CN111172194B - Construction method and application of chimeric animal model based on hNPY and hAgRP gene overexpression - Google Patents

Abstract

The invention discloses a method for constructing a gene over-expression chimeric animal model based on hNPY and hAgRP and application thereof, wherein the method comprises the steps that Synapsin constructed is a promoter-driven NPY and hAgRP lentiviral expression vector; injecting the vector into the paraventricular nucleus of macaque; the hNPY and hAgRP are transcribed into the paraventricular nuclear cell gene to realize the overexpression of the hNPY and hAgRP genes, induce phenotype generation and form a gene overexpression chimeric animal model based on the hNPY and hAgRP. The method can be used for screening, verifying, testing, preclinical relevant research and the like of pathogenesis research of related diseases and clinical treatment means research of therapeutic drugs. The method has the characteristics of short construction period of the animal model, high efficiency, low cost, high phenotype display rate, good synchronism, high repeatability, small difficulty, convenience for large-scale application and the like.

Description

Construction method and application of chimeric animal model based on hNPY and hAgRP gene overexpression

Technical Field

The invention belongs to the technical field of biology, relates to a construction technology of an animal model, and particularly relates to a construction method of the animal model realized in somatic cells by using a transgenic technology and application thereof.

Background

Animal disease models (animal models) are mainly important tools for research in experimental physiology, experimental pathology and experimental therapeutics, including new drug screening. The development of human diseases is very complex, the pathogenesis of the diseases is deeply studied by taking a human body as an experimental object, the development of medicines is promoted slowly, and the clinical accumulated experience has limitations in time and space, and a plurality of experiments are limited in sense and method. By means of indirect research of animal models, factors which cannot be or are not easy to exclude under natural conditions can be intentionally changed, so that experimental results of the models can be accurately observed and compared with human diseases for research, and the method is beneficial to more conveniently and effectively recognizing occurrence and development rules of human diseases and researching prevention and treatment measures.

An animal model (animal model of human disease) of human disease refers to an animal with a simulated representation of human disease established in various medical science studies. The inventor finds that in the process of completing the invention, the current animal model building mode mainly adopts:

The method for constructing the hyperuricemia animal model by drug induction, such as the construction of the hyperuricemia animal model by Chinese patent application 2014100899525 and the application thereof, discloses a method for constructing the non-human primate hyperuricemia animal model, which comprises the following steps: healthy adult rhesus monkeys with normal uric acid levels, uric acid levels of 1.54+/-1.05 umol/L, are selected as model animals; selecting a pharmaceutical composition comprising ethambutol and uric acid as a molding agent; the specific dosage of the medicine is ethambutol: 10-50mg/kg and uric acid: 20-50mg/kg, and can be taken after molding, drinking water, once daily administration, and continuous administration for more than 3 days to obtain the non-human primate hyperuricemia model. The inventor finds that the method can not be widely applied even because a large amount of medicines are forcedly infused into animals for establishing models, so that the formed animal models have limited application range of later animal models due to medicines in bodies.

And, constructing a corresponding animal model using genetic engineering, as disclosed in U.S. patent application US10/179639, a method of developing an animal model to create a plurality of mutant species having the same genotype, phenotype, performance, and genetic background, comprising the steps of: inducing superovulation in sexually immature mutation-initiating animals (G0); fertilising a superovulated sexually immature mutant starting animal; obtaining a first generation mutant animal (F1) by completing the gestation process; confirming the identity of the mutation, genotype, phenotype, performance, and genetic background in the first generation mutant animal; and repeating the above steps for at least 20 generations, if necessary, with one or more progeny of the mutant animal, wherein all animals are monitored and strictly guaranteed to be identical in each step, and wherein the stability of the mutation in each generation, as well as the identity of genotype, phenotype, performance, and genetic background, is subject to planned genetic monitoring and spot checking of environmental factors; and, wherein the mutant is fertilized only if the mutation is confirmed to be stable in each generation of the mutant by planned genetic monitoring and spot check, and the genotype, phenotype, performance, and genetic background of the mutant are identical to those of the mutant-initiating animal. The inventor finds that the technical scheme has long period, high cost, narrow adaptation range and relatively low success rate in the process of completing the invention, and is inconvenient for large-scale application.

And, an animal model constructed by specific feeding enhancement, such as chinese patent application 201610793182.1 discloses a method for constructing an animal model of alcoholic liver disease, comprising the steps of: selecting 8-10kg male cynomolgus monkeys as a study object, and allowing the male cynomolgus monkeys to drink the drinking water containing aspartame by themselves in a1 st week adaptation stage except for normal feeding of basic feed, adding 10% alcohol to the drinking water for free drinking every week from the 2 nd week, and screening out the cynomolgus monkeys which drink 30% alcohol more than 50mL daily as a test animal by the 4 th weekend; 10% sucrose is used for replacing aspartame in the alcohol solution from the 5 th week, and 20% lard is additionally added into the basic feed except for continuously feeding 30% alcohol; establishing an alcoholic fatty liver model: feeding the raw materials by using high-sugar and high-fat feed and under the condition of free drinking of 30% alcohol, feeding the raw materials on the 12 th weekend ①, performing liver biopsy by B ultrasonic induction, staining liver tissues by HE, and enabling the cynomolgus monkey to show obvious fatty liver symptoms; establishing an alcoholic hepatitis and liver fibrosis model: on the basis, the high-sugar and high-fat feed is continuously used for feeding, the free drinking condition of 30% alcohol is adopted, and carbon tetrachloride solution is injected into the abdominal cavity for 2 times per week, and the injection amount is 0.25mL/kg each time; on the 16 th weekend, liver biopsy is conducted through B ultrasonic induction, liver tissues are dyed by HE, and the cynomolgus monkey shows obvious symptoms of hepatitis caused by rupture and infiltration of liver cell membranes; on the 24 th weekend, liver biopsy was performed by B-ultrasonic induction, liver tissue was stained with HE, and cynomolgus monkey showed obvious liver fibrosis symptoms. The inventor finds that the method has long period for constructing the animal model, is greatly influenced by animal individuals, environment and the like, has poor character stability and is not beneficial to large-scale application in the process of completing the invention.

Disclosure of Invention

The technical problem to be solved by the invention is to overcome the defects and provide a method for constructing a gene overexpression chimeric animal model based on hNPY and hAgRP and application of the method.

The method for constructing the gene over-expression chimeric animal model comprises the following steps of,

Synapsin constructed is a promoter driven hNPY and hAgRP lentiviral expression vector;

Injecting the vector into the paraventricular nucleus of macaque;

The hNPY and hAgRP are transcribed into the paraventricular nuclear cell gene to realize the overexpression of the hNPY and hAgRP genes, induce phenotype generation and form a gene overexpression chimeric animal model based on the hNPY and hAgRP.

Preferably, the vectors are Lenti-hsyn-NPY-2A-mcherry and Lenti-hsyn-hAgRP-2A-mcherry; or, lenti-hsyn-NPY-2A-agRP-2A-mcherry.

Preferably, the NPY in the carrier is hNPY, and the AgRP is hAgRP; further, the vector comprises WPRE as a transcription enhancing element.

Preferably, the vectors are flag-tagged hNPY and HA-tagged hAgRP lentiviral expression vectors.

Preferably, the vector is injected into the mouse brain to examine expression efficiency prior to injection into macaques.

Preferably, frozen sections of mouse brain are obtained 3-6 weeks after injection of the vector, which vector can be used for cynomolgus injection if hNPY and hAgRP are shown to be expressed at high levels.

Preferably, the method comprises the steps of,

Vector construction, wherein Synapsin is constructed to drive hNPY and hAgRP lentiviral expression vectors by a promoter;

selecting animals, namely selecting 1-2 years old macaques, preferably 1 year old male macaques;

The carrier is imported, and is injected into the paranuclei of the macaque by adopting a microinjection mode under the assistance of nuclear magnetic resonance;

Animal nursing, namely nursing the macaque injected with the carrier;

The gene is transcribed through overexpression, the hNPY and hAgRP are transcribed into the periventricular nuclear cell gene, the overexpression of the hNPY and hAgRP genes is realized, and the phenotype generation is induced;

And (3) detecting the character of the macaque by using animal model character detection, and forming a gene overexpression chimeric animal model based on hNPY and hAgRP if the phenotype meets the character requirement.

Preferably, the number of injected carrier particles is much greater than the number of paracellular cells; further, the carrier particle concentration of the carrier is 109/μl, and 2 μl is injected.

Preferably, the sequence of the vector Lenti-hsyn-hNPY-2A-mcherry is the sequence of SEQ ID NO.1 in the sequence table, and the sequence of the vector Lenti-hsyn-hAgRP-2A-mcherry is the sequence of SEQ ID NO.2 in the sequence table.

The animal model construction method is used for researching pathogenesis of obesity/nervous system diseases; and/or the number of the groups of groups,

Is used for researching pathogenesis of diabetes and other diseases; and/or the number of the groups of groups,

Clinical therapeutic approach research for obesity/neurological diseases; and/or the number of the groups of groups,

Is used for researching clinical treatment means of diabetes and other diseases; and/or the number of the groups of groups,

Screening, verifying, testing and preclinical related researches for obesity/nervous system disease treatment drugs; and/or the number of the groups of groups,

Is used for verification, test and preclinical related research of diabetes and other diseases treatment medicines.

The method has the characteristics of short construction period, high efficiency, low cost, high phenotype display rate, good synchronism, high repeatability, small difficulty, convenient large-scale application and the like of the animal model, and the constructed animal model has stable properties, and particularly has stronger construction stability for chimeric gene animal models of tissues, organs and the like without cell regeneration function. Because the chimeric animal model is adopted, the method has no genetic characteristic, low biological risk, high safety and strong controllability.

