Gene overexpression chimeric animal model and engineered monkey model based on hNPY and their applications

Abstract

The present invention discloses a chimeric animal model based on hNPY gene overexpression, an engineered monkey and its application. The chimeric animal model based on gene overexpression is characterized in that the NPY gene is overexpressed in the paraventricular nucleus cell gene of the animal to induce the phenotype, thereby forming a chimeric animal model based on NPY gene overexpression. The chimeric genetic engineered monkey model is characterized in that the hNPY gene is overexpressed in the paraventricular nucleus cell gene of the macaque to induce the phenotype, thereby forming a chimeric genetic engineered monkey model based on hNPY gene overexpression. The model can be used for the study of the pathogenic mechanism of related diseases, the screening, verification, and testing of therapeutic drugs for clinical treatment methods, and related preclinical research. The present invention has the characteristics of short construction cycle of animal models, high efficiency, low cost, high phenotypic penetrance, good synchronization, high repeatability, low difficulty, and convenience for large-scale application.

Description

[0001] This application is a divisional application based on the invention patent application "Method for constructing a gene overexpression chimeric animal model based on hNPY and its application" (application number 2019111075856) submitted by the applicant to the State Intellectual Property Office of China on November 13, 2019. Technical Field

[0002] The present invention belongs to the field of biotechnology and relates to the technology of constructing animal models, in particular to an animal model realized in somatic cells using gene technology and its application. Background Art

[0003] Animal disease models (animal models) are important tools for experimental physiology, experimental pathology and experimental therapeutics (including new drug screening). The development of human diseases is very complex. Using humans as experimental subjects to explore the mechanism of disease and promote the development of medicine is slow. The experience accumulated in clinical practice is not only limited in time and space, but many experiments are also limited in morality and methods. With the help of indirect research on animal models, factors that are impossible or difficult to eliminate under natural conditions can be consciously changed, so as to more accurately observe the experimental results of the model and conduct comparative studies with human diseases, which will help to more conveniently and effectively understand the occurrence and development of human diseases and study prevention and control measures.

[0004] Animal models of human disease refer to animals that have simulated manifestations of human diseases established in various medical and scientific research. In the process of completing the present invention, the inventors found that the current methods for establishing animal models mainly adopt:

[0005] Drug-induced construction of corresponding animal models, such as the establishment of hyperuricemia animal models and applications in Chinese patent application 2014100899525, discloses a method for establishing a non-human primate hyperuricemia model, comprising the following steps: selecting healthy adult rhesus monkeys with normal blood uric acid levels: uric acid levels of 1.54±1.05umol / L as model animals; selecting a pharmaceutical composition including ethambutol and uric acid as a modeling drug; gavage administration, the specific dosage is ethambutol: 10-50mg / kg and uric acid: 20-50mg / kg, free food and water after modeling, once a day, continuous administration for more than 3 days, to obtain a non-human primate hyperuricemia model. The inventor found in the process of completing the present invention that the method is forced to infuse a large amount of drugs into the animal used to establish the model, resulting in the formation of an animal model due to the drugs in the body, resulting in the application scope of the later animal model being limited, and even unable to be widely used.

[0006] Also, using genetic engineering to construct corresponding animal models, such as U.S. patent application US10 / 179639 discloses a method for developing an animal model to establish multiple mutant animal species with the same genotype, phenotype, performance type and genetic background, which includes the following steps: inducing superovulation in sexually immature mutant starting animals (G0); fertilizing the superovulated sexually immature mutant starting animals; obtaining the first generation of mutant animals (F1) by completing the pregnancy process; confirming the stability of the mutation, the identity of the genotype, phenotype, performance type, and genetic background in the first generation of mutant animals; and if necessary, using mutant animals Repeat the above steps for at least 20 generations of one or more generations of offspring, wherein the genetic and environmental factors of all animals are monitored and strictly guaranteed to be the same in each step, wherein the stability of the mutation and the identity of the genotype, phenotype, performance type, and genetic background in each generation are subject to planned genetic monitoring and spot checks of environmental factors; and, wherein the mutant is fertilized only when it is confirmed by planned genetic monitoring and spot checks that the mutation is stable in each generation of mutants, and the genotype, phenotype, performance type, and genetic background of the mutant are the same as the genotype, phenotype, performance type, and genetic background of the starting animal of the mutation. The inventors found in the process of completing the present invention that the technical solution has a long cycle, high cost, narrow adaptability, and a relatively low success rate, and is not convenient for large-scale application.

[0007] Also, animal models constructed by specific feeding reinforcement, such as Chinese patent application 201610793182.1 discloses a method for constructing an animal model of alcoholic liver disease, including the following steps: selecting male 8-10kg crab-eating macaques as research subjects, in addition to normal feeding of basic feed, allowing them to drink drinking water containing aspartame on their own during the first week of adaptation, and from the second week, increasing 10% of alcohol in the drinking water every week for free drinking, and by the end of the fourth week, selecting crab-eating macaques that drink more than 50mL of 30% alcohol per day as experimental animals; from the fifth week, replacing aspartame in the alcohol solution with 10% sucrose, and adding an additional 20% of pig fat to the basic feed in addition to continuing to feed 30% alcohol; alcoholic fatty liver Model establishment: under the condition of feeding with high sugar and high fat feed and free drinking of 30% alcohol, at the 12th week of feeding①, liver biopsy was performed by B-ultrasound induction, liver tissue was stained with HE, and cynomolgus monkeys showed obvious fatty liver symptoms; Alcoholic hepatitis and liver fibrosis model establishment: on the basis of the above, high sugar and high fat feed were continued to be used, 30% alcohol was freely drunk, and carbon tetrachloride solution was injected intraperitoneally twice a week, each injection volume was 0.25mL / kg; at the 16th week, liver biopsy was performed by B-ultrasound induction, liver tissue was stained with HE, and cynomolgus monkeys showed obvious hepatitis symptoms of liver cell membrane rupture and infiltration; at the 24th week, liver biopsy was performed by B-ultrasound induction, liver tissue was stained with HE, and cynomolgus monkeys showed obvious liver fibrosis symptoms. The inventor found in the process of completing the present invention that the method has a long cycle for constructing animal models, is greatly affected by individual animals and the environment, has poor trait stability, and is not conducive to large-scale application. Summary of the invention

[0008] In view of the above-mentioned deficiencies, the technical problem to be solved by the present invention is to overcome the above-mentioned defects and provide a gene overexpression chimeric animal model and an engineered monkey model based on hNPY, so as to achieve the purpose of efficiently constructing the corresponding animal models; and also includes related applications of the models.

[0009] In the chimeric genetically engineered monkey model, the hNPY gene is overexpressed in the paraventricular nucleus cell gene of the macaque to induce the occurrence of phenotypes, thereby forming a chimeric genetically engineered monkey model based on the overexpression of the hNPY gene.

[0010] Preferably, the hNPY gene overexpression is achieved by the following steps:

[0011] Construct a flag-tagged hNPY lentiviral expression vector driven by Synapsin promoter;

[0012] injecting the vector into the paraventricular nucleus of macaques;

[0013] The hNPY is transcribed into the paraventricular nucleus cell gene, achieving overexpression of the hNPY gene, inducing phenotype occurrence, and forming a chimeric genetic engineering monkey model based on hNPY gene overexpression.

[0014] Preferably, the vector is Lenti-hsyn-hNPY-2A-mcherry.

[0015] Preferably, the sequence of the vector is the SEQ ID NO.1 sequence in the sequence list.

[0016] Preferably, the injection is performed by microinjection to inject the vector into the paraventricular nucleus of the macaque.

[0017] Preferably, the microinjection is performed by microinjection assisted by nuclear magnetic resonance to inject the vector into the paraventricular nucleus of the macaque.

[0018] A gene overexpression chimeric animal model, in which the NPY gene is overexpressed in the paraventricular nucleus cell genes of the animal to induce phenotype production, thereby forming a gene overexpression chimeric animal model based on NPY.

[0019] Gene overexpression chimeric animal model, the overexpression of the NPY gene is achieved by the following steps,

[0020] Vector construction: constructing a lentiviral expression vector with Synapsin as the promoter driving NPY;

[0021] vector introduction, injecting the vector into the paraventricular nucleus of the animal;

[0022] Gene overexpression transcription, the NPY is transcribed into the paraventricular nucleus cell gene to achieve overexpression of the NPY gene;

[0023] A chimeric animal model is formed, wherein the overexpression of the gene induces the production of a phenotype, thereby forming a chimeric animal model based on NPY gene overexpression.

[0024] Preferably, the vector is Lenti-hsyn-hNPY-2A-mcherry.

[0025] Preferably, the sequence of the vector Lenti-hsyn-hNPY-2A-mcherry is the SEQ ID NO.1 sequence in the sequence listing.

[0026] Preferably, the injection is performed by microinjection to inject the vector into the paraventricular nucleus of the animal.

[0027] In the paraventricular nucleus cell genes of the engineered monkey, the hNPY gene is overexpressed to induce the occurrence of phenotypes, thereby forming a chimeric genetically engineered monkey model based on the overexpression of the hNPY gene.

[0028] The model or engineered monkey is used for studying the pathogenic mechanism of obesity / neurological diseases.

[0029] The model or engineered monkey is used for studying the pathogenesis of diabetes.

[0030] The model or engineered monkey is used for the research of clinical treatment methods and drugs for obesity / neurological diseases.

[0031] The model or engineered monkey is used for the research of clinical treatment methods and drugs for diseases such as diabetes.

[0032] The model or engineered monkey is used for the screening, verification, testing and preclinical related research and / or experiments of drugs for treating obesity / neurological diseases.

[0033] Models or engineered monkeys are used for the validation, testing and preclinical research and / or experiments of diabetes therapeutic drugs.

[0034] Models or engineered monkeys as described above for studying the pathogenic mechanisms of obesity / neurological diseases.

[0035] Models or engineered monkeys as described above for studying the pathogenesis of diabetes.

[0036] Models or engineered monkeys as described above for use in clinical treatments and drug research on obesity / neurological diseases.

[0037] Model constructs or engineered monkeys as described above are used for clinical treatment methods and drug research for diseases such as diabetes.

