluorescence microscope that the spermatogonial stem cells successfully integrated APP K670N / M671L genes expressed red fluorescent protein, and the successfully integrated Combined with PSEN1 M139V / M146L / H163R genes expressed green fluorescent protein.

2. The method for establishing an APP / PSEN1 double transgenic Alzheimer's disease tree shrew model according to claim 1, wherein: The male tree shrews described above were obtained by intraperitoneal administration of busulfan at a concentration of 35 mg / kg to remove endogenous spermatogenic cells of the tree shrews.

3. The method for establishing an APP / PSEN1 double transgenic Alzheimer's disease tree shrew model according to claim 1, wherein: The transfected spermatogonial stem cells were resuspended in PBS and then injected into the seminiferous tubules of recipient tree shrews. Two months later, the recipient male tree shrews were mated with wild - type female tree shrews to obtain F1 - generation AD transgenic tree shrews.

4. The method for establishing an APP / PSEN1 double transgenic Alzheimer's disease tree shrew model according to claim 3, wherein: The F1 - generation AD transgenic tree shrews were used as male and female parents for mating to obtain F2 - generation AD transgenic tree shrews. The obtained F2 - generation AD transgenic tree shrews were used as male and female parents for mating to obtain F3 - generation AD transgenic tree shrews. The above - mentioned mating steps were continued 5 - 10 times to obtain Fn - generation AD transgenic tree shrews.

5. The method for establishing an APP / PSEN1 double transgenic Alzheimer's disease tree shrew model according to claim 4, characterized in that: The obtained F1 - to Fn - generation AD transgenic tree shrews were identified, including the molecular, pathological, and behavioral characteristics of AD.

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