Drawings

In order to more clearly describe the relevant technical solutions to which the invention relates, the figures to which it relates will be briefly described below, it being obvious that the figures in the following description are only some embodiments of the invention, from which other figures can be obtained, without the inventive effort, for a person skilled in the art.

FIG. 1 is a flow chart of a chimeric animal model construction method;

FIG. 2 is a flow chart of a carrier injection process;

FIG. 3 shows a schematic diagram of a vector structure and a vector introduction, wherein (a) shows a schematic diagram of a constructed Lenti-hNPY lentiviral expression vector structure (only a specific part is shown in the figure), (a) shows a constructed Lenti-hRgAp lentiviral expression vector structure (only a specific part is shown in the figure), (c) shows a schematic diagram of a Lenti control vector structure without hNPY and hAgRP, and (c) shows a schematic diagram of injection of a nuclear magnetic resonance scanned macaque double-sided paraventricular virus;

FIG. 4 is a schematic representation of a vector injection site;

FIG. 5 is a fluorescent image one of the periventricular nuclei of macaque after virus injection (background is nuclear marker Hoechst staining);

FIG. 6 is a second fluorescent image of the periventricular nucleus of macaque after virus injection (background is nuclear marker Hoechst staining);

FIG. 7 is a fluorescent image of virus expression in mouse brain;

FIG. 8 is a nuclear magnetic resonance scan of experimental macaque;

FIG. 9 is a graph showing the change in body weight gain of experimental macaques caused by overexpression of hNPY and hAgRP in paranuclei of macaques;

FIG. 10 is the effect on feeding behavior, food intake and serum insulin of macaques after virus injection, wherein a-d are the curves of change after 7 months of virus injection;

FIG. 11 is the effect of human NPY and hAgRP nuclear overexpression at the side of macaque on experimental macaque body temperature and other metabolic indicators;

FIG. 12 shows the NPY level, agRP and leptin change in macaque blood after virus injection;

FIG. 13 is a schematic diagram of a vector structure comprising two genes of interest.

Reference numerals illustrate: in the figure, the brain 1, the paraventricular nucleus 2, the injection site 3, the injector 4, the carrier injection needle point 31, the auxiliary injection needle point 32.

Detailed Description

For the purpose of facilitating further understanding of the present application and for clearly understanding the technical solutions described in the present application, a complete and thorough disclosure of the relevant technical contents of the present application will become apparent to those skilled in the art from the following detailed description of the embodiments of the present application taken in conjunction with the accompanying drawings, wherein the detailed description is given by way of example only of a part of the present application to assist in understanding the present application and its core ideas.

Based on the embodiments of the present invention, all other embodiments obtained by a person having ordinary skill in the art without making creative efforts and/or without departing from the spirit and essence of the present invention, even if the order of the steps is changed, and various corresponding changes and modifications are made according to the present invention, all such corresponding changes and modifications shall fall within the scope of protection of the present invention.

The invention relates to the definition or supplementary explanation of the relevant terms as follows:

NPY: i.e., neuropeptide Y, collectively referred to as neuropeptide tyrosine, english Neuropeptide Y, abbreviated NPY. NPY is a polypeptide composed of 36 amino acid residues, belongs to pancreatic polypeptide family, is widely distributed in the central and peripheral nervous systems of mammals, and is one of the most abundant neuropeptides. By binding to different receptors, different effects are produced in vivo. NPY is based on tyrosine synthesized by "NPY gene", but in the present application, the gene name of "NPY gene" is denoted by NPY in the case of different specific descriptions for the sake of simplicity of description.

HNPY: namely human NPY, which is also known as human neuropeptide tyrosine (there are also uses of "human neuropeptide Y"), english human Neuropeptide Y, abbreviated hNPY. In the present document, the NPY is simply referred to as hNPY unless otherwise specified. Of course, other NPY may be applied to achieve the technical solution of the experiment, and based on the corresponding biological characteristics, the same or similar effects may be achieved. In the present application, however, as a preferred embodiment, human-derived NPY is selected for obtaining a clinically (human) -relevant anthropomorphic animal model; of course, other aspects may be used to select the desired NPY. The hNPY is based on tyrosine synthesized from the "hNPY gene", but in the present application, the gene name of the "hNPY gene" is denoted by hNPY in the case of different specific descriptions for the sake of simplicity of description.

AgRP: the protein is called guinea pig related protein (Agouti related protein is also used), and the English Agouti-related protein is abbreviated as AgRP. AgRP is a protein synthesized based on the "AgRP gene", but in the present application, the gene name of the "AgRP gene" is denoted by AgRP in the case of different specific descriptions for the sake of simplifying the description.

HAgRP: namely, human AgRP, a fully-known human guinea pig related protein (there are also uses of "human Agouti related protein"), english human Agouti-related protein, abbreviation hAgRP. In the present document, the AgRP is simply referred to as hAgRP unless otherwise indicated. Similar to hNPY in the selection of the application, other AgRP can be applied to realize the technical scheme of the experiment, so as to achieve the same or similar effects. However, in the present application, as a preferable mode, in order to obtain a clinically (human) -relevant anthropomorphic animal model, a human-derived AgRP is selected; of course, it is also possible to use the AgRP as desired, based on other requirements. hAgRP A protein synthesized based on "hAgRP gene", however, in the present application, the gene name of "hAgRP gene" is denoted by hAgRP in the case of different specific descriptions for the sake of simplicity of description.

PVN: the hypothalamic periventricular nucleus is called fully, the English Paraventricular nucleus of the hypothalamus, the abbreviation PVN, and PVH is also known, but PVN is more commonly used, and PVN is used in the abbreviation mode in the application.

Hemagglutinin, english hemagglutinin, abbreviated HA.

FLAG-tag, i.e., FLAG tag, corresponds to an 8 amino acid peptide DYKDDDDK (peptide sequence DYKDDDDK) that serves to label the target protein. In the protein expression and localization research, a target gene to be researched and a FLAG-tag gene sequence can be connected through a genetic engineering technology means, and can be connected at the C end or the N end of the target protein, and then the integrated gene is transferred into cells or embryonic stem cells or fertilized eggs.

Amino acid (amino acid), which constitutes the basic unit of a protein, is a class of compounds having both amino and carboxyl groups in the molecule.

Tyrosine (Tyr), chemical name 2-amino-3-p-hydroxy phenyl propionic acid, is an aromatic polar a-amino acid containing phenolic hydroxyl group. Is a conditionally essential amino acid and ketogenic glycogenic amino acid of the human body.

Genes (genetic factors), DNA fragments with genetic messages, other DNA sequences, some of which act directly as self-constructs and some of which are involved in the regulation of the expression of genetic messages. A gene is the entire nucleotide sequence required to produce a single polypeptide chain or functional RNA. Genes support the basic architecture and performance of life. Genes are both materialized (presence mode) and informative (root attribute).

DNA (deoxyribonucleic acid ) is an organic compound with a complex molecular structure. Is present in the nucleus as a component of the chromosome. The function is to store genetic information. DNA molecules are large and consist of nucleotides.

Chromosome (chromosome), which is a substance with genetic information in the nucleus, is cylindrical or rod-shaped under a microscope, mainly composed of DNA and protein, mainly DNA and protein, and is a vector of genetic information.

Proteins (proteins) are nitrogen-containing macromolecular organic compounds which are connected by peptide bonds by taking amino acids as basic units. Is an organic macromolecule, is a basic organic substance constituting cells, and is a main undertaker of vital activities. It is generally considered that: proteins are the executors of gene function.

The guide ribonucleic acid, i.e. guide RNA (gRNA), is also called small guide RNA (sgRNA). Is a post-transcriptional modification process RNA called RNA editing (RNA EDITING) acting on the plastid (kinetoplastid) in vivo. Is also a small non-coding RNA. Can be paired with pre-mRNA and have some uracil (U) inserted therein, resulting in mRNA with effect. The guide RNA edited RNA molecule, which is approximately 60-80 nucleotides in length, is transcribed from a single gene, has a3 'oligo U tail, has a sequence in the middle that is precisely complementary to the mRNA being edited, and has an anchor sequence at the 5' end that is complementary to the sequence of the non-edited mRNA.

Animal models (i.e., animal disease models) are used primarily for experimental physiology, experimental pathology, and experimental therapeutics (including new drug screening) research. The development of human diseases is very complex, the pathogenesis of the diseases is deeply studied by taking a human body as an experimental object, the development of medicines is promoted slowly, and the clinical accumulated experience has limitations in time and space, and a plurality of experiments are limited in sense and method. By means of indirect research of animal models, factors which cannot be or are not easy to exclude under natural conditions can be intentionally changed, so that experimental results of the models can be accurately observed and compared with human diseases for research, and the method is beneficial to more conveniently and effectively recognizing occurrence and development rules of human diseases and researching prevention and treatment measures.

An animal model (animal model of human disease) of human disease refers to an animal with a simulated representation of human disease established in various medical science studies.

A chimeric animal model (i.e., chimeric gene animal model, which may also be referred to as gene chimeric animal model, the former is used in the present application) is a non-germ line genetic (non-germline) animal model, and is produced by purposefully modifying genes in local tissues or organs of animals (genes in somatic cells) to induce phenotype generation, thereby forming a related pathological model (animal model). Chimeric animal models are a continuation and supplement of stabilizable genetic animal models. The genetic engineering technique used in the present application is gene overexpression, and belongs to one of the means of transgenic technology. Of course, other genetic engineering techniques may be used. Furthermore, the application discloses a non-human primate chimeric gene animal model based on gene overexpression, in particular to a chimeric gene engineering monkey based on gene overexpression.