[0038] Models or engineered monkeys as described above for use in the screening, validation, testing and preclinical research and / or experiments of drugs for treating obesity / neurological diseases.

[0039] Models or engineered monkeys as described above for use in validation, testing and preclinical research and / or experiments of diabetes therapeutic drugs.

[0040] The hNPY gene was overexpressed in the paraventricular nucleus cells of macaques for use in constructing a chimeric genetically engineered monkey model.

[0041] The NPY gene is overexpressed in the paraventricular nucleus cells of animals for use in constructing a gene overexpression chimeric animal model.

[0042] The present invention has the characteristics of short animal model construction cycle, high efficiency, low cost, high phenotypic penetrance, good synchronization, high repeatability, low difficulty, and easy large-scale application. The constructed animal model has stable traits, especially for tissues and organs without cell regeneration function, the construction of chimeric gene animal models is more stable. Since the chimeric animal model does not have genetic characteristics, it has low biological risks, high safety, and strong controllability. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] In order to more clearly describe the relevant technical solutions involved in the present invention, the accompanying drawings involved are briefly described below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative work.

[0044] Figure 1 Flowchart of methods for constructing chimeric animal models;

[0045] Figure 2 Flow chart of the vector injection process;

[0046] Figure 3 Schematic diagram of vector structure and vector introduction, where (a) is a schematic diagram of the constructed Lenti-hNPY lentiviral expression vector structure (only the specific part is shown in the figure), (b) is a schematic diagram of the Lenti control vector structure without hNPY, and (c) is a schematic diagram of the virus injection into the bilateral paraventricular nuclei of macaques scanned by nuclear magnetic resonance imaging;

[0047] Figure 4 Schematic diagram of the vector injection site;

[0048] Figure 5 This is the first fluorescent image of the paraventricular nucleus of the macaque after virus injection (the background is the cell nucleus marker Hoechst staining);

[0049] Figure 6 This is the second fluorescent image of the paraventricular nucleus of the macaque after virus injection (the background is the cell nucleus marker Hoechst staining);

[0050] Figure 7 This is a fluorescent picture of the virus expression in the mouse brain;

[0051] Figure 8 This is an MRI scan image of an experimental macaque;

[0052] Fig. 9 This is a curve diagram showing the changes in weight gain in experimental macaques caused by overexpression of hNPY in the paraventricular nucleus of macaques;

[0053] Fig.10The effects of virus injection on feeding behavior, food intake and serum insulin in macaques, where ad is the change curve 7 months after virus injection;

[0054] Fig.11 The effect of overexpression of human NPY in the paraventricular nucleus of macaques on the body temperature and other metabolic indicators of experimental macaques;

[0055] Fig.12 Shown are the changes in NPY levels, AgRP and leptin in the blood of macaques after virus injection.

[0056] Explanation of the accompanying drawings: In the figure, brain 1, paraventricular nucleus 2, injection site 3, syringe 4, vector injection needle point 31, auxiliary injection needle point 32. DETAILED DESCRIPTION

[0057] In order to facilitate further understanding of the present invention by those skilled in the art and to clearly recognize the technical solutions described in the present application, the relevant technical contents of the present invention are fully and comprehensively disclosed, and the specific implementation modes of the present invention are described in detail below in conjunction with the accompanying drawings. Obviously, the described specific implementation modes only give a part of the embodiments of the present invention in an enumerated manner to help understand the present invention and its core ideas.

[0058] Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative work, and / or without departing from the spirit and essence of the present invention, even if the order of each step is changed, and various corresponding changes and deformations are made according to the present invention, these corresponding changes and deformations should all fall within the scope of protection of the present invention.

[0059] The definitions or supplementary explanations of the relevant terms involved in the present invention are as follows:

[0060] NPY: Neuropeptide Y, the full name of which is neuropeptide tyrosine, in English Neuropeptide Y, abbreviated as NPY. NPY is a polypeptide composed of 36 amino acid residues, belonging to the pancreatic polypeptide family, widely distributed in the central and peripheral nervous systems of mammals, and is one of the most abundant neuropeptides. It produces different effects in the body by binding to different receptors. NPY is a tyrosine synthesized based on the "NPY gene", but in this application, in order to simplify the description, NPY is used to represent the gene name of the "NPY gene" in different special cases.

[0061] hNPY: human NPY, the full name is human neuropeptide tyrosine (also known as "human neuropeptide Y"), English human Neuropeptide Y, abbreviated as hNPY. In this application document, unless otherwise specified, the NPY is the abbreviation of hNPY. Of course, other NPYs can be used to implement the technical scheme of this experiment, and based on the corresponding biological characteristics, the same or similar effects can be achieved. However, in the present invention, as a preferred scheme, in order to obtain an anthropomorphic animal model related to clinical (human) conditions, human NPY was selected; of course, the required NPY can also be selected based on other needs. hNPY is tyrosine synthesized based on the "hNPY gene", but in this application, in order to simplify the description, hNPY is used to represent the gene name of the "hNPY gene" in different special cases.

[0062] AgRP: AgRP is the abbreviation of guinea pig related protein (also known as "Agouti-related protein"). AgRP is a protein synthesized based on the "AgRP gene", but in this application, in order to simplify the description, AgRP is used to represent the gene name of the "AgRP gene" in different special cases.

[0063] hAgRP: human AgRP, the full name of which is human guinea pig-related protein (also known as "human Agouti-related protein"), in English human Agouti-related protein, abbreviated as hAgRP. In this application document, unless otherwise specified, the AgRP is the abbreviation of hAgRP. Similar to the choice of hNPY in the present invention, other AgRPs can of course be used to implement the technical scheme of this experiment to achieve the same or similar effects. However, in the present invention, as a preferred solution, in order to obtain an anthropomorphic animal model related to clinical (human) conditions, human AgRP was selected; of course, the required AgRP can also be selected based on other needs. hAgRP is a protein synthesized based on the "hAgRP gene", but in this application, in order to simplify the description, hAgRP is used to represent the gene name of the "hAgRP gene" in different special cases.

[0064] PVN: full name Paraventricular nucleus of the hypothalamus, abbreviated as PVN, also known as PVH, but PVN is more commonly used. In this application, the abbreviation PVN is used.

[0065] Hemagglutinin, abbreviated as HA.

[0066] FLAG-tag, that is, FLAG label, FLAG corresponds to an 8-amino acid peptide segment DYKDDDDK (peptidesequence DYKDDDDK), which is used to mark the target protein. In the study of protein expression and localization, the target gene to be studied and the FLAG-tag gene sequence can be connected by genetic engineering technology, which can be connected to the C-terminus or N-terminus of the target protein, and then the integrated gene is transferred into cells or embryonic stem cells or fertilized eggs. Subsequent detection is mainly achieved through the specific binding of the immune determinant cluster formed by the FLAG-tag peptide chain and its monoclonal antibody. The detection methods include immunofluorescence, Western Blotting, etc.

[0067] Amino acids, the basic units of proteins, are a class of compounds that have both amino and carboxyl groups in their molecules.

[0068] Tyrosine (Tyr), chemically known as 2-amino-3-p-hydroxyphenylpropionic acid, is an aromatic polar α-amino acid containing a phenolic hydroxyl group. It is a conditionally essential amino acid and a ketogenic and glucogenic amino acid for the human body.

[0069] Genes (genetic factors) are DNA fragments that carry genetic information. Other DNA sequences, some of which play a role directly through their own structure, and some of which participate in regulating the expression of genetic information. Genes are all the nucleotide sequences required to produce a polypeptide chain or functional RNA. Genes support the basic structure and performance of life. Genes have materiality (existence mode) and information (fundamental attributes).

[0070] DNA (deoxyribonucleic acid) is an organic compound with a complex molecular structure. It exists in the cell nucleus as a component of chromosomes. Its function is to store genetic information. DNA molecules are huge and are composed of nucleotides.

[0071] Chromosome (chromosome), chromosome is the substance in the cell nucleus that carries genetic information. It is cylindrical or rod-shaped under a microscope and is mainly composed of DNA and protein. It is the carrier of genetic information.

[0072] Protein is a type of nitrogen-containing macromolecular organic compound that is connected by peptide bonds and has amino acids as basic units. It is an organic macromolecule, the basic organic matter that constitutes cells, and the main bearer of life activities. It is generally believed that protein is the executor of gene function.

[0073] Guide RNA, also known as guide RNA (gRNA), is also called small guide RNA (sgRNA). It is a post-transcriptional modification RNA called RNA editing in kinetoplastids. It is also a small non-coding RNA. It can pair with pre-mRNA and insert some uracil (U) into it to produce mRNA with function. The RNA molecule edited by guide RNA is about 60 to 80 nucleotides in length. It is transcribed from a separate gene and has a 3' oligo U tail. There is a sequence in the middle that is precisely complementary to the edited mRNA, and the 5' end is an anchor sequence that is complementary to the non-edited mRNA sequence.

[0074] Animal models (i.e. animal disease models) are mainly used in experimental physiology, experimental pathology and experimental therapeutics (including new drug screening) research. The development of human diseases is very complex. Using humans as experimental subjects to deeply explore the mechanism of disease occurrence and promote the development of medicine is slow. The accumulated clinical experience is not only limited in time and space, but many experiments are also limited in morality and methods. With the help of indirect research on animal models, factors that are impossible or difficult to eliminate under natural conditions can be consciously changed, so as to more accurately observe the experimental results of the model and conduct comparative studies with human diseases, which will help to more conveniently and effectively understand the occurrence and development of human diseases and study prevention and control measures.

[0075] Animal models of human disease refer to animals that have simulated manifestations of human diseases established in various medical and scientific research.

[0076] Chimeric animal model (i.e., chimeric gene animal model, also referred to as gene chimeric animal model, the former is used uniformly in this application), as a non-germline animal model, is a model that purposefully modifies the genes (genes in somatic cells) of local tissues or organs of animals to induce phenotypes and form relevant pathological models (animal models). Chimeric animal models are a continuation and supplement of stable genetic animal models. The genetic modification technology used in this application is gene overexpression, which is one of the means of transgenic technology. Of course, other genetic modification technologies can also be used to achieve this. In addition, the present invention discloses a non-human primate chimeric gene animal model based on gene overexpression, in particular a chimeric genetically engineered monkey based on gene overexpression.

[0077] Animal behavior refers to the actions and activities of animals, including their movement, limb movements, eating, courtship, fighting, and color changes. It is the sum of all reactions to meet the basic needs of animal survival and reproduction and to adapt to changes in the internal and external environment. All animal behaviors are basically determined by three factors: external and internal stimuli, the functional state of the animal's sensory organs and nervous system, and the structure and physiological characteristics of the animal's body.

[0078] Animal behavior models are animal models established based on animal behavior, including various external and internal stimuli (such as physical stimulation, chemical stimulation, biological stimulation, etc.), interventions, etc., so that the behavior of the animal develops according to the needs of the model builder, and based on the changes in the behavior, the physiology of the animal is affected, thereby constructing an animal disease model that meets the requirements of the model builder. The present invention discloses the construction of a specific animal behavior model based on the corresponding specific target gene. Based on the same principle, those skilled in the art can fully select other suitable target genes as needed, and adopt the technical solution disclosed in the present invention to achieve the establishment of their animal behavior model.

[0079] Transcription is the process of genetic information flowing from DNA to RNA. It is the process of synthesizing RNA under the catalysis of RNA polymerase, using a certain strand of double-stranded DNA (the template strand is used for transcription, and the coding strand is not used for transcription) as a template and four nucleoside triphosphates, ATP, CTP, GTP, and UTP, as raw materials. As the first step in protein biosynthesis, during transcription, a gene will be read and copied into mRNA, that is, a specific DNA fragment is used as a template for genetic information, and the DNA-dependent RNA polymerase is used as a catalyst to synthesize the precursor mRNA according to the principle of base complementarity. RNA polymerase forms a dynamic complex with a series of components to complete the processes of transcription initiation, extension, and termination. The generated mRNA carries the codons, which can achieve protein synthesis after entering the ribosome. Transcription uses only one strand of DNA as a template. The single strand selected as the template is called the template strand, also known as the nonsense strand; the other single strand is called the non-template strand, that is, the coding strand. Because the coding strand is consistent with the RNA sequence generated by transcription, it is also called the sense strand. The transcription region on DNA is called a transcription unit.

[0080] Gene overexpression: Gene overexpression is the use of transgenic technology to make the expression of a specific gene exceed the original level. The basic principle of gene overexpression is to obtain a target gene plasmid containing upstream regulatory elements by artificial construction, and transduce the above plasmid into cells or tissues and organs through transgenic operations to achieve large-scale transcription and translation of the target gene, thereby achieving overexpression of the gene product. Gene overexpression does not involve gene editing. It is to randomly insert the gene (target gene) carried by the vector into the chromosome, independently of the original gene sequence, and transcribe the above-mentioned target gene fragment alone, so that the expression of the target gene in the cell exceeds the normal level, but this fragment is free from the original DNA. For example, the target gene hNPY gene in this application is introduced into macaques in large quantities, so that this part of the target gene is transcribed beyond its original level, thereby forming a corresponding pathological model.

[0081] The present invention effectively constructs a corresponding animal model through the above technical solution, as well as a method for constructing the animal model, and related applications based on the animal model and method. Since overeating is the main cause of all factors causing obesity, the inventors found in their research that the hypothalamus is the main area in the brain that controls food intake. The hypothalamus is involved in sensing nutrition and energy levels, regulating food intake and energy consumption, and thus maintaining normal metabolic balance. Multiple neural nuclei in the hypothalamus, such as the arcuate nucleus, paraventricular nucleus, ventromedial nucleus, and lateral hypothalamic area, participate in regulating energy metabolic balance. The neurons in these neural nuclei regulate food intake through different chemical signal molecules, such as transmitters, cytokines, neuropeptides, etc. However, there are many difficulties and obstacles in how to establish animal models with stable traits in batches using controllable and effective technical solutions, which also leads to the inability to use simple solutions to achieve it in existing research and exploration. After repeated theoretical analysis and experiments, the inventors of the present invention found that since NPY in the hypothalamus mainly comes from neurons in the arcuate nucleus, the axons of NPY neurons directly project to the paraventricular nucleus, and the paraventricular nucleus is the main target area of ​​NPY neurons, the neural signal pathway from the arcuate nucleus to the paraventricular nucleus plays an important role in regulating feeding behavior. Based on the above findings, as well as related research and experiments, an overexpression of NPY in the hypothalamus was constructed by (transgenic) technology, a corresponding animal model was constructed, especially a chimeric gene animal model, and a non-human primate chimeric gene animal model was constructed based on non-human primates-macaques, especially a chimeric gene engineering monkey model; based on the technical solution, it can also be used to construct an animal behavior model.

[0082] In terms of the specific implementation of the technical solution, in the present invention, as a preferred technical solution, the hNPY lentivirus (Lenti-hNPY) containing specific expression is accurately injected into the bilateral paraventricular nuclei of young experimental macaques using nuclear magnetic resonance scanning technology. The experimental results showed that the weight and feeding behavior of hNPY overexpressing macaques increased significantly (changing their eating behavior, promoting increased food intake, and thus leading to weight gain). Obviously, the non-genetic chimeric genetically engineered macaque model of the present invention can be used as a clinical translational obesity model for obesity research and exploration of new clinical treatment strategies for obesity; of course, it can also be further converted into a diabetes model.

[0083] In addition, as one of the inventive points of the present invention, the present invention provides a chimeric gene animal model and optimizes the gene sequence of the vector used, especially the target gene hNPY used (see the Lenti-hNPY sequence table for details) to improve the construction effect of the chimeric gene animal model, especially for the construction of the chimeric gene engineering monkey model.

[0084] Some examples of specific implementation and application of the present invention are given below by way of example:

[0085] The inventors of the present invention found in the process of realizing the present invention that in order to solve the problem of affecting the health of the general public of men, women, old and young due to the gradual increase in the incidence of obesity, it is necessary to continuously develop new obesity treatment methods, and study related pathogenic mechanisms, etc., and it is necessary to use suitable animal models for preclinical research. At present, a large number of animal models of dogs, pigs, mice and rats have been used to study obesity and health problems related to obesity. In order to construct corresponding animal models, there are animal models and genetic engineering models caused by feeding in the prior art. Rodent model research has greatly promoted the exploration of the causes of human obesity. However, the findings of rodent experiments often fail in the process of clinical transformation. How to achieve a more effective preclinical animal research model is a technical problem that needs to be solved at present. Because of the similarity with humans in genetics and physiology, the non-human primate model will be a better model for valuable translational medicine and will become a bridge linking rodent experiments with human clinics. The present invention provides an animal model constructed by overexpression of human NPY / AgRP in the paraventricular nucleus mediated by lentivirus, specifically, a macaque animal model constructed by overexpression in the macaque paraventricular nucleus. The animal model and macaque can be used for related pathological research, new drug screening, etc. Specifically, the present invention is implemented through the following embodiments.

[0086] like Figure 1As shown, according to the needs of the animal model construction goal, animal selection and vector construction are carried out, the vector is introduced into the animal, the animal is cared for, and the target gene in the vector is transcribed in the animal body (cell) to achieve gene overexpression, so that the animal forms the traits that meet the animal model construction goals, and the animal model traits are tested, and the animal that meets the construction template becomes the corresponding animal model. The specific instructions are as follows:

[0087] In the present application, in order to construct a chimeric genetically engineered monkey model for regulating feeding behavior, macaques were used as the subjects, hNPY was selected as the target gene, and the paraventricular nucleus of the macaque was subjected to corresponding genetic modification through transgenic technology - gene overexpression to achieve the purpose of the invention.

[0088] 1. Animal Selection

[0089] Using macaques, of course, other types of animals can be selected as needed. In the present invention, in order to achieve similarity with humans in genetics and physiology, selecting a non-human primate model will be a better model for valuable translational medicine and better simulate the relevant pathological model of humans. In the present invention, it is considered that spontaneous obesity in monkeys is gradually generated and developed in adulthood. In order to explore whether genetic engineering of young macaques will lead to obesity in experimental animals, the inventors of the present invention selected male macaques about 1 and a half years old for experiment (macaques of other age groups can also be selected, preferably 1-2 years old macaques). In order to facilitate experimental control, a 1-year-old male macaque was selected during the experimental process of the present invention. Of course, the present invention can also be implemented by selecting macaques or primates of other primates or other age groups. And based on the technical ideas of the present invention, different selection criteria and conditions can be adopted according to different applications.

[0090] In the verification experiment of the present invention, 24 1-year-old male macaques were used as experimental subjects, of which 8 were injected with control virus (as a control group), 8 were injected with NPY gene overexpression virus Lenti-hNPY (for constructing a chimeric animal model), and 8 were injected with AgRP gene overexpression virus Lenti-hAgRP (for constructing a chimeric animal model). This application only discusses the experimental subjects of the corresponding type of gene overexpression virus, and all relevant information is given here to further ensure the integrity and objectivity of the experimental process of the present invention. The subsequent operating conditions and steps (including the dosage of various required instruments, drugs, reagents, etc. are also determined based on the type and quantity of the aforementioned experimental subjects, which can be adaptively adjusted and changed according to different subjects) are all explained on this basis. However, the selection of the experimental subjects does not represent a limitation to the technical solution of the present invention, especially not a limitation to the present invention and the scope of application and protection of the present invention, but is only convenient for scientific and objective comparison and experiment to illustrate the scientificity, feasibility, practicality, and corresponding technical effects of the present invention.

[0091] 2. Vector construction (virus preparation).

[0092] As a preferred solution in a specific application, the gene overexpression is determined to use Lenti virus as the basis for constructing a vector (of course, other corresponding viruses can also be selected as the basis for constructing a vector according to the needs of the application). At the same time, since the organ targeted by the invention of the present invention is the brain, a subtype virus with stronger infectivity to brain neurons is used. Figure 3 As shown, in the experiments to verify the present invention, the names of the viral vectors used are as follows:

[0093] Lenti virus vector overexpressing the target gene: Lenti-hsyn-NPY-2A-mcherry (abbreviated as Lenti-hNPY) (such as Figure 3 (a)); Lenti-hsyn-AgRP-2A-mcherry (Lenti-hAgRP for short); viral vector sequences are listed in the corresponding sequence table attached to this application. Unless otherwise specified, the vectors or viruses described in this application are all Lenti virus vectors that overexpress the target gene.