Animal behavior refers to actions and activities performed by animals, including animal position movement, limb movement, feeding, puppet, fight, body color change, and the like; is the sum of all reactions which are suitable for the in-vivo and in-vitro environmental changes and meet the basic living and reproduction requirements of animals. All behavior of animals depends essentially on three factors: external and internal stimuli, functional states of the animal's sensory organs and nervous system, and structural and physiological characteristics of the animal's body.

Animal behavior models are established based on animal behaviors, and include external and internal various stimuli (such as physical stimulus, chemical stimulus, biological stimulus and the like), intervention and the like, so that the animal behaviors develop according to the needs of a model constructor, and based on the changes of the behaviors, the animal behaviors are influenced on the physiology and the like, so that an animal disease model meeting the requirements of the model constructor is constructed. The invention discloses construction of a specific animal behavior model based on corresponding specific target genes, and based on the same principle, a person skilled in the art can completely select other suitable target genes according to requirements.

The invention discloses a technical scheme for constructing a related animal model (animal behavior model), in particular a chimeric gene animal model, by taking hNPY and hAgRP as target genes at the same time, and in fact, the target genes can be independently used to achieve the corresponding aim and technical effects. Of course, other target genes can be selected according to the needs, and local somatic genes of different organs or tissues can be modified, so that corresponding chimeric gene animal models can be constructed. As another specific application of the invention, the corresponding chimeric gene animal model is formed by genetically modifying different brain tissues (such as sleep, exercise and the like) of the animal brain, and the animal behavior is correspondingly changed due to the modification of somatic genes of the corresponding brain tissues, so that the corresponding animal behavior model is constructed according to the requirement.

Transcription (Transcription), the process by which genetic information flows from DNA to RNA. Namely, a process of synthesizing RNA under the catalysis of RNA polymerase using a specific strand (template strand is used for transcription and coding strand is not used for transcription) in double-stranded DNA as a template and ATP, CTP, GTP, UTP kinds of nucleoside triphosphates as raw materials. As a first step in protein biosynthesis, a gene is read and copied into mRNA, i.e., a specific DNA fragment is used as a template for genetic information, and a DNA-dependent RNA polymerase is used as a catalyst to synthesize pre-mRNA by the principle of base complementation. RNA polymerase completes transcription initiation, extension, termination and the like by forming a dynamic complex with a series of components. The generated mRNA carries codons, and can realize the synthesis of protein after entering ribosome. Transcription uses only one strand of DNA as a template, and the single strand selected as the template is called the template strand, also called the nonsense strand; the other single strand is called the non-template strand, i.e., the coding strand, and is also called the sense strand because the coding strand is identical to the transcribed RNA sequence. The transcribed region on DNA is called a transcription unit.

Overexpression of genes: gene overexpression is the use of transgenic technology to allow a particular gene to be expressed beyond its original level. The basic principle of gene overexpression is that a target gene plasmid containing an upstream regulatory element is obtained by an artificial construction mode, and the plasmid is transduced into cells or tissue organs through transgenic operation, so that a large amount of transcription and translation of the target gene are realized, and the overexpression of a gene product is realized. Gene overexpression does not involve gene editing, which is to randomly insert a gene (target gene) carried by a vector into a chromosome, and separately transcribe the target gene fragment independently of the original gene sequence so that the target gene in a cell is expressed beyond a normal level, but the fragment is episomal from the original DNA. For example, the target genes hNPY and hAgRP are introduced into macaque in large quantity, so that the target genes are transcribed beyond the original level of the target genes, and a corresponding pathological model is formed.

According to the technical scheme, the corresponding animal model is effectively constructed, the method for constructing the animal model is effectively constructed, and the related application is realized based on the animal model and the method. Since excessive feeding is the main cause of all factors causing obesity epidemic, the inventors found in the study that the hypothalamus is the main area of the brain controlling feeding, and the hypothalamus is involved in sensing nutrition and energy levels, regulating food intake and energy consumption, and thus maintaining normal metabolic balance. Multiple neuronucles in the hypothalamus, such as the arciform nucleus, the paraventricular nucleus, the ventral medial nucleus, and the lateral hypothalamic region, are involved in regulating energy metabolic balance. Neurons in the nuclei carry out ingestion regulation and control through different chemical signal molecules, such as transmitters, cytokines, neuropeptides and the like, but a plurality of difficulties and barriers exist in establishing animal models with stable characters in batches by adopting a controllable and effective technical scheme, and the problems also result in that the prior research and exploration cannot be realized by adopting simple scheme transfer and the like. Through repeated theoretical analysis and experiments, the inventor finds that since NPY and hAgRP in hypothalamus mainly originate from neurons in arciform nuclei, axons of the NPY and hAgRP neurons are directly projected to paraventricular nuclei, and the paraventricular nuclei are main target areas of the NPY and hAgRP neurons, a nerve signal path from the arciform nuclei to the paraventricular nuclei plays an important role in regulating feeding behaviors. Based on the findings described above, and related studies and experiments, it was constructed to achieve overexpression of NPY and hAgRP in the hypothalamus by (transgenic) techniques, to construct corresponding animal models, in particular chimeric gene animal models, and to construct non-human primate chimeric gene animal models, in particular chimeric gene engineering monkey models, based on non-human primate cynomolgus monkey; based on the technical scheme, the method can also be used for constructing animal behavior models.

In the specific implementation aspect of the technical scheme, as a preferable technical scheme, the invention utilizes a nuclear magnetic resonance scanning technology to accurately inject the hNPY lentivirus (Lenti-hNPY) and hRgAp lentivirus (Lenti-hAgRP) respectively containing specific expression into bilateral paraventricular nuclei of young experimental macaques. Experimental results show that the body weight and the feeding behavior of the hNPY and hAgRP over-expressed macaques are obviously increased (the eating behavior of the macaques is changed, the food quantity is promoted to be increased, and thus the body weight is increased). Obviously, the non-genetic chimeric genetic engineering macaque model of the invention can be used as a clinical transformation obesity model for obesity research and exploration of new clinical treatment strategies for obesity; of course, it can be further converted into a diabetes model.

Furthermore, as one of the inventions of the present invention, the present invention provides a chimeric gene animal model, and optimizes the gene sequence using a vector, particularly optimizes the target genes hNPY and hAgRP etc. used (see the sequence listing in particular), to improve the construction effect of the chimeric gene animal model, particularly has a remarkable effect on the construction of the chimeric gene engineering monkey model.

Some examples of specific implementations and applications of the invention are given below by way of example:

The inventor of the present invention has found that in order to solve the problem of influence on health of people of all ages and both sexes due to gradual increase of occurrence rate of obesity, new treatment means for obesity should be developed continuously, relevant pathogenesis should be studied, and preclinical study should be performed by using a proper animal model. At present, animal models of dogs, pigs, mice and rats have been used in large numbers to study obesity, and the health problems associated with obesity. In order to construct a corresponding animal model, there are animal models and genetic engineering models caused by feeding in the prior art. Rodent model studies have greatly driven the search for etiology of human obesity. However, the findings of rodent experiments often fail in performing clinical transformations, and how to achieve a more efficient preclinical animal research model is a technical problem that currently needs to be solved. Because of the genetic and physiological similarity to humans, the non-human primate model will be a better way to carry out valuable transformed medical models and will be a bridge linking rodent experiments with human clinics. The invention provides the construction of an animal model by the overexpression of lentivirus-mediated humanized NPY and AgRP in paraventricular nuclei, in particular to the construction of a macaque animal model by the overexpression of the paraventricular nuclei of macaque. The animal model and the macaque can be used for relevant pathological research, new drug screening and the like. Specifically, the present invention is achieved by the following examples.

As shown in fig. 1, animal screening and vector construction are performed according to the requirements of animal model construction targets, the vector is introduced into animals and the animals are kept, and target genes in the vector are transcribed in animal bodies (cells) to realize gene overexpression, so that the animals form characters meeting the animal model construction targets, and the animals meeting the construction templates become corresponding animal models by checking the animal model characters. The concrete explanation is as follows:

In the application, in order to construct a chimeric gene engineering monkey model for regulating feeding behavior, macaque is taken as a target, hNPY and hAgRP are selected as target genes, and the purpose of the application is realized by carrying out corresponding genetic modification on paraventricular nuclei of macaque through transgenic technology-gene overexpression.

1. Animal screening

With macaque, of course, other types of animals can be selected as desired, and in the present invention, to achieve genetic and physiological similarity to humans, the selection of a non-human primate model would be a better performing valuable transformed medical model, better simulating the relevant pathological model of humans. In the present invention, it is considered that spontaneous obesity of monkeys is gradually developed and progressed in the adult period. To investigate whether genetic engineering of young macaques would result in obesity in experimental animals, the inventors of the present invention selected male macaques of about 1 year half to perform experiments (macaques of other ages may be selected, preferably macaques of 1-2 years). For ease of experimental control, 1 year old male macaque was selected during the experiments of the present invention. Of course, other primates or macaques or primates of other ages may be selected to practice the invention. Based on the technical idea of the invention, different screening standards and conditions can be adopted according to different applications.