[0094] Control viral vector: Lenti-hSyn-mCherry (eg Figure 3 (b) shows), using the same lentivirus as the above-mentioned Lenti-hNPY and Lenti-hAgRP, the difference being that the target genes hNPY and hAgRP are not included. In practical applications, the control viral vector has no practical significance. In this application, the injection of the vector is intended to be compared with the macaques injected with the aforementioned viral vectors Lenti-hNPY and Lenti-hAgRP to verify the actual technical effect of the present invention.

[0095] Description of the functions of the elements in the viral vector:

[0096] Lenti is a name prefix used to distinguish virus types, such as Lenti (i.e., Lenti virus); hsyn is the name of the promoter that drives the expression of different genes; NPY, AgRP, etc. are the names of target genes or sgRNAs; 2A is the connection sequence between two genes; mcherry is a fluorescent protein gene that forms a fluorescent detection marker to indicate the position of virus expression in vivo; flag, HA is usually fused with the target gene and is a tag sequence used to detect the target gene. It should be noted that due to writing habits, in this application, for each element of the vector, and except for the measurement unit (international unit), unless otherwise specified, uppercase and lowercase letters have the same meaning.

[0097] Lentivirus (generally referred to as Lenti) is a widely used transgenic vector that can effectively deliver exogenous genetic material to both dividing and non-dividing mammalian cells and eventually integrate into the cell genome. The present invention uses Lentivirus as a vector to deliver the target gene to the somatic cell gene that needs to be modified. Figure 3 (a) The structure of the Lenti-NPY lentiviral expression vector of the corresponding vector is schematically shown (only the relevant elements of the specific part are shown in the figure, and other elements are not drawn. The complete structure is in a closed-circular plasmid.)

[0098] Furthermore, in order to verify safety and effectiveness, the virus should be tested in vivo in mice before use. The specific test method is a well-known technical solution. Figure 7 The fluorescent images of the above virus (Lenti-hNPY virus vector) expressed in the mouse brain are given. The images show that the human NPY lentivirus is highly expressed in the mouse brain, and the virus can achieve the purpose of genetic modification and is safe.

[0099] like Figure 3 As shown in (a), the present invention first constructs Synapsin (Chinese name: neuronal synaptophysin, abbreviated as Syn) as a promoter-driven lentiviral expression vector of flag-tagged hNPY and HA-tagged hAgRP (FLAG and HA are two commonly used tags. In order to distinguish the two target proteins NPY and AgRP in future experiments, different tags are used for hNPY and hAgRP in the experiments of the present invention to facilitate the distinction of the overexpression of hNPY and hAgRP. In specific applications, other tags can be selected, or the two can be exchanged, that is, the flag and HA can be selected as other tags as needed. Of course, the relevant tags may not be required in the application). In order to detect the expression efficiency of the vector, the two lentiviral vectors were injected into the mouse brain to detect the expression efficiency (this step is not a necessary step and can be omitted in specific applications). Three weeks after microinjection, the inventors of the present invention obtained frozen sections of the mouse brain and detected the red fluorescence signal of mCherry co-expressed with NPY or AgRP. The results showed that both lentiviruses could produce a large amount of red fluorescence signals in the dentate gyrus region of the mouse hippocampus (such as Figure 7 As shown in Figure 2, 4-6 weeks after injection into the mouse hypothalamus, the Lenti-hNPY virus showed high levels of hNPY expression. Obviously, these two lentiviruses can be used for the next experimental operation of injection into the macaque brain. Preferably, the present invention uses the hSynapsin (humanSynapsin) promoter, whose Chinese name is human neuronal synaptic protein promoter, abbreviated as hSYN (both uppercase and lowercase). Specifically, in Figure 3In the characteristic partial structure of the vector shown in (a) and (b), LTR (abbreviation of Long Terminal Repeat, which is the long terminal repeat sequence present on both sides of the LTR retrotransposon (LTRs) and is part of the plasmid) is a fixed sequence in the viral vector; hSynapsin is the promoter; hNPY is the target gene to be overexpressed; 2a is the linker used to co-express mcherry; mcherry is a red fluorescent protein used to indicate viral expression; WPRE is a transcriptional enhancer element. Figure 3 (a) and (b) are vector structures expressed in a linear plasmid format, and the closed-circular plasmid connects the LTRs at both ends of the linear plasmid.

[0100] The expression of each gene is controlled by its own related regulatory elements. Common regulatory elements include: 1. Promoter. The promoter in the present invention is hSynapsin (i.e. human Synapsin, abbreviated as hSyn); 2. Enhancer (not shown in the figure).

[0101] In order to ensure that the virus can be accurately microinjected into the paraventricular nucleus, the inventors of the present invention used nuclear magnetic resonance technology to determine the location of the paraventricular nucleus of each macaque before virus injection (e.g. Figure 8 Three lentiviruses expressing hNPY or hAgRP or control red mCherry fluorescent protein were injected into the paraventricular nuclei on both sides (as shown). Figure 3 (c) Figure 4 as shown).

[0102] By analyzing the brain slices of a euthanized macaque, the inventors of the present invention found that the expression of AgRP lentivirus can be detected in the paraventricular nucleus region, and the expression efficiency is high (e.g. Figure 5 , 6 as shown). Figure 5 The fluorescence image of the macaque paraventricular nucleus after virus injection is shown (the background is the cell nucleus marker Hoechst staining). The left side of the picture is a schematic diagram of the macaque brain structure, and the four pictures on the right are sequential slice fluorescence images of the macaque PVN. Due to experimental reasons (to reduce experimental costs and manage surviving macaques, only slices of Lenti-hAgRP vectors were made to detect the status of the experiment), in fact, due to the use of the same lentivirus, the Lenti-hNPY vector also has the same expression.

[0103] In the carrier reagent comprising the above-mentioned Lenti-hAgRP carrier and Lenti-hNPY carrier, the carrier particle concentration of the Lenti-hAgRP carrier or Lenti-hNPY carrier is 10 9 Pieces / µl.

[0104] The vector and the vector reagent containing the vector can be prepared according to conventional standard procedures, and the introduced vector is injected into the paraventricular nucleus 2 of the macaque in the form of a vector reagent. The related reagents used can also be prepared by conventionally known reagents.

[0105] 3. Vector introduction

[0106] The vector is introduced into the body of a macaque, especially into an appropriate position of the brain. The introduction can be achieved by existing technical means such as direct injection, microinjection, and interventional introduction, or by new introduction means that will appear in the future. In the present invention, the following precise positioning introduction scheme is preferably adopted, specifically including:

[0107] Required instruments: Magnetic resonance imaging (MRI, such as 3.0T, other specifications can also be used), monkey brain locator, brain locator operating arm, microinjection pump, cranial drill, surgical light, surgical instruments, macaque special transport vehicle (set), etc.

[0108] Reagents, drugs and disinfectants: the vectors obtained above, such as Lenti-hNPY, Lenti-hAgRP overexpression virus, some PBS, physiological saline, some ketamine 0.1g / tube, some midazolam 10mg / tube, some nikethamide 0.375mg / tube, some cod liver oil Φ5mm, physiological saline 0.9% (250ml), some iodine disinfectant 500ml / bottle, and injectable veterinary antibiotics. In addition to the relevant vector viruses, other drugs can be selected as auxiliary drugs according to needs.

[0109] Consumables: surgical gowns, surgical caps, several masks, ten pairs of sterile surgical gloves, 24 leather preparation bags, 24 debridement and suture kits, 24 packs of gelatin hemostatic sponges, 24 No. 7 indwelling needles, and empty needles (1ml×24, 2ml×24, 20ml×24).

[0110] Steps (such as Figure 1 , and combined with Figure 2 shown):

[0111] 1. Anesthesia

[0112] 1) Method: General anesthesia, access: intravenous indwelling needle to establish access, normal saline to seal the tube, anesthetic dose: ketamine 2mg slow push. Then 1.5mg ketamine is used to maintain the dose until the end of the experiment according to the ciliary reflex or tail and limb reflex. (The first dose of ketamine can be increased).

[0113] 2) Remove the hair on the head to expose the entire cranial scalp layer.

[0114] 3) Creation: Take the point 3cm behind the eyebrow arch as the coordinate insertion point, the iodine disinfection range is 3cm to 5cm, make a coronal incision of about 4cm with the point as the center, and peel off the periosteum to fully expose the parietal bone and the point coordinates. Drill a small hole at the suture point of the macaque skull to place a cod liver oil particle as the base point of the MRI scan below (origin: 0,0,0). The purpose of the cod liver oil particle is to use the imaging characteristics of fat on the nuclear magnetic resonance imaging (MRI) that are different from the monkey brain tissue (cod liver oil is white imaging) to facilitate positioning as a reference point. Of course, other materials can also be selected as positioning reference points according to different scenes and scanning equipment, such as metal particles when CT, etc. Figure 8 Some MRI scans of experimental macaques are given. The original scan of experimental macaque number C1406067 is lost. The white bright spot on the top of the head of the experimental macaque in the picture is a mineral oil tablet (cod liver oil granules), which is the part indicated by the white arrow. The white arrow in the picture is for the convenience of display and is considered to be added later. It is used to determine the three-dimensional coordinate parameters of the paraventricular nucleus of the experimental animal.

[0115] 4) Fixation: The macaques were fixed in a magnetic resonance compatible brain localizer using ear bars and tongue plates.