In the verification experiment of the application, 24 male macaques with the age of 1 year old are adopted as experimental objects, wherein 8 male macaques with the age of 1 year old are injected with control viruses (used as a control group), 8 male macaques with the age of 1 year old are injected with NPY genes to over-express viruses Lenti-hNPY (used for constructing a chimeric animal-based model), 8 male macaques with AgRP genes to over-express viruses Lenti-hAgRP (used for constructing a chimeric animal-based model), and the application only discusses the experimental objects of the over-expressed viruses of the genes of the corresponding types, and all relevant information is given herein so as to further ensure the integrity and objectivity of the experimental process of the application. The subsequent operation conditions and steps (including the amounts of various instruments, medicines, reagents and the like required are also determined based on the types and the amounts of the foregoing experimental objects, which can be adaptively adjusted and changed according to different objects) are all described based on the foregoing experimental objects, but the selection of the experimental objects does not represent the limitation of the technical scheme of the present application, especially the limitation of the application range and the protection range of the present application, and only the scientific and objective comparison and experiments are convenient to be performed to describe the scientificity, feasibility, practicability and corresponding technical effects of the present application.

2. Vector construction (virus preparation).

In specific applications, the vector is constructed based on the Lenti virus (of course, corresponding other viruses can be selected as the vector based on the application according to the requirement). Meanwhile, as the organ positioned by the invention is the brain, subtype viruses with stronger infectivity on brain neurons are used. As shown in fig. 3, in the experiment for verifying the present invention, the following viral vector names were used:

A Lenti virus vector overexpressing a gene of interest: lenti-hsyn-NPY-2A-mcherry (abbreviated as Lenti-hNPY) (shown in FIG. 3 (a)); lenti-hsyn-AgRP-2A-mcherry (abbreviated as Lenti-hAgRP) (shown in FIG. 3 (b)); the sequence of the virus vector is shown in the corresponding sequence table attached to the application. The vector or virus according to the application is, unless otherwise specified, a Lenti virus vector over-expressing the gene of interest.

Control viral vector: lenti-hsyn-mcherry (FIG. 3 (b)) uses the same lentivirus as those of Lenti-hNPY and Lenti-hAgRP described above, except that the target genes hNPY and hAGRP are not included. In practical applications, the control viral vector is not practically significant, and in the present application, the injection of the vector is aimed at comparison with the injection of the aforementioned viral vectors Lenti-hNPY, lenti-hAgRP in macaques for checking the practical technical effects of the present application.

Description of the role of the elements in the viral vector:

Lenti is a name prefix for distinguishing virus types, such as Lenti (i.e., lenti virus); hsyn is the name of the promoter driving the expression of different genes; NPY, agRP, etc. are target genes or sgRNA names; 2A is the junction sequence between two genes; mcherry is a fluorescent protein gene, and forms a fluorescent detection mark for indicating the expression position of the virus in the body; flag, HA is usually expressed in fusion with the target gene and is a tag sequence for detecting the target gene. In the present application, the terms "upper" and "lower" refer to the same meaning for each element of the carrier, except for the unit of measurement (international unit), unless otherwise specified, due to the writing habit.

Lentiviruses (Lentivirus, generally abbreviated as Lenti) are a widely used transgene vector that is effective in delivering exogenous genetic material to both dividing and non-dividing mammalian cells and eventually integrating into the cell genome. The invention uses the Lenti slow virus as a vector to transfer the target gene into somatic genes to be modified. FIG. 3 (a) (b) schematically shows the structures of the Lenti-NPY lentiviral expression vector and the Lenti-AgRP lentiviral expression vector of the corresponding vectors (only the relevant elements of the specific part are shown, no other elements are drawn, the complete structure is in a closed-loop plasmid.)

Furthermore, for safety and effectiveness verification, the virus should be subjected to mouse in-vitro expression test experiments before use, and the specific test experiment mode is a known technical scheme. FIG. 7 shows fluorescence images of the expression of the above viruses (Lenti-hNPY viral vector, lenti-AgRP viral vector) in the brain of mice. The pictures show that hNPY and hAgRP lentivirus are highly expressed in the brain of mice, and the virus can achieve the purpose of gene modification and has safety.

As shown in FIG. 3 (a), synapsin (Chinese name: neuronal synaptosin, abbreviated Syn) of the present invention was first constructed as a slow virus expression vector of a promoter-driven FLAG-tag hNPY and HA-tag hAgRP (FLAG and HA are two commonly used tags, and in order to distinguish between two target proteins of NPY and AgRP in future experiments, different tags are used in verifying that hNPY and hAGRP in the experiments of the present invention in order to distinguish between overexpression of hNPY and hAgRP, other tags may be selected in specific applications or exchanged, i.e. the FLAG and HA may select other tags as needed. To test the expression efficiency of the vector, the expression efficiency was tested by injecting two lentiviral vectors into the mouse brain, respectively (this link is not a necessary link and may be omitted in specific applications). Three weeks after microinjection, the inventors obtained frozen sections of mouse brain and examined mCherry red fluorescence signal co-expressed with NPY or AgRP. The results showed that both lentiviruses produced a large amount of red fluorescent signal in the dentate gyrus region of the hippocampal nerve of mice (as shown in FIG. 7), and that Lenti-hNPY virus and Lenti-hAgRP virus showed high levels of expressed hNPY and hAGRP 4-6 weeks after hypothalamic injection of mice. Obviously, these two lentiviruses can be used for the next experimental procedure of cynomolgus brain injection. Preferably, the present invention employs HSYNAPSIN (HUMAN SYNAPSIN) -promoters, where the text name is the human neuronal synapsin promoter, abbreviation hSYN (both cases). Specifically, in the characteristic part structure of the vector shown in fig. 3 (a) and (b), LTR (abbreviation of Long TERMINAL REPEAT, chinese Long terminal repeat, which is a Long terminal repeat present on both sides of LTR retrotransposons (LTRs), which is a part of plasmid) is a fixed sequence in the viral vector; HSYNAPSIN is a promoter; hNPY is the target gene that needs to be overexpressed; 2a is a joined embryo for co-expression of mcherry; mcherry is a red fluorescent protein used to indicate virus expression; WPRE is a transcription enhancing element. FIGS. 3 (a) and (b) show the structure of a vector expressed as a linear plasmid, and the closed-loop plasmid is only required to join LTRs at both ends of the linear plasmid.

The expression of each gene is controlled by self-associated regulatory elements, common regulatory elements including: 1, promoter (promoter), the promoter in the present invention is HSYNAPSIN (i.e., human Synapsin, abbreviation hSyn); 2, enhancer (enhancer) (not shown).

To ensure that the virus was accurately microinjected into the paraventricular nucleus, the inventors of the present invention determined the location of each macaque paraventricular nucleus prior to virus injection using nuclear magnetic resonance techniques (as shown in figure 8). Three lentiviruses expressing hNPY or hAgRP or control red mCherry fluorescent protein were injected into the lateral paraventricular nuclei (as shown in fig. 3 (c), fig. 4), respectively.

By analyzing brain slices of one cynomolgus monkey euthanized, the inventors of the present invention found that the expression of AgRP lentivirus could be detected in the paraventricular nuclear region with high expression efficiency (as shown in fig. 5, 6). The fluorescence image of the paraventricular nucleus of the macaque (the background is the nuclear marker Hoechst staining) after virus injection shown in fig. 5, wherein the left side of the image is a schematic diagram of the brain structure of the macaque, and the right 4 images are sequentially slice fluorescence images of PVN parts of the macaque. For experimental reasons (reduced cost of experiments and management of surviving macaques, only sections of the Lenti-hAgRP vector were made to detect the status of the experiment), in fact, the Lenti-hNPY vector had the same expression due to the use of the same lentivirus.

In the carrier reagent containing the Lenti-hAgRP carrier and the Lenti-hNPY carrier, the concentration of carrier particles of the Lenti-hAgRP carrier or the Lenti-hNPY carrier is 10 ⁹/. Mu.l.

The vector and vector reagent containing the vector can be prepared according to conventional standard procedures, and the introduced vector is injected into the periventricular nucleus 2 of the macaque in the form of the vector reagent. The related reagents and the like used can be prepared by the conventional known reagents.

In addition, as a modification, FIG. 13 provides a schematic diagram of the structure of a vector comprising two genes of interest (hNPY and hAgRP in the drawing), and by using the vector with the duplex structure, one-time injection can be realized, which is convenient for operation and application. The target gene can be replaced by one or more other target genes according to the requirement.

3. Vector introduction

The vector is introduced into the body of macaque, especially at the appropriate location of the brain. The introduction can be realized by adopting the prior art means such as direct injection, microinjection, interventional way introduction and the like, and can also adopt the new introduction means which appear in the future. In the invention, the following precise positioning and guiding scheme is preferably adopted, and specifically comprises the following steps:

The required instrument: nuclear magnetic resonance (MRI for acronym, e.g., 3.0T, other specifications are also applicable), monkey brain locator, brain locator manipulator, microinjection pump, cranial drill, operating lamp, surgical instrument, cynomolgus monkey-dedicated transfer vehicle (table), etc.

Reagents, medicines and disinfectant: the vectors obtained in the foregoing, such as Lenti-hNPY, lenti-hAgRP overexpressed virus, PBS, physiological saline, ketamine 0.1 g/min, midazolam 10 mg/min, nikometer 0.375 mg/min, cod liver oil Φ5mm, physiological saline 0.9% (250 ml), iodophor disinfectant 500 ml/bottle, and antibiotics for injection into animals. Other drugs besides the related vector viruses can be selected as auxiliary drugs according to the needs.

Consumable: surgical gowns, surgical caps, a plurality of masks, ten pairs of sterile surgical gloves, 24 bags for skin preparation, 24 bags for debridement and suture, 24 bags for gelatin hemostatic sponge, 24 sets of seventh indwelling needles, and empty needles (1 ml×24 sets, 2ml×24 sets, 20ml×24 sets).

The steps (as shown in fig. 1, in conjunction with fig. 2):

1. Anesthesia

1) The method comprises the following steps: general anesthesia, passage: the venous indwelling needle establishes a channel, the physiological saline is used for sealing a tube, and the anesthetic dosage is as follows: ketamine 2mg is slowly pushed. Then, the maintenance amount of the conditions of the trichiasis reflex or the tail and limb reflex of the ketamine is 1.5mg until the experiment is finished. (the amount of ketamine can be increased for the first time).

2) Head hair is removed, exposing the entire craniocerebral scalp layer.

3) Opening: taking the position 3cm behind the eyebrow arch as a coordinate needle insertion point, carrying out iodophor disinfection within a range of 3cm to 5cm, taking the point as a center, making a coronary incision about 4cm, and stripping periosteum to fully expose the parietal bone and the point coordinate. A small hole was drilled in the kiwi skull suture site for placement of a cod liver oil pellet as the underlying MRI scan base (origin: 0, 0). The cod liver oil particles are aimed at forming imaging characteristics (cod liver oil is white imaging) different from that of brain tissue of a monkey on a magnetic resonance apparatus (MRI) by using grease so as to be used as a reference point for positioning, and of course, other materials can be selected as positioning reference points according to different scenes and scanning equipment, such as CT and the like, and metal particles and the like can be adopted. Figure 8 shows a nuclear magnetic resonance scan of a portion of the experimental macaque. The original scan of experimental macaque number C1406067 was lost. The white bright spots on the top of the experimental kiwi head in the figure are mineral oil tablets (cod liver oil particles), namely the parts indicated by white arrows, the white arrows in the figure are marks which are convenient to display and are considered to be added later and are used for determining the three-dimensional coordinate parameters of the paraventricular nuclei of the experimental animals.

4) Fixing: the macaque is fixed in a magnetic resonance compatibility vertical positioning instrument through an ear rod and a tongue plate.

5) MRI scanning and target spot calculation: the kiwi-shaped wearing locator performs magnetic resonance scanning. Scanning by adopting a 3.0T magnetic resonance head coil to obtain thin-layer axial T1 and T2 images, and performing sagittal T1 imaging, wherein scanning data are as follows: TE100-120, TR3800-6000; the layer thickness is 2mm; interlayer spacing 0; FOV = 280 x 280mm; and (3) placing a cod liver oil particle serving as a base point at a small hole at the joint point of the kiwi skull bone, and confirming the coordinates of the injection site. Three cod liver oil particles are stuck on the horizontal position of the positioning instrument and are placed in a right triangle for confirming the horizontal plane during scanning and scanning a base line, and the scanning range is from the skull base to the skull top and comprises all magnetic resonance positioning points. And determining the lateral nuclei of the two side chambers by referring to the brain magnetic resonance imaging of the macaque and the contrast map. And determining a coordinate needle insertion point, measuring and calculating target point coordinate values, selecting 6 target points before and after the target points, and 1mm above and below the target points, thereby finishing accurate positioning of the paraventricular nucleus of the hypothalamus. The whole process is about 1-1.5hr after improvement.

The purpose of this step is to determine the exact injection site for each individual, because the exact location of the respective organ and operating site for each individual is different because of individual differences. Other measurement modes or direct operation can be adopted for other objects and other visually revealed positions.

Meanwhile, the above 1) -5) aims at facilitating fixation of macaque, accurate positioning and operation of injection position, and the technical scheme can be realized in other modes, and can be replaced or omitted as required.

6) And (3) hole opening: the kiwi belt positioning instrument is carefully taken down from the magnetic resonance instrument, and the head is ensured not to be displaced in the positioning instrument in the process. A bone window of about 6mm to 8mm in size was opened centered on the injection origin to fully expose the meninges. In the tapping process, the temperature is reduced by using normal saline, and meanwhile, the skull drilling force is lightened when approaching to periosteum, so that the skull and the meninges are prevented from being penetrated instantaneously, and the cortex injury is caused. Finally, the remaining thin layer of skull is removed with a needle or forceps, exposing the meninges.

The whole drilling process time is about: 10-15 min/hole.

7) Injecting a reagent: and (5) assembling a mechanical arm, a microinjection pump and an injection needle.

One of the experimental macaque is taken as, for example, the following:

The needle head of the injection needle is just placed at the right center of the skull cod liver oil particles through the positioning arm, and the X, Y and Z values obtained on the coordinate instrument are the coordinates of the injection origin.

The coordinates of the actual injection point are generated by adding or subtracting the distance between the room-side nuclei converted by the previous magnetic resonance scan and the origin point in the front-back direction and the depth direction to the origin point coordinates. The movement of the positioning arm and the needle insertion operation are performed according to the coordinates, and if an obvious blood vessel exists right below the needle insertion point, the positioning arm is moved a certain distance forwards or backwards to avoid the blood vessel and then the needle is inserted down, and the distance is recorded.

Injection time per site: 2 minutes/μl virus volume (i.e. carrier reagent), needle is left for 7-8min per site. Slowly withdrawing the syringe requires 3-4min. Due to the 10 ⁹/μl concentration of carrier particles in the injected viral (carrier) reagent, the number of carrier particles entering the paraventricular nucleus 2 is significantly greater than the number of cells of the paraventricular nucleus 2 and is more than 2 times the number of cells, to ensure the achievement of overexpression.

For ease of understanding, reference is made to the following description by way of example, and the specific coordinate values shown below are merely illustrative, and in practice, it is necessary to collate and record the relevant data as the case may be.

In-situ coordinates: x=7.08 cm, y= -2.19cm, z=6.32 cm;

Right: x=1 mm, y= -2.19cm, z=6.32 cm;

Left: x=1 mm, y= -2.19cm, z=6.32 cm (30 cm real standard)

As shown in fig. 4, the whole macaque brain is schematically divided into four parts (the broken line is the information demarcation of four parts in the figure) in the upper, lower, left and right directions by the sagittal plane and the coronal plane: the carrier injection needle insertion point 31 is located centrally and the auxiliary injection needle insertion points 32, which are located around the changing point, include upper left (point a), lower right (point c), upper right (point d), lower left (point b).

Carrier injection needle insertion point 31: injecting 2 μl of carrier;

point a: injecting a purple coloring agent 2 μl;

X=7.38 cm，Y=0.29cm，Z=3.32cm；

origin correction: x=7.08 cm, y= -2.19cm, z=6.68 cm.

Point b: injecting 5 μl of grease;

X=7.08-0.1=6.98cm；

Y=-2.19cm，Z=6.68-2.8=3.88cm。

and c, point: injecting a green coloring agent 2 [ mu ] l;

origin correction: x=7.08 cm, y= -2.19cm, z=5.85 cm

C, coordinates: x=7.38 cm, y=0.09 cm, z=5.85-3.0=2.85 cm

D point: 2 mu l of injection regulation virus

X=6.98 cm, y= -2.19cm, z=0.4 cm subcutaneously.

7) Post-injection magnetic resonance imaging scans (deriving imaging data maps for subsequent analytical verification of experimental results).

8) Suture incision

Sterilizing wound surface and surrounding 3-5 cm, filling open bone wound with gelatin sponge, and suturing scalp. Clearing the suture wound surface and supplementing physiological saline and antibiotics in time.

Of particular note, the macaque diet prior to anesthesia is a diet that is fasted for 8 hours and is forbidden for 6 hours; the breathing and heart rate of the macaque are closely observed during anesthesia, and a special person observes that if the macaque has an abnormal condition, the macaque is rescued immediately; the incision is to stop bleeding and to open the hole, and the outflow of the cranial contents caused by pressure or posture change is to be paid attention to; the vital signs of the macaque should be closely observed during the transportation of the macaque; the whole experiment process is to pay attention to the constant warmth retention and external temperature of the macaque; the operation is rapid and accurate, the progress of each step of the experiment is not delayed, and each operation is skilled to fix the reading and take medicine; the method is to record that the iodophor disinfectant is not wrongly put into the cranium; timely recording and recording magnetic resonance imaging in the scanning process; nursing records of special nursing personnel after operation; in particular to avoid post-operative infections, and later on to observe relevant behaviour by specific personnel.

In practical application, besides the mode, direct positioning injection can be adopted, and the initiation of skull is not needed, for example, a positioning high-speed steel needle is adopted, high-speed penetration is performed under the conditions of disinfection and anesthesia, viruses are injected, and the like, or other prior art or new technology is adopted for related operation.

Although the above-mentioned technical solution for introducing the virus microinjection has the purpose of accurately introducing the vector into a proper position (e.g., the paraventricular nucleus) of the brain of the macaque while performing the traumatic treatment on the head portion of the macaque by using medical equipment, instruments and medicines, so as to minimize the injury to the macaque and the influence of other medicines and the like.

4. Animal care (nursing and feeding).

As shown in fig. 2, for the carrier introduction realized by the above-mentioned incision method, the macaque after the carrier is introduced should be sent into the observation chamber, and after the macaque is completely awakened, the vital signs are normal and stable, the incision is free from bleeding, etc., the antibiotic treatment is started, preferably, the treatment can be injected, and the specific injection metering can be implemented according to the scheme disclosed in the prior art. In the process of wound healing of macaque, the macaque is subjected to corresponding wound treatment by adopting the prior art scheme, and medicines can be used according to the needs to promote the wound healing of the macaque. Subsequently, the macaque is fed by adopting the existing common feeding mode, the change condition of vital signs of the macaque is noted, and corresponding observation records are made. The macaque can be basically completely recovered after two weeks of careful care. After carrier injection, the condition of the macaque must be monitored in real time (BW monitoring) during the macaque resuscitation and wound healing period, and after about two weeks (day 14) following carrier injection, it can be managed and fed in a conventional manner.