[0116] 5) MRI scanning and target calculation: The macaques wore a locator for magnetic resonance scanning. The 3.0T magnetic resonance head coil was used for scanning to obtain thin-layer axial T1 and T2 images and sagittal T1 imaging. The scanning data were: TE100-120, TR3800-6000; layer thickness 2mm; layer spacing 0; FOV = 280×280mm; a cod liver oil particle was placed in the small hole at the suture point of the macaque skull as a base point to confirm the coordinates of the injection site. Three cod liver oil particles were pasted on the horizontal position of the locator in a right triangle to confirm the horizontal plane during scanning and the scanning baseline. The scanning range was from the base of the skull to the top of the skull, including all magnetic resonance positioning points. The bilateral paraventricular nuclei were determined by referring to the macaque brain magnetic resonance imaging and the reference atlas. The coordinates of the needle insertion point were determined and the target coordinates were measured and calculated, and 6 targets were selected before and after the target, left and right, and 1mm above and below, to complete the accurate positioning of the hypothalamic paraventricular nucleus. After the improvement, the whole process takes about 1-1.5 hours.

[0117] The purpose of this step is to determine the exact injection site for each individual. Due to individual differences, the exact location of the corresponding organs and operation sites of each individual is different. For other objects and other sites that can be intuitively displayed, other measurement methods or direct operation can be used.

[0118] At the same time, the above 1)-5) are intended to facilitate the fixation of macaques, accurate positioning and operation of the injection site, and the technical solution can also be implemented in other ways, and can also be replaced or omitted as needed.

[0119] 6) Opening a hole: Carefully remove the macaque with the locator from the MRI machine, and make sure that the head does not move in the locator. Open a bone window of about 6mm to 8mm with the injection origin as the center to fully expose the meninges. During the opening process, use physiological saline to cool down, and reduce the force of the skull drill when approaching the periosteum to prevent instantaneous penetration of the skull and meninges, causing cortical damage. Finally, use a needle or tweezers to peel off the remaining thin layer of skull to expose the meninges.

[0120] The entire drilling process takes approximately 10-15 minutes per hole.

[0121] 7) Injection reagent: Assemble the robotic arm, microinjection pump and injection needle.

[0122] Take one of the experimental macaques as an example:

[0123] The positioning arm is used to place the injection needle tip exactly at the center of the skull cod liver oil particles. At this time, the X, Y, and Z values ​​obtained on the coordinate meter are the injection origin coordinates.

[0124] The coordinates of the actual injection point are generated by adding and subtracting the distance between the paraventricular nucleus and the origin in terms of front and back and depth obtained by the previous MRI scan from the origin coordinates. The positioning arm is moved and the needle is inserted according to this coordinate. If there is an obvious blood vessel directly below the needle insertion point, the needle is inserted by moving slightly forward or backward a certain distance to avoid the blood vessel and the distance is recorded.

[0125] Injection time at each site: 2 minutes / µl virus volume (i.e. vector reagent), leaving the needle in each site for 7-8 minutes. It takes 3-4 minutes to slowly withdraw the syringe. Since the concentration of vector particles in the injected virus (vector reagent) is 10 9 / µl, so that the number of vector particles entering the PVN 2 is significantly greater than the number of cells in the PVN 2, and is more than twice the number of cells to ensure overexpression.

[0126] For ease of understanding, the following reference is given by way of example. The specific coordinate values ​​shown below are only for illustrative purposes. During specific implementation, it is necessary to check and record relevant data according to the specific situation.

[0127] Original position coordinates: X=7.08 cm, Y=-2.19 cm, Z=6.32 cm;

[0128] Right: X=1mm, Y=-2.19cm, Z=6.32cm;

[0129] Left: X=1mm, Y=-2.19cm, Z=6.32cm (30cm actual mark)

[0130] like Figure 4As shown, it is a schematic diagram of the entire macaque brain being divided into four parts, upper, lower, left and right, by the sagittal plane and the coronal plane respectively (the dotted lines in the figure serve as the information dividing lines of the four parts): the vector injection needle point 31 is located in the middle, and the auxiliary injection needle points 32 separated around this point include the upper left (point a), the lower right (point c), the upper right (point d), and the lower left (point b).

[0131] Vector injection into the needle point 31: inject 2µl of vector;

[0132] Point a: 2 µl of purple dye was injected;

[0133] X=7.38 cm, Y=0.29cm, Z=3.32cm;

[0134] Origin correction: X=7.08 cm, Y=-2.19 cm, Z=6.68 cm.

[0135] Point b: 5µl of oil was injected;

[0136] X=7.08-0.1=6.98cm;

[0137] Y=-2.19cm, Z=6.68-2.8=3.88cm.

[0138] Point c: injection of 2 µl of green dye;

[0139] Origin correction: X=7.08 cm, Y=-2.19 cm, Z=5.85 cm

[0140] Point C coordinates: X=7.38 cm, Y=0.09 cm, Z=5.85-3.0=2.85 cm

[0141] Point d: Inject 2µl of regulatory virus

[0142] X=6.98cm, Y=-2.19cm, Z=0.4cm below the skin.

[0143] 8) Post-injection magnetic resonance imaging scan (export imaging data for subsequent analysis and verification of experimental results).

[0144] 9) Suturing the incision

[0145] Disinfect the wound surface and the surrounding 3 to 5 cm, fill the bone wound with gelatin sponge, suture the scalp, clean and suture the wound surface, and replenish physiological saline and antibiotics in time.

[0146] Special attention should be paid to the diet of macaques before anesthesia: fasting for 8 hours and drinking for 6 hours; closely observing the breathing and heart rate of the macaques during anesthesia, and having someone observe and rescue immediately if any abnormality occurs; paying attention to stopping bleeding during the incision and paying attention to the outflow of cranial contents due to pressure or position changes when opening the hole; closely observing the vital signs of the macaques during transportation; paying attention to keeping the macaques warm and keeping the external temperature constant during the entire experiment; operating quickly and accurately without delaying the progress of each step of the experiment, being proficient in fixing the readings and using medicines for each operation; remember not to accidentally inject iodine disinfectant into the skull; timely recording during the scanning process and the magnetic resonance imaging record; having a dedicated person to take care of and record postoperative care; especially to avoid postoperative infection, specific personnel will be needed to observe related behaviors in the future.

[0147] In practical applications, in addition to the above methods, direct positioning injection can also be used without opening the skull, such as using a positioning high-speed steel needle, inserting it at high speed under disinfection and anesthesia, and injecting the virus, etc., or using other existing or new technologies to perform related operations.

[0148] It should be noted that although the above-mentioned technical scheme for introduction of virus microinjection involves the use of medical equipment, instruments and drugs to perform local traumatic treatment on the head of the macaque, the purpose is to accurately introduce the vector into the appropriate position of the macaque's brain (such as the paraventricular nucleus) while minimizing the damage to the macaque and the impact of other drugs on the macaque, the treatment does not involve the diagnosis and treatment of the disease.

[0149] 4. Animal care (nursing and feeding).

[0150] like Figure 2 As shown, for the vector introduction achieved by the above-mentioned incision method, the macaque after the introduction of the vector should be sent to the observation room, and when the macaque is fully awake, and the vital signs are normal and stable, and there is no bleeding in the wound, antibiotic treatment is started, preferably by injection, and the specific injection dosage can be implemented according to the scheme disclosed in the prior art. During the wound healing process of the macaque, the corresponding wound treatment is performed on it using the existing technical scheme, and drugs can be used as needed to promote its wound healing. Subsequently, the macaque is fed using the existing common feeding method, and the changes in the vital signs of the macaque are noted, and corresponding observation records are made. After two weeks of careful care, the macaque can be basically completely recovered. After the vector injection, the state of the macaque must be monitored in real time (BW monitoring) during the recovery and wound healing of the macaque. After about two weeks (the 14th day) after the vector injection, it can be managed and raised in a conventional manner.

[0151] For macaques that have been introduced with vectors by other means, they should be sent to the observation room for appropriate observation until their vital signs return to normal and stable.

[0152] 5. Gene Overexpression Transcription

[0153] After the vector is introduced into the paraventricular nucleus 2 of the macaque, due to biological activity, the injected Lenti-hNPY vector is transcribed into the gene in the somatic cells of the paraventricular nucleus 2, achieving overexpression of hNPY in the paraventricular nucleus 2, inducing phenotypes, and forming relevant pathological models. Its implementation forms include:

[0154] hNPY is free from the original target DNA and is not randomly integrated into the chromatin DNA. It is only transiently expressed in the cell. As the cell divides, the overexpressed gene fragment hNPY is gradually diluted. However, due to the non-renewability of brain cells, hNPY will not be diluted, ensuring the stability of the chimeric gene animal model. Alternatively, hNPY is free from the original target DNA, but is randomly integrated into the chromatin DNA. The overexpressed gene fragment can be transferred to the new cells formed during cell division (it will also have an effect on other renewable tissues or organs, and maintain the stability of the chimeric gene animal model to a certain extent). Since the inventor of the present invention overexpresses in somatic cells rather than germ cells, it will not be inherited to the next generation of animals, which is beneficial to the stability of the animal's own genetic characteristics; of course, other chimeric gene animal models can be constructed again for its offspring without being affected by the chimeric gene animal model constructed in the previous generation.

[0155] Of course, the random integration of overexpressed gene fragments into chromatin DNA will lead to loss or gain of function of genes or elements related to the insertion site, depending on the location of the insertion site and whether the insertion site is functional. However, since its principle is random, the process is usually defined as transgenic operation rather than gene editing. Relatively speaking, gene editing is fixed-point, and the results of editing can be predicted before the experiment begins.

[0156] VI. Animal model trait testing

[0157] By analyzing the brain slices of a euthanized macaque, the inventors of the present invention found that the expression of AgRP lentivirus can be detected in the paraventricular nucleus region, and the expression efficiency is high (e.g. Figure 5 , 6 as shown). Figure 5 The fluorescence image of the macaque paraventricular nucleus after virus injection is shown (the background is the cell nucleus marker Hoechst staining). The left side of the picture is a schematic diagram of the macaque brain structure, and the four pictures on the right are sequential slice fluorescence images of the macaque PVN. Due to experimental reasons (to reduce experimental costs and manage surviving macaques, only slices of Lenti-hAgRP vectors were made to detect the status of the experiment), in fact, due to the use of the same lentivirus, the Lenti-hNPY vector also has the same expression.

[0158] Furthermore, combined with Figure 9-12The experimental results shown are described in detail below.