For macaque with carrier introduced in other modes, the macaque should be sent into an observation chamber for corresponding observation until vital signs of the macaque are normal and stable.

5. Gene overexpression transcription

After the vector is introduced into the periventricular nucleus 2 of the macaque, the injected Lenti-hNPY vector and the Lenti-hAgRP are transcribed into genes in somatic cells of the periventricular nucleus 2 due to biological activity, so that the overexpression of hNPY and hAgRP in the periventricular nucleus 2 is realized, phenotype generation is induced, and a related pathological model is formed. The implementation forms include:

The hNPY and hAgRP are dissociated outside the original target DNA, are not randomly integrated and inserted into the genome DNA, are only transiently expressed in cells, and the over-expressed gene fragments hNPY and hAgRP are gradually diluted along with cell division, but the hNPY and hAgRP are not diluted because of the nonrenewability of brain cells, so that the stability of the characteristics of the chimeric gene animal model is ensured. Alternatively, hNPY and hAgRP are isolated from the original target DNA, but randomly integrated into the genomic DNA, the over-expressed gene fragments can be transferred into new cells formed by division during cell division (which also act on other regenerable tissues or organs and maintain the stability of the animal model traits of the chimeric genes to some extent). Because the over-expression of the inventor is carried out in somatic cells, but not germ cells, the over-expression is not inherited to the next-generation animal, and the stability of the self genetic characteristic of the animal is not affected; of course, other chimeric gene animal models can be built again for their offspring without being affected by the chimeric gene animal model built in the previous generation.

Of course, the over-expressed gene fragments are randomly integrated into the genomic DNA, and depending on the location of the insertion site, and whether the insertion site is functional, it is determined whether the functional deletion or gain of the gene or element associated with the insertion site will result. However, since its principle is random, the process is generally defined as a transgenic operation, not gene editing. Relatively speaking, gene editing is a fixed point, and the edited result can be predicted before the experiment starts.

6. Animal model trait testing

By analyzing brain slices of one cynomolgus monkey euthanized, the inventors of the present invention found that the expression of AgRP lentivirus could be detected in the paraventricular nuclear region with high expression efficiency (as shown in fig. 5, 6). The fluorescence image of the paraventricular nucleus of the macaque (the background is the nuclear marker Hoechst staining) after virus injection shown in fig. 5, wherein the left side of the image is a schematic diagram of the brain structure of the macaque, and the right 4 images are sequentially slice fluorescence images of PVN parts of the macaque. For experimental reasons (reduced cost of experiments and management of surviving macaques, only sections of the Lenti-hAgRP vector were made to detect the status of the experiment), in fact, the Lenti-hNPY vector had the same expression due to the use of the same lentivirus.

Further, the experimental results shown in FIGS. 9-12 are described in detail below.

The effect of hNPY or hAgRP overexpression on experimental macaque body weight was as follows:

After the virus microinjection experiment was completed, the experimental macaque was carefully attended for two weeks and was essentially completely recovered. The inventors of the present invention performed weight weighing once every two weeks for each experimental macaque. Previous experimental results in adult rats show that viral-mediated overexpression of NPY in the paraventricular nucleus will lead to weight gain. When experimental macaques were 2 years old, the body weight of the NPY over-expressed macaques was significantly increased compared to control macaques. Interestingly, the NPY group experimental macaques showed significant weight gain at month 6 after virus injection; while the AgRP group did not show significant weight gain until month 19 after injection. The hNPY overexpression at the rhesus chamber side core as shown in fig. 9 caused a graph of the experimental rhesus weight gain change, which showed that the NPY group experimental rhesus weight began to increase significantly after 6 months of virus injection. Monthly body weight change charts show that the AgRP group experimental macaque weight began to increase significantly after 19 months of virus injection. Arrows indicate three blood sampling periods in the experiment. Statistical analysis, p < 0.05, p < 0.01, p < 0.0001. Data: average standard error (error bars are average s.e.m. (standard error), i.e. average standard error, the same applies below). Obviously, in the early development process of the macaque, the functional mechanisms of regulating and controlling the weight change of NPY and AgRP are different, so that in practical application, the method can be adopted, and simultaneously, the virus vectors containing two target genes of NPY and AgRP are injected into the same macaque to enhance the effect.

The effect on feeding behavior and body temperature of experimental macaques on hNPY overexpression was as follows:

FIG. 10 shows the effect of the 7 th month after virus injection (analyzed on the previous 3 days) on feeding behavior, feeding amount and serum insulin of macaques, showing that the feeding time and feeding amount of macaques in NPY group experiments were both increased at the 7 th month after virus injection (figures a, b); locomotor activity and sleep time were not affected by virus injections (c, d in the figure). The blood glucose concentration of the cynomolgus monkey was not changed in NPY group experiments, but serum insulin levels were increased at 10 months after virus injection; serum insulin levels returned to normal after 12 months post virus injection (e, f in the figure). The blood glucose concentration and serum insulin levels of the macaques in the AgRP group experiments were unchanged at both month 10 and 12 after virus injection (g, h in the figure). Data: average standard error. In fig. 11 d, no significant difference was found between the continuous monitoring of three days of body temperature change in the control group, NPY experimental group and AgRP experimental group, 7 months after virus injection.

The influence of the overexpression of human NPY or AgRP on the feeding behavior of experimental macaques is detected through video recording analysis and feeding monitoring. As shown in fig. 9, the body weight of the NPY over-expressed experimental group of macaques increased significantly compared to the control macaques after 7 months of virus injection; as shown in fig. 10, the feeding time and food intake of the NPY over-expressed experimental group macaques were both increased during the recording period of 3 consecutive days. In the same period, the body weight, feeding time and food intake of the AgRP over-expression experimental group macaque are similar to those of the control group macaque, and no abnormality is found. In addition, the exercise amount and sleep time of the experimental macaques in the contemporaneous NPY or AgRP overexpression group were similar to those of the control group (fig. 10c, d), suggesting that the weight gain and the prolongation of 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 the exercise capacity change. The inventors of the present invention further measured the body temperature of experimental animals, and found that the body temperature of experimental macaques in NPY or AgRP overexpressing group was not abnormal 7 months after virus injection, compared to that of macaques in control group (fig. 11 e).

The effect of hNPY or hAgRP over-expression on experimental cynomolgus monkey endocrine and metabolic biochemical indicators is as follows:

fig. 11 shows the effect of hNPY overexpression of paranuclei in cynomolgus monkeys on experimental cynomolgus body temperature and other metabolic indicators, wherein: concentration of triglyceride, cholesterol, high density lipoprotein, low density lipoprotein, non-esterified lipid in control group, NPY experimental group and AgRP experimental group before virus injection. b, concentration of triglycerides, cholesterol, high density lipoproteins, low density lipoproteins, non-esterified lipids in control group, NPY experimental group and AgRP experimental group 10 months after virus injection. c, concentration of triglyceride, cholesterol, high density lipoprotein, low density lipoprotein, non-esterified lipid in control group, NPY experimental group and AgRP experimental group 12 months after virus injection.

The inventor of the invention respectively analyzes partial endocrine and metabolic biochemical indexes of collected serum of experimental macaques in 10 th and 12 th month periods before virus injection (0 point) and after NPY or AgRP virus injection. As shown in e, g-corresponding AgRP of fig. 10, blood glucose levels were normal in the NPY or AgRP group experimental macaques at three periods; at 10 months after injection, the serum insulin concentration of the experimental macaques in the NPY group was significantly higher than that in the control group; however, at month 12, with the experiments in winter NPY group, the change in body weight was not significant, nor was the change in serum insulin concentration (fig. 10 f). In all three phases, consistent with no significant difference in body weight, no abnormality was seen in the serum insulin concentration of the AgRP group experimental macaque compared to the control macaque (fig. 10 g). The change in serum insulin concentration was correlated with the body weight of the experimental animals. Other blood biochemical index detection results show that the concentration of triglyceride, cholesterol, high density lipoprotein, low density lipoprotein and non-esterified lipid in the serum of the experimental macaque in the over-expression is not abnormal and is not influenced by the over-expression of NPY or AgRP.

The effect of lentivirus-mediated overexpression of hNPY or hAgRP at paraventricular nuclei on the body weight, feeding behavior and endocrine metabolism index of young macaques was examined. Overexpression of human NPY in the paranucleuses of experimental macaques resulted in an increase in body weight, feeding time and food intake at month 6 after virus injection. This finding is consistent with the experimental results in rats. However, overexpression of human AgRP in the paranucleuses of experimental macaques, at month 19 after virus injection, caused weight gain; this finding is different from the experimental results of AgRP in rodents. Since lentiviruses expressing hNPY or hAgRP respectively expressed at near levels in animals, the inventors analyzed that in early macaque development, the regulation of body weight by hNPY and hAgRP was mediated by different mechanisms, resulting in hNPY or hAgRP regulation at different times.

The NPY group experimental macaques developed weight gain after 10 months of virus injection, accompanied by an increase in serum insulin levels. When the body weight of the NPY group experimental macaque was reduced to be similar to that of the control group macaque in winter, the serum insulin level was also restored to normal. This finding is consistent with the known fact that weight loss in humans is a determinant of blood insulin levels. In previous studies, the effects of NPY overexpression on blood glucose concentration and body temperature were not consistent. The inventors of the present invention studied that no virus was observed before and after the virus injection.

Blood glucose concentrations for the hNPY or hAgRP group of experimental macaques differed from body temperature from the control group as follows:

The inventors of the present invention have not observed other biochemical indicators of blood such as concentration anomalies in serum of experimental macaque in overexpression of triglycerides, cholesterol, high density lipoproteins, low density lipoproteins, non-esterified lipids and the like. This observation is consistent with the reported effect of long-term NPY perfusion on fatty acids.

In addition, FIG. 12 shows the change of NPY level, agRP and leptin in macaque blood after virus injection, and analysis of warp variance and post-hoc test show that at 10, 12 and 19 months after virus injection, the NPY level of blood, agRP and leptin are not changed in macaques injected with Lenti-hAgRP and Lenti-hNPY (data: average standard error), and it is seen that the Lenti-hAgRP and Lenti-hNPY injected in paraventricular nucleus 2 only affect genes of local tissues of macaque, thus forming a chimeric gene animal model.

Taken together, the experimental data of the inventors of the present invention show that overexpression of hNPY or hAgRP in the paraventricular nucleus of young macaques will result in weight gain. The feeding behavior, the feeding amount and the change of serum insulin level are positively correlated with the weight change of experimental macaques. In future studies, the inventors of the present invention have also continued to investigate and analyze the long-term effects of human NPY or AgRP overexpression at paraventricular nuclei on experimental animals. Obviously, the non-hereditary chimeric gene engineering monkey is a very good transformed medical animal model, and can provide a new clinical treatment means for preventing and treating diseases including obesity/nervous system diseases and the like.

The technical scheme (comprising a method, an animal model and the like) provided by the invention can be used for researching and preventing pathogenesis of diseases such as obesity/nervous system diseases, diabetes and the like, clinical treatment means, verification and experiment of related treatment medicaments, other related researches before clinic, exploration of new clinical treatment strategies for diseases such as obesity, diabetes and the like. And raising other animals according to specific requirements, such as increasing the weight of test animals, promoting the feeding of animals, etc., to provide the efficiency and quality of animal raising, and the modified genes are in specific tissues or organs, so that other tissues or organs of animals are not involved, and the public is not caused to worry about the safety of transgenic technology in the food field.

In the above scheme, for convenience of comparison, the injection of the human NPY or the human AgRP is implemented on different individuals, and further, the human NPY and the human AgRP can be injected on the same individual at the same time, and the complementation of the human NPY and the human AgRP is implemented, so that the corresponding animal model is quickly and efficiently built.

Since the organ in which the present invention operates is the brain. The brain nerve cells do not have the ability of continuing division and regeneration, so that in theory, no new cells can be replaced after gene knockout is performed in the brain, the knockout or over-expression effect is permanent, the characteristics of a related model cannot be lost due to the self-repairing ability of an organism, the stability and the persistence of the related characters are good, and the reproducibility degree is high. Meanwhile, the animal model obtained by the scheme has no modification or editing of individual genes (for the part of the pathological model, which occurs), no other factors are introduced, the influence of other uncontrollable factors is reduced, and the scientificity and accuracy of related research and experiments are ensured. In operation, the number of the virus vectors is far greater than the number of brain cells of tissues or organs at corresponding positions, so that all brain cells of corresponding areas can be ensured to be infected, and interference to the brain cells is realized. Thereby realizing the over-expression of genes in local brain areas or nerve nuclei and forming a chimeric gene animal model.

Compared with the traditional animal model capable of editing genetic genes, the chimeric animal model has the advantages of short period, high phenotype display rate, high synchronism, high repeatability, easy large-scale batch acquisition, low cost and the like. The technical means can be diversified, for example, the genetically modified stem cells or the genetically modified vectors (such as viruses) can be locally injected into the animal body to locally cause genetic modification and induce phenotype generation. In addition, cell-specific diseases such as those represented by neoplastic diseases are usually developed under the premise of local gene mutations, and diseased cells are in the surrounding normal cellular microenvironment. Clearly, systemic heritable gene mutant animal models do not fully mimic the progression of cell-specific diseases in the actual physiological state of their occurrence. The chimeric animal model can truly simulate the occurrence and development of cell-specific diseases in a normal physiological microenvironment, rather than the abnormal mutation background of the traditional model. More importantly, the scheme solves the restriction of high cost, long time, small number, large difficulty and the like of establishing the germline genetic engineering monkey. In particular to the characteristic that a plurality of human diseases are organ, tissue and cell heterogeneous local lesions, the related gene technology can be utilized to carry out accurate gene modification in related animal tissue and organ somatic cells, and a chimeric animal model can be established. Therefore, the technical scheme of the invention can be widely applied to the construction of different animal models and has wide application range.

The technical scheme of the invention can be applied to various models, and related models can be established according to the needs (by using the technical scheme, the operation can be realized by selecting proper viral vectors and gene segments and operating corresponding parts, organs and tissues according to the technical scheme).

The model is characterized in that: a, in adult primates; b, the invention is local gene modification, can be a single gene locus, can also be a combination of a plurality of gene loci; the model of the invention can be used for screening clinical medicines.

The invention discloses a technical scheme for constructing a related animal model (animal behavior model) by taking hNPY and hAgRP as target genes simultaneously, and in fact, the target genes can be independently used to achieve corresponding invention aims and technical effects.

The application is based on the priority of ' application number 201811343685.4, filed by applicant in 2018, 11 and 13 to China national intellectual property agency, namely ' animal model construction method, animal breeding method, animal model and application '. In the present application, some terms and descriptions are slightly different from the above-described expression on the basis of priority in order to make the related expression clearer, more accurate and complete, but are sufficient for discrimination by those skilled in the art, and enable the related content of the present application to be supported.

In the description and claims, if a method described herein comprises a series of steps, the order in which the steps appear is not necessarily the only order in which the steps are performed, and some of the mentioned steps may be omitted and/or some other steps not described herein may be added to the method. Furthermore, the terms "comprises," "comprising," "includes," "including," "having," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements, but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.

The foregoing has outlined the detailed description of the embodiments of the present invention, and the detailed description of the principles and embodiments of the present invention is provided herein by way of example only to facilitate the understanding of the method and core concepts of the present invention; meanwhile, as those skilled in the art will have variations in the specific embodiments and application scope in accordance with the ideas of the present invention, the present description should not be construed as limiting the present invention in view of the above.

Sequence listing

<110> Sichuan horizontal and vertical biotechnology Co., ltd

<120> Construction method and application of chimeric animal model based on hNPY and hAgRP gene overexpression

<141> 2019-11-13

<150> 2018113436854

<151> 2018-11-13

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 7664

<212> DNA

<213> Artificial sequence Lenti-hNPY plasmid (plasmid)

<400> 1

caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac 60

attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa 120

aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat 180

tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc 240

agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga 300

gttttcgccc 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 ccgctttttt 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 gatattgtat 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

gaagaactta gatcattata taatacagta gcaaccctct attgtgtgca tcaaaggata 3060

gagataaaag acaccaagga agctttagac aagatagagg aagagcaaaa caaaagtaag 3120

accaccgcac agcaagcggc cgctgatctt cagacctgga ggaggagata tgagggacaa 3180

ttggagaagt gaattatata aatataaagt agtaaaaatt gaaccattag gagtagcacc 3240

caccaaggca aagagaagag tggtgcagag agaaaaaaga gcagtgggaa taggagcttt 3300

gttccttggg ttcttgggag cagcaggaag cactatgggc gcagcctcaa tgacgctgac 3360

ggtacaggcc agacaattat tgtctggtat agtgcagcag cagaacaatt tgctgagggc 3420

tattgaggcg caacagcatc tgttgcaact cacagtctgg ggcatcaagc agctccaggc 3480

aagaatcctg gctgtggaaa gatacctaaa ggatcaacag ctcctgggga tttggggttg 3540

ctctggaaaa ctcatttgca ccactgctgt gccttggaat gctagttgga gtaataaatc 3600

tctggaacag attggaatca cacgacctgg atggagtggg acagagaaat taacaattac 3660

acaagcttaa tacactcctt aattgaagaa tcgcaaaacc agcaagaaaa gaatgaacaa 3720

gaattattgg aattagataa atgggcaagt ttgtggaatt ggtttaacat aacaaattgg 3780

ctgtggtata taaaattatt cataatgata gtaggaggct tggtaggttt aagaatagtt 3840

tttgctgtac tttctatagt gaatagagtt aggcagggat attcaccatt atcgtttcag 3900

acccacctcc caaccccgag gggacccgac aggcccgaag gaatagaaga agaaggtgga 3960

gagagagaca gagacagatc cattcgatta gtgaacggat ctcgacggtt aacttttaaa 4020

agaaaagggg ggattggggg gtacagtgca ggggaaagaa tagtagacat aatagcaaca 4080

gacatacaaa ctaaagaatt acaaaaacaa attacaaaaa ttcaaaattt tatcgatacg 4140

cgtgtgtcta gactgcagag ggccctgcgt atgagtgcaa gtgggtttta ggaccaggat 4200

gaggcggggt gggggtgcct acctgacgac cgaccccgac ccactggaca agcacccaac 4260

ccccattccc caaattgcgc atcccctatc agagaggggg aggggaaaca 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 gggggcacgg gcgcgaccat ctgcgctgcg gcgccggcga 4560

ctcagcgctg cctcagtctg cggtgggcag cggaggagtc gtgtcgtgcc tgagagcgca 4620

gtcgagaagg taccgccacc atgctgggct ccaaacggct ggggctgagt gggctgactc 4680

tggctctgtc cctgctggtc tgcctggggg ctctggctga ggcttatccc 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 aacttttccc 4980

tgctgaagca ggctggcgat gtggaggaaa acccaggccc cgtcagcaaa ggggaggagg 5040

acaacatggc catcattaag gaattcatgc gctttaaagt gcacatggag ggcagcgtca 5100

atggccatga gttcgaaatc gagggcgaag gggagggacg accatacgag ggcacacaga 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 6660

tattcagtat ttataacttg caaagaaatg aatatcagag agtgagagga acttgtttat 6720

tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt 6780

tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg 6840

gctctagcta tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga 6900

ctaatttttt ttatttatgc agaggccgag gccgcctcgg cctctgagct attccagaag 6960

tagtgaggag gcttttttgg aggcctaggc ttttgcgtcg agacgtaccc aattcgccct 7020

atagtgagtc gtattacgcg cgctcactgg ccgtcgtttt acaacgtcgt gactgggaaa 7080

accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta 7140

atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat 7200

ggcgcgacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg 7260

tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc 7320

tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc 7380

gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta 7440

gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta 7500

atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg 7560

atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa 7620

aatttaacgc gaattttaac aaaatattaa cgtttacaat ttcc 7664

<210> 2

<211> 7772

<212> DNA

<213> Artificial sequence Lenti-hAgRP plasmid (plasmid)