[0159] The effects of hNPY overexpression on the body weight of experimental macaques are as follows:

[0160] After the virus microinjection experiment was completed, the experimental macaques recovered completely after two weeks of intensive care. The inventors of the present invention weighed each experimental macaque every two weeks. Previous experimental results in adult rats showed that virus-mediated overexpression of NPY in the paraventricular nucleus will lead to weight gain. When the experimental macaques were 2 years old, the weight of the macaques in the NPY overexpression group increased significantly compared with the macaques in the control group. Interestingly, the experimental macaques in the NPY group showed significant weight gain in the 6th month after the virus injection. Fig. 9 The hNPY overexpression in the paraventricular nucleus of macaques causes the experimental macaques to gain weight. The monthly weight change chart shows that 6 months after virus injection, the weight of the experimental macaques in the NPY group began to increase significantly. The arrows indicate the three blood collection periods in the experiment. Statistical analysis, *p < 0.05, **p < 0.01, ****p < 0.0001. Data: standard error of the mean (error bars are mean SEM (standard error), i.e. standard error of the mean, the same below).

[0161] The effects of hNPY overexpression on feeding behavior and body temperature of experimental macaques are as follows:

[0162] Fig.10 The effects of virus injection on macaques' feeding behavior, food intake, and serum insulin at 7 months after virus injection (analysis of the first 3 days) are given. The feeding time and food intake of macaques in the NPY group increased at 7 months after virus injection (a and b in the figure); exercise behavior and sleep time were not affected by virus injection (c and d in the figure). The blood glucose concentration of macaques in the NPY group did not change, but the serum insulin level increased at 10 months after virus injection; after 12 months after virus injection, the serum insulin level returned to normal (e and f in the figure). Data: standard error of the mean. Fig.11 Figure d shows the body temperature changes of the control group and the NPY experimental group monitored continuously for three days at 7 months after virus injection, and there were no significant differences.

[0163] The effects of human NPY overexpression on feeding behavior of experimental macaques were examined by video recording analysis and feeding monitoring. Fig. 9 As shown, 7 months after virus injection, the weight of the macaques in the NPY overexpression experimental group increased significantly compared with the control macaques; Fig.10 As shown in the figure, during the three-day video recording period, the feeding time and food intake of the NPY overexpression group macaques increased. In addition, the amount of exercise and sleep time of the NPY overexpression group macaques during the same period were similar to those of the control group ( Fig.10 c, d), indicating that the weight gain and prolonged feeding time of the experimental macaques in the NPY group were caused by the overexpression of NPY in the paraventricular nucleus of the hypothalamus, and were not related to changes in motor ability. The inventors of the present invention further measured the body temperature of the experimental animals and found that compared with the macaques in the control group, the body temperature of the experimental macaques in the NPY or AgRP overexpression group was normal 7 months after virus injection ( Fig.11 e).

[0164] The effects of hNPY overexpression on endocrine and metabolic biochemical parameters in experimental macaques are as follows:

[0165] Fig.11 The effects of hNPY overexpression in the paraventricular nucleus of macaques on the body temperature and other metabolic indicators of experimental macaques are given, including: a, the concentrations of triglycerides, cholesterol, high-density lipoprotein, low-density lipoprotein, and non-esterified lipids in the control group and the NPY experimental group before virus injection. b, the concentrations of triglycerides, cholesterol, high-density lipoprotein, low-density lipoprotein, and non-esterified lipids in the control group, the NPY experimental group, and the AgRP experimental group at 10 months after virus injection. c, the concentrations of triglycerides, cholesterol, high-density lipoprotein, low-density lipoprotein, and non-esterified lipids in the control group, the NPY experimental group, and the AgRP experimental group at 12 months after virus injection.

[0166] The inventors of the present invention collected serum from experimental macaques before virus injection (0 o'clock), 10 and 12 months after NPY or AgRP virus injection, and analyzed some endocrine and metabolic biochemical indicators. Fig.10 As shown in Figure 5, the blood glucose levels of the experimental macaques in the NPY or AgRP groups were normal in the three periods; in the 10th month after injection, the serum insulin concentration of the experimental macaques in the NPY group was significantly higher than that in the control group; however, in the 12th month, as the weight of the experimental macaques in the NPY group did not change significantly in winter, the serum insulin concentration did not change significantly (as shown in Figure 5). Fig.10 f). In all three periods, there was no significant difference in their body weight. The changes in serum insulin concentration were related to the body weight of the experimental animals. The results of other blood biochemical indexes showed that the concentrations of triglycerides, cholesterol, high-density lipoprotein, low-density lipoprotein, and non-esterified lipids in the serum of the overexpression experimental macaques were not abnormal and were not affected by NPY overexpression.

[0167] The effects of lentivirus-mediated hNPY overexpression in the paraventricular nucleus on body weight, feeding behavior and endocrine metabolic indicators in young macaques were examined. Overexpression of human NPY in the paraventricular nucleus of experimental macaques caused an increase in body weight, feeding time and food intake at 6 months after virus injection. This finding is consistent with the experimental results in rats. However, overexpression of human AgRP in the paraventricular nucleus of experimental macaques caused weight gain at 19 months after virus injection; this finding is different from the experimental results of AgRP in rodents. Since the expression levels of lentiviruses expressing hNPY in animals are similar, the inventors of the present invention analyzed that in the early development of macaques, hNPY regulates body weight through different mechanisms, resulting in hNPY regulating and mediating changes in macaque weight at different periods.

[0168] Ten months after the virus injection, the experimental macaques in the NPY group gained weight, accompanied by an increase in serum insulin levels. When the weight of the experimental macaques in the NPY group dropped to a level similar to that of the macaques in the control group in winter, the serum insulin level also returned to normal. This finding is consistent with the known fact that weight loss in humans is a determining factor in blood insulin levels. Previous studies have shown inconsistent effects on blood glucose concentrations and body temperature regarding NPY overexpression. The inventors of the present invention did not observe this before and after the virus injection.

[0169] The differences in blood glucose concentration and body temperature between the hNPY group and the control group are as follows:

[0170] The inventors of the present invention did not observe abnormal concentrations of other blood biochemical indicators, such as triglycerides, cholesterol, high-density lipoprotein, low-density lipoprotein, non-esterified lipids, etc. in the serum of the overexpression experimental macaques. This observation is consistent with the reported results that the effect of long-term infusion of NPY on fatty acids is temporary.

[0171] also, Fig.12 The changes in NPY levels, AgRP and leptin in the blood of macaques after virus injection are given. Transverse variance analysis and post hoc tests show that at 10, 12 and 19 months after virus injection, the blood NPY levels, AgRP and leptin did not change in macaques injected with Lenti-hNPY (data: standard error of the mean). It can be seen that Lenti-hNPY injected into the paraventricular nucleus 2 only affects the genes of the local tissues of the macaques, forming a chimeric gene animal model.

[0172] In summary, the experimental data of the inventors of the present invention show that overexpression of hNPY in the paraventricular nucleus of young macaques will lead to weight gain. Changes in feeding behavior, food intake, and serum insulin levels are positively correlated with weight changes in experimental macaques. In future studies, the inventors of the present invention need to continue to explore and analyze the long-term effects of overexpression of human NPY or AgRP in the paraventricular nucleus on experimental animals. Obviously, non-genetic chimeric genetically engineered monkeys are very good animal models for translational medicine, and can provide new clinical treatments for diseases including the prevention and treatment of obesity / neurological diseases.

[0173] The technical solution (including methods, animal models, etc.) provided by the present invention can be used to study the pathogenic mechanism of diseases including obesity / neurological diseases, diabetes, etc., clinical treatment methods, verification and testing of related therapeutic drugs, and other related preclinical research, and explore new clinical treatment strategies for diseases such as obesity and diabetes, etc. In addition, other animals can be raised according to specific needs, such as increasing the weight of experimental animals, promoting animal feeding, etc., to improve the efficiency and quality of animal breeding, and because the modified genes are in specific tissues or organs, other tissues or organs of animals are not involved, it will not cause public concerns about the safety of transgenic technology in the field of food.

[0174] Since the organ operated by the present invention is the brain. Brain nerve cells do not have the ability to continue to divide and regenerate, so in theory, there will be no new cells to replace after gene knockout in the brain, and the effect of knockout or overexpression should be permanent, and the characteristics of the relevant model will not be lost due to the self-repair ability of the body. The stability and sustainability of the relevant traits are good, and the degree of reproducibility is high. At the same time, the animal model obtained by the above scheme does not modify or edit its individual genes (for the location where the pathological model occurs itself), and no other factors are introduced, which reduces the influence of other uncontrollable factors and ensures the scientificity and accuracy of related research and experiments. In operation, the number of viral vectors is much larger than the number of brain cells in the corresponding position tissue or organ, which can ensure that all brain cells in the corresponding area are infected, thereby achieving interference with it. Thereby achieving gene overexpression in the local brain area or nerve nucleus to form a chimeric gene animal model.

[0175] Compared with traditional heritable gene-edited animal models, chimeric animal models have many advantages, such as short cycle, high phenotypic penetrance, high synchronization, high repeatability, easy large-scale batch acquisition, and low cost. Moreover, the technical means of implementation can be diversified. For example, by local injection of genetically modified stem cells or genetically modified vectors (such as viruses), genetic modification is caused locally in the animal body to induce phenotypes. In addition, cell-specific diseases such as tumor diseases usually occur under the premise of local gene mutations, and the diseased cells are in the surrounding normal cell microenvironment. Obviously, systemic heritable gene mutation animal models cannot fully simulate the actual physiological process of cell-specific diseases. Chimeric animal models can truly simulate the occurrence and development of cell-specific diseases in normal physiological microenvironments, rather than the abnormal mutation background of traditional models. More importantly, this solution solves the constraints of high cost, long time, small number, and difficulty in establishing germline genetic engineering monkeys. In particular, for many human diseases that are local lesions of heterogeneous organs, tissues, and cells, relevant gene technologies can be used to accurately modify genes in relevant animal tissues and organ somatic cells to establish chimeric animal models. Therefore, the technical solution of the present invention can be widely applied to the construction of different animal models and has a wide range of adaptability.

[0176] The technical solution of the present invention can be applied to a variety of models, and relevant models can be established as needed (using this technical solution, after selecting appropriate viral vectors and gene fragments, and operating on the corresponding parts, organs, and tissues according to this technical solution, this can be achieved).

[0177] The main features of the model are: a. It is carried out in adult primates; b. The present invention is a local gene modification, which can be a single gene site or a combination of multiple gene sites; c. The model of the present invention can be used for clinical drug screening.

[0178] It should be noted that this application is based on the priority of "the application number 201811343685.4 submitted by the applicant to the State Intellectual Property Office of China on November 13, 2018, and the invention name is 'Animal model construction method, animal breeding method, animal model and its application'". In this application, in order to make the relevant expressions clearer, more accurate and complete, some terms and descriptions are slightly different from the above expressions as the basis of priority. However, as a person skilled in the art, it is sufficient to make a judgment and enable the relevant contents of this application to be supported.

[0179] In the specification and claims, if a method is described herein comprising a series of steps, the order in which the steps appear is not necessarily the only order in which the steps may be performed, and certain steps mentioned may be omitted and / or certain other steps not described herein may be added to the method. In addition, the terms "comprises," "comprising," "including," "having," and any variation thereof, are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus comprising a series of elements is not necessarily limited to only those elements, but may include other elements not specifically listed or inherent to such processes, methods, articles, or apparatuses.

[0180] The technical solutions provided by the embodiments of the present invention are introduced in detail above. Specific examples are used herein to illustrate the principles and implementation methods of the present invention. The description of the above embodiments is only used to help understand the methods and core ideas of the present invention. At the same time, for those skilled in the art, according to the ideas of the present invention, there will be changes in the specific implementation methods and application scopes. In summary, the content of this specification should not be understood as limiting the present invention. Sequence Listing <110> Sichuan Hengshu Biotechnology Co., Ltd. <120> Gene overexpression chimeric animal model and engineered monkey model based on hNPY and their applications <141> 2019-11-13 <150> 2018113436854 <151> 2018-11-13 <160> 1 <170> SIPOSequenceListing 1.0 <210> 1 <211> 7664 <212> DNA <213> Artificial sequence Lenti-hNPY plasmid <400> 1 caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac 60 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa 120 aaaggaag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat 180 tttgccttcc tgtttttgct cacccagaaa cgctgggtgaa agtaaagat gctgaagaatc 240 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga 300 gttttcgcc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg 360 cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc 420 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag 480 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc 540 tgacaacgat cggaggaccg aaggagctaa ccgcttttt gcacaacatg ggggatcatg 600 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg 660 acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac 720 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac 780 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg 840 agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg 900 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg 960 agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac 1020 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg 1080 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg 1140 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc 1200 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc 1260 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt 1320 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc 1380 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact 1440 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac 1500 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag 1560 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg 1620 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg 1680 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga 1740 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt 1800 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct 1860 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg 1920 aggaagcgga agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt 1980 aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta 2040 atgtgagtta gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta 2100 tgttgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt 2160 acgccaagcg cgcaattaac cctcactaaa gggaacaaaa gctggagctg caagcttaat 2220 gtagtcttat gcaatactct tgtagtcttg caacatggta acgatgagtt agcaacatgc 2280 cttacaagga gagaaaaagc accgtgcatg ccgattggtg gaagtaaggt ggtacgatcg 2340 tgccttatta ggaaggcaac agacgggtct gacatggatt ggacgaacca ctgaattgcc 2400 gcattgcaga gatatgtat ttaagtgcct agctcgatac ataaacgggt ctctctggtt 2460 agaccagatc tgagcctggg agctctctgg ctaactaggg aacccactgc ttaagcctca 2520 ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa 2580 ctagagatcc ctcagaccct tttagtcagt gtggaaaatc tctagcagtg gcgcccgaac 2640 agggacttga aagcgaaagg gaaaccagag gagctctctc gacgcaggac tcggcttgct 2700 gaagcgcgca cggcaagagg cgaggggcgg cgactggtga gtacgccaaa aattttgact 2760 agcggaggct agaaggagag agatgggtgc gagagcgtca gtattaagcg ggggagaatt 2820 agatcgcgat gggaaaaaat tcggttaagg ccagggggaa agaaaaaata taaattaaaa 2880 catatagtat gggcaagcag ggagctagaa cgattcgcag ttaatcctgg cctgttagaa 2940 acatcagaag gctgtagaca aatactggga cagctacaac catcccttca gacaggatca 3000 gagaactta gatcattata window gcaaccctct attgtgtgca tcaaggata 3060 gagataaag ahaaaagga agctttagac agatagagg agaaaaaaaagtaag 3120 accaccgcac agcaagcggc cgctgatctt cagacctgga ggaggata tgagggacaa 3180 ttggagagt gattatatta atataagt agtaaaaatt gaaccattag gagtagcacc 3240 caccaaggca agagagag tggtgcagag agaaaaaga gcagtggga taggagcttt 3300 gttccttggg ttctgggag cagcaggaag cactatggc gcagcctca tgacgctgac 3360 ggtacaggcc agacattat tgtctggtat agtgcagcag cagaacatt tgctgaggc 3420 tattgaggcg cacagcatc tgttgaggcact cacagtgg gggcatcagc agctcaggc 3480 aagaatcctg gctgtggaaa gatacctaa ggatcacag ctcctgggga tttggggttg 3540 ctctggaaaa ctcattgca ccactgctgt gccttggaat gctagttgga gtaataaatc 3600 tctggacag attggaatca cacgacctgg atggagtggg acagagaat taacaattac 3660 acagcttaa tacactcctt aattgaagaa tcgcaaaacc agcagaaa gatgaacaa 3720 gatttattgg aattagaata atgggcaagt ttgtggaatt ggtttaacat aacaattgg 3780 ctgtgtata taaaattatt cataatgata gtaggaggct tggtaggttt agaatagtt 3840 tttgctgtac tttctatagt gatagagtt agggcaggt attcaccatt atcgttttcag 3900 acccacctcc acccaccgag gggacccgac aggccgag agaagaga agaggtgga 3960 gagagagaca gagacagatc cattcgatta gtgaacggat ctcgacggtt aacttttaaa 4020 agaaaagggg ggattggggg gtacagtgca gggaaagaa tagtagacat atagcaaca 4080 gaatacaa ctaagaatt aaaaaaaa ttcaaattt tatcgaacg 4140 cgtgtgtcta gactgcagag ggccctgcgt atgagtgcaa gtgggtttta ggaccaggat 4200 gaggcggggt gggggtgcct acctgacgac cgaccccgac ccactggac agcacccac 4260 ccccattccc caattgcgc atcccctatc aggaggggg agggaaaca ggatgcggcg 4320 aggcgcgtgc gcactgccag cttcagcacc gcggacagtg ccttcgcccc cgcctggcgg 4380 cgcgcgccac cgccgcctca gcactgaagg cgcgctgacg tcactcgccg gtcccccgca 4440 aactcccctt cccggccacc ttggtcgcgt ccgcgccgcc gccggcccag ccggaccgca 4500 ccacgcgagg cgcgagatag ggggcacgg gcgcgaccat ctgcgctgcg gcgccggcga 4560 ctcagcgctg cctcagtctg cggtgggcag cggaggagtc gtgtcgtgcc tgagagcgca 4620 gtcgagaagg taccgccacc atgctggggct ccaaacggct ggggctgagt gggctgactc 4680 tggctctgtc cctgctggtc tgcctggggg ctctggctga ggcttaatccc agtaagcctg 4740 acaacccagg cgaagatgca cccgccgagg acatggctcg gtactattca gcactgagac 4800 actacatcaa tctgattact cgacagcggt atgggaaacg aagctcccct gaaaccctga 4860 tctctgatct gctgatgcgg gaaagtactg agaacgtgcc cagaaccagg ctggaggacc 4920 ccagcatgtg ggactacaag gacgatgacg ataaaggatc cggagccaca aacttttcc 4980 tgctgaagca ggctggcgat gtggagaaa acccaggccc cgtcagcaaa ggggaggagg 5040 acaacatggc catcattaag gaattcatgc gctttaaagt gcacatggag ggcagcgtca 5100 5160 ctgcaaagct gaaagtgaca aaaggaggac ctctgccatt cgcctgggat atcctgtcac 5220 ctcagtttat gtacggcagc aaggcatatg tcaaacatcc cgccgacatt cctgattatc 5280 tgaagctgag cttcccagaa gggtttaaat gggagagagt gatgaacttt gaggacggcg 5340 gcgtggtcac cgtcacacag gattcaagcc tgcaggacgg agagttcatc tacaaggtga 5400 aactgagagg gacaaatttt ccatccgatg gacccgtcat gcagaagaaa actatgggct 5460 gggaggccag cagcgagaga atgtatcccg aggacggcgc cctgaaggga gagatcaagc 5520 agaggctgaa gctgaaagat gggggacact acgacgctga ggtgaaaacc acctataaag 5580 caaagaaacc tgtgcagctg ccaggcgcct acaacgtcaa tatcaagctg gatattacaa 5640 gccataatga ggactatact atcgtggagc agtatgagag agccgagggg agacattcaa 5700 ccggggggat ggacgagctg tataaatgaa cggcttaagt cgacaatcaa cctctggatt 5760 acaaaatttg tgaaagattg actggtattc ttaactatgt tgctcctttt acgctatgtg 5820 gatacgctgc tttaatgcct ttgtatcatg ctattgcttc ccgtatggct ttcattttct 5880 cctccttgta taaatcctgg ttgctgtctc tttatgagga gttgtggccc gttgtcaggc 5940 aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc cactggttgg ggcattgcca 6000 ccacctgtca gctcctttcc gggactttcg ctttccccct ccctattgcc acggcggaac 6060 tcatcgccgc ctgccttgcc cgctgctgga caggggctcg gctgttgggc actgacaatt 6120 ccgtggtgtt gtcggggaag ctgacgtcct ttccatggct gctcgcctgt gttgccacct 6180 ggattctgcg cgggacgtcc ttctgctacg tcccttcggc cctcaatcca gcggaccttc 6240 cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga 6300 cgagtcggat ctccctttgg gccgcctccc cgcgtcgact ttaagaccaa tgacttacaa 6360 ggcagctgta gatcttagcc actttttaaa agaaaagggg ggactggaag ggctaattca 6420 ctcccaacga agacaagatc tgctttttgc ttgtactggg tctctctggt tagaccagat 6480 ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc aataaagctt 6540 gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta actagagatc 6600 cctcagaccc ttttagtcag tgtggaaaat ctctagcagt agtagttcat gtcatcttat tattcagtat ttataacttg caaagaaatg aatatcagag acttgtttat tgcagcttat aatggttaca aataagcaa tagcatcaca aatttcacaa aataagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg gctctagcta tcccgcccct aactccgccc agttccgcc attctccgcc ccatggctga 6900. ctaatttttt ttatttatgc agaggccgag gccgcctcgg cctctgagct attccagag 6960 tagtgaggag gcttttttgg aggcctagc ttttgcgtcg agacgtaccc aattcgccct 7020. atagtgagtc gtattacgcg cgctcactgg ccgtcgtttt acaacgtcgt gactgggaa 7080 accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta 7140 atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat ggcgcgacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg 7260 tgaccgctac acttgccagc gccctagcgc ccgctcctttt cgctttcttc ccttcctttc 7320 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc 7380 gattagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta 7440 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta 7500 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg 7560 atttataagg gatttgccg atttcggcct attggttaaaa aaatgagctg atttaacaaa 7620 aatttaacgc gaattttaac aaaatattaa cgtttacaat ttcc 7664