<400> 2

caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac 60

attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa 120

aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat 180

tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc 240

agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga 300

gttttcgccc 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 ccgctttttt 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 gatattgtat 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

gaagaactta gatcattata taatacagta gcaaccctct attgtgtgca tcaaaggata 3060

gagataaaag acaccaagga agctttagac aagatagagg aagagcaaaa caaaagtaag 3120

accaccgcac agcaagcggc cgctgatctt cagacctgga ggaggagata tgagggacaa 3180

ttggagaagt gaattatata aatataaagt agtaaaaatt gaaccattag gagtagcacc 3240

caccaaggca aagagaagag tggtgcagag agaaaaaaga gcagtgggaa taggagcttt 3300

gttccttggg ttcttgggag cagcaggaag cactatgggc gcagcctcaa tgacgctgac 3360

ggtacaggcc agacaattat tgtctggtat agtgcagcag cagaacaatt tgctgagggc 3420

tattgaggcg caacagcatc tgttgcaact cacagtctgg ggcatcaagc agctccaggc 3480

aagaatcctg gctgtggaaa gatacctaaa ggatcaacag ctcctgggga tttggggttg 3540

ctctggaaaa ctcatttgca ccactgctgt gccttggaat gctagttgga gtaataaatc 3600

tctggaacag attggaatca cacgacctgg atggagtggg acagagaaat taacaattac 3660

acaagcttaa tacactcctt aattgaagaa tcgcaaaacc agcaagaaaa gaatgaacaa 3720

gaattattgg aattagataa atgggcaagt ttgtggaatt ggtttaacat aacaaattgg 3780

ctgtggtata taaaattatt cataatgata gtaggaggct tggtaggttt aagaatagtt 3840

tttgctgtac tttctatagt gaatagagtt aggcagggat attcaccatt atcgtttcag 3900

acccacctcc caaccccgag gggacccgac aggcccgaag gaatagaaga agaaggtgga 3960

gagagagaca gagacagatc cattcgatta gtgaacggat ctcgacggtt aacttttaaa 4020

agaaaagggg ggattggggg gtacagtgca ggggaaagaa tagtagacat aatagcaaca 4080

gacatacaaa ctaaagaatt acaaaaacaa attacaaaaa ttcaaaattt tatcgatacg 4140

cgtgtgtcta gactgcagag ggccctgcgt atgagtgcaa gtgggtttta ggaccaggat 4200

gaggcggggt gggggtgcct acctgacgac cgaccccgac ccactggaca agcacccaac 4260

ccccattccc caaattgcgc atcccctatc agagaggggg aggggaaaca 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 gggggcacgg gcgcgaccat ctgcgctgcg gcgccggcga 4560

ctcagcgctg cctcagtctg cggtgggcag cggaggagtc gtgtcgtgcc tgagagcgca 4620

gtcgagaagg taccgccacc atgctgacag ccgctctgct gagttgtgca ctgctgctgg 4680

ctctgcctgc aacacagggc gcccagatgg gactggctcc actggaaggc attcggagac 4740

ccgatcaggc cctgtttcct gagctgcctg gactgggcct gagggctcca ctgaagaaaa 4800

ccacagccga gctggctgag gaggacctgc tgcaggaagc tcaggcactg gccgaggtgc 4860

tggactccca ggatcgcgaa ccccgatcta gtaggcgctg cgtgcgcctg cacgagtctt 4920

gtctggggca gcaggtccca tgctgtgacc cctgcgcaac ttgctactgt aggttcttta 4980

acgccttctg ctattgtcgc aagctgggaa ccgccatgaa tccttgttct cggacatacc 5040

cttatgacgt gccagattac gcaggatccg gagccacaaa cttttccctg ctgaagcagg 5100

ctggcgatgt ggaggaaaac ccaggccccg tcagcaaagg ggaggaggac aacatggcca 5160

tcattaagga attcatgcgc tttaaagtgc acatggaggg cagcgtcaat ggccatgagt 5220

tcgaaatcga gggcgaaggg gagggacgac catacgaggg cacacagact gcaaagctga 5280

aagtgacaaa aggaggacct ctgccattcg cctgggatat cctgtcacct cagtttatgt 5340

acggcagcaa ggcatatgtc aaacatcccg ccgacattcc tgattatctg aagctgagct 5400

tcccagaagg gtttaaatgg gagagagtga tgaactttga ggacggcggc gtggtcaccg 5460

tcacacagga ttcaagcctg caggacggag agttcatcta caaggtgaaa ctgagaggga 5520

caaattttcc atccgatgga cccgtcatgc agaagaaaac tatgggctgg gaggccagca 5580

gcgagagaat gtatcccgag gacggcgccc tgaagggaga gatcaagcag aggctgaagc 5640

tgaaagatgg gggacactac gacgctgagg tgaaaaccac ctataaagca aagaaacctg 5700

tgcagctgcc aggcgcctac aacgtcaata tcaagctgga tattacaagc cataatgagg 5760

actatactat cgtggagcag tatgagagag ccgaggggag acattcaacc ggggggatgg 5820

acgagctgta taaatgaacg gcttaagtcg acaatcaacc tctggattac aaaatttgtg 5880

aaagattgac tggtattctt aactatgttg ctccttttac gctatgtgga tacgctgctt 5940

taatgccttt gtatcatgct attgcttccc gtatggcttt cattttctcc tccttgtata 6000

aatcctggtt gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg 6060

tgtgcactgt gtttgctgac gcaaccccca ctggttgggg cattgccacc acctgtcagc 6120

tcctttccgg gactttcgct ttccccctcc ctattgccac ggcggaactc atcgccgcct 6180

gccttgcccg ctgctggaca ggggctcggc tgttgggcac tgacaattcc gtggtgttgt 6240

cggggaagct gacgtccttt ccatggctgc tcgcctgtgt tgccacctgg attctgcgcg 6300

ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc ggaccttcct tcccgcggcc 6360

tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg ccctcagacg agtcggatct 6420

ccctttgggc cgcctccccg cgtcgacttt aagaccaatg acttacaagg cagctgtaga 6480

tcttagccac tttttaaaag aaaagggggg actggaaggg ctaattcact cccaacgaag 6540

acaagatctg ctttttgctt gtactgggtc tctctggtta gaccagatct gagcctggga 6600

gctctctggc taactaggga acccactgct taagcctcaa taaagcttgc cttgagtgct 6660

tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc tcagaccctt 6720

ttagtcagtg tggaaaatct ctagcagtag tagttcatgt catcttatta ttcagtattt 6780

ataacttgca aagaaatgaa tatcagagag tgagaggaac ttgtttattg cagcttataa 6840

tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca 6900

ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctggc tctagctatc 6960

ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt 7020

atttatgcag aggccgaggc cgcctcggcc tctgagctat tccagaagta gtgaggaggc 7080

ttttttggag gcctaggctt ttgcgtcgag acgtacccaa ttcgccctat agtgagtcgt 7140

attacgcgcg ctcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta 7200

cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg 7260

cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg cgcgacgcgc 7320

cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac 7380

ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg 7440

ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt 7500

tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc 7560

cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct 7620

tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga 7680

ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga 7740

attttaacaa aatattaacg tttacaattt cc 7772

Claims (5)

1. The animal model construction method is characterized in that lentivirus is used for mediating human NPY and human AgRP to be over expressed at the paraventricular nucleus of the brain of an animal, so as to change the feeding behavior of the animal and form a corresponding animal model; the animal is macaque, the sequences of two vectors adopted by the slow virus mediation are SEQ ID NO.1 and SEQ ID NO.2 respectively, and the vectors are injected into the paraventricular nucleus of the macaque in a microinjection mode.

2. The model building method of claim 1, wherein said altering feeding behavior in an animal comprises increasing feeding time, increasing food intake.

3. The method for constructing a model according to any one of claims 1 to 2, wherein the vectors are respectively microinjected into the brains of mice to examine the expression efficiency thereof before the vectors are injected into the macaques.

4. The method of claim 1-2, wherein the microinjection is performed by injecting the vector into the paraventricular nucleus of the macaque by means of microinjection with the aid of a nuclear magnetic resonance scanning technique.

5. The method of constructing a model according to claim 4, wherein the macaque is a 1 year old macaque.