Claims

1. Application of chimeric genetically engineered monkey models in the study of the pathogenic mechanism of obesity / neurological diseases, The method for constructing the chimeric genetic engineering monkey model is to use lentivirus to mediate overexpression of human NPY in the paraventricular nucleus of the macaque brain, thereby changing the feeding behavior of the macaque and forming a corresponding chimeric genetic engineering monkey model; The vector sequence used in the lentivirus-mediated therapy is SEQ ID NO.1, and the vector is injected into the paraventricular nucleus of macaques by microinjection; There were no significant changes in the monkey's exercise volume and sleep time, as well as body temperature, blood sugar concentration, serum insulin level, and blood biochemical index test results.

2. The use according to claim 1, characterized in that The changing of the feeding behavior of macaques includes increasing the feeding time and increasing the food intake.

3. The use according to any one of claims 1 to 2, characterized in that: Before the vector was injected into macaques, the vector was first microinjected into the mouse brain to detect its expression efficiency.

4. The use according to claim 3, characterized in that Three weeks after the vector was microinjected, frozen sections of the mouse brain were obtained to detect the red fluorescence signal of mCherry co-expressed with NPY.

5. The use according to any one of claims 1 to 2, characterized in that: The microinjection adopts a microinjection method assisted by nuclear magnetic resonance scanning technology to inject the vector into the paraventricular nucleus of the macaque.

6. The use according to claim 5, characterized in that The macaque was 1 year old.

7. Application of chimeric genetically engineered monkey models in the study of the pathogenesis of diabetes, The method for constructing the chimeric genetic engineering monkey model is to use lentivirus to mediate overexpression of human NPY in the paraventricular nucleus of the macaque brain, thereby changing the feeding behavior of the macaque and forming a corresponding chimeric genetic engineering monkey model; The vector sequence used in the lentivirus-mediated therapy is SEQ ID NO.1, and the vector is injected into the paraventricular nucleus of macaques by microinjection; There were no significant changes in the monkey's exercise volume and sleep time, as well as body temperature, blood sugar concentration, serum insulin level, and blood biochemical index test results.

8. The use according to claim 7, characterized in that The changing of the feeding behavior of macaques includes increasing the feeding time and increasing the food intake.

9. The use according to any one of claims 7 to 8, characterized in that: Before the vector was injected into macaques, the vector was first microinjected into the mouse brain to detect its expression efficiency.

10. The use according to claim 9, characterized in that Three weeks after the vector was microinjected, frozen sections of the mouse brain were obtained to detect the red fluorescence signal of mCherry co-expressed with NPY.

11. The use according to any one of claims 7 to 8, characterized in that: The microinjection adopts a microinjection method assisted by nuclear magnetic resonance scanning technology to inject the vector into the paraventricular nucleus of the macaque.

12. The use according to claim 11, characterized in that The macaque was 1 year old.

13. Application of chimeric genetically engineered monkey models in clinical treatments and drug research for obesity / neurological diseases. The method for constructing the chimeric genetic engineering monkey model is to use lentivirus to mediate overexpression of human NPY in the paraventricular nucleus of the macaque brain, thereby changing the feeding behavior of the macaque and forming a corresponding chimeric genetic engineering monkey model; The vector sequence used in the lentivirus-mediated therapy is SEQ ID NO.1, and the vector is injected into the paraventricular nucleus of macaques by microinjection; There were no significant changes in the monkey's exercise volume and sleep time, as well as body temperature, blood sugar concentration, serum insulin level, and blood biochemical index test results.

14. The use according to claim 13, characterized in that The changing of the feeding behavior of macaques includes increasing the feeding time and increasing the food intake.

15. The use according to any one of claims 13 to 14, characterized in that: Before the vector was injected into macaques, the vector was first microinjected into the mouse brain to detect its expression efficiency.

16. The use according to claim 15, characterized in that Three weeks after the vector was microinjected, frozen sections of the mouse brain were obtained to detect the red fluorescence signal of mCherry co-expressed with NPY.

17. The use according to any one of claims 13 to 14, characterized in that: The microinjection adopts a microinjection method assisted by nuclear magnetic resonance scanning technology to inject the vector into the paraventricular nucleus of the macaque.

18. The use according to claim 17, characterized in that The macaque was 1 year old.

19. Application of chimeric genetically engineered monkey models in the clinical treatment of diabetes and drug research. The method for constructing the chimeric genetic engineering monkey model is to use lentivirus to mediate overexpression of human NPY in the paraventricular nucleus of the macaque brain, thereby changing the feeding behavior of the macaque and forming a corresponding chimeric genetic engineering monkey model; The vector sequence used in the lentivirus-mediated therapy is SEQ ID NO.1, and the vector is injected into the paraventricular nucleus of macaques by microinjection; There were no significant changes in the monkey's exercise volume and sleep time, as well as body temperature, blood sugar concentration, serum insulin level, and blood biochemical index test results.

20. The use according to claim 19, characterized in that The changing of the feeding behavior of macaques includes increasing the feeding time and increasing the food intake.

21. The use according to any one of claims 19 to 20, characterized in that: Before the vector was injected into macaques, the vector was first microinjected into the mouse brain to detect its expression efficiency.

22. The use according to claim 21, characterized in that Three weeks after the vector was microinjected, frozen sections of the mouse brain were obtained to detect the red fluorescence signal of mCherry co-expressed with NPY.

23. The use according to any one of claims 19 to 20, characterized in that: The microinjection adopts a microinjection method assisted by nuclear magnetic resonance scanning technology to inject the vector into the paraventricular nucleus of the macaque.

24. The use according to claim 23, characterized in that The macaque was 1 year old.

25. Application of chimeric genetically engineered monkey models in the screening, validation, testing and preclinical research and / or experiments of drugs for the treatment of obesity / neurological diseases. The method for constructing the chimeric genetic engineering monkey model is to use lentivirus to mediate overexpression of human NPY in the paraventricular nucleus of the macaque brain, thereby changing the feeding behavior of the macaque and forming a corresponding chimeric genetic engineering monkey model; The vector sequence used in the lentivirus-mediated therapy is SEQ ID NO.1, and the vector is injected into the paraventricular nucleus of macaques by microinjection; There were no significant changes in the monkey's exercise volume and sleep time, as well as body temperature, blood sugar concentration, serum insulin level, and blood biochemical index test results.

26. The use according to claim 25, characterized in that The changing of the feeding behavior of macaques includes increasing the feeding time and increasing the food intake.

27. The use according to any one of claims 25 to 26, characterized in that: Before the vector was injected into macaques, the vector was first microinjected into the mouse brain to detect its expression efficiency.

28. The use according to claim 27, characterized in that Three weeks after the vector was microinjected, frozen sections of the mouse brain were obtained to detect the red fluorescence signal of mCherry co-expressed with NPY.

29. The use according to any one of claims 25-26, characterized in that: The microinjection adopts a microinjection method assisted by nuclear magnetic resonance scanning technology to inject the vector into the paraventricular nucleus of the macaque.

30. The use according to claim 29, characterized in that The macaque was 1 year old.

31. Application of chimeric genetically engineered monkey models in the validation, testing and preclinical research and / or experiments of diabetes therapeutic drugs. The method for constructing the chimeric genetic engineering monkey model is to use lentivirus to mediate overexpression of human NPY in the paraventricular nucleus of the macaque brain, thereby changing the feeding behavior of the macaque and forming a corresponding chimeric genetic engineering monkey model; The vector sequence used in the lentivirus-mediated therapy is SEQ ID NO.1, and the vector is injected into the paraventricular nucleus of macaques by microinjection; There were no significant changes in the monkey's exercise volume and sleep time, as well as body temperature, blood sugar concentration, serum insulin level, and blood biochemical index test results.

32. The use according to claim 31, characterized in that The changing of the feeding behavior of macaques includes increasing the feeding time and increasing the food intake.

33. The use according to any one of claims 31 to 32, characterized in that: Before the vector was injected into macaques, the vector was first microinjected into the mouse brain to detect its expression efficiency.

34. The use according to claim 33, characterized in that Three weeks after the vector was microinjected, frozen sections of the mouse brain were obtained to detect the red fluorescence signal of mCherry co-expressed with NPY.

35. The use according to any one of claims 31 to 32, characterized in that: The microinjection adopts a microinjection method assisted by nuclear magnetic resonance scanning technology to inject the vector into the paraventricular nucleus of the macaque.

36. The use according to claim 35, characterized in that The macaque was 1 year old.

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