CN114047468A - Method, system and application for positioning target in animal body - Google Patents

Abstract

The invention discloses a method, a system and application for positioning an object in an animal body. The method is used for accurately positioning the position of organs in the brain of the monkey, and comprises the steps of setting a reference point, and placing a cod-liver oil particle at the skull bone seam of the monkey to serve as the reference point; fixing, namely fixing the macaque in the magnetic resonance compatible brain positioning instrument through an ear stick and a tongue plate; determining a scanning baseline, and respectively sticking three cod-liver oil particles on the horizontal position of the positioning instrument for confirming the horizontal plane during scanning and the scanning baseline; the three cod liver oil particles are distributed in the horizontal position in a right triangle shape. The positioning accuracy of the present invention can exceed millimeter (mm) levels.

Description

Method, system and application for positioning target in animal body

The application is a divisional application based on the invention patent application 'hNPY-based gene overexpression chimeric animal model construction method and application thereof' (application number 2019111075856) which is submitted to the intellectual property office of China by the applicant in 2019, 11, 13.

Technical Field

The invention belongs to the field of biotechnology, and relates to an accurate positioning technology of organs and tissues in an animal body and application thereof.

Background

In various scientific research institutions, medical colleges and medical health institutions, in the processes of biology and related scientific research, experiments, animal living body detection, medical operations and the like, animals are often required to be positioned, and animal brain stereotaxic apparatuses are generally used. For example, chinese patent application 201120190928.2 discloses an animal brain stereotaxic apparatus, which comprises: the syringe fixing column, the syringe fixing ring, the syringe fixing nut, the fixing foot, the ear rod, the fixing supporting plate, the ear rod fixing frame, the lower limb fixing button, the upper limb fixing button, the ear rod fixing nut and the ear rod fixing clamp. The fixed supporting plate is arranged on the ear pole fixing frame, the ear pole is fixed on the ear pole fixing frame through an ear pole fixing nut for an ear pole fixing clamp, the lower limb fixing button, the upper limb fixing button and the injector fixing column are respectively arranged on the fixed supporting plate, the injector fixing ring is arranged on the injector fixing column, the injector fixing nut is arranged on the injector fixing ring, and the fixing foot is arranged at the lower end of the ear pole fixing frame. Because the experimental animal binding table is additionally arranged, the animal can be completely fixed during the experiment, the stability of the experimental result is ensured, the dosage of the medicament during the injection of the medicament can be strictly controlled, and the possibility of causing experimental errors is eliminated.

However, on the one hand, the differences of each animal individual and the accurate positions of the corresponding organs and operation parts of each individual cannot be absolutely relative, and if a certain accurate position and parameter (position information, coordinate information relative to a reference system) in the animal individual are determined, the accurate positioning of the corresponding target position of each individual cannot be carried out even if accurate anatomical information exists. On the other hand, the technical solution mainly solves the problems of fixation of the brain of the animal, and the like, and it is still difficult to accurately locate internal organs, and it is often difficult to obtain approximate location information of organs only by scanning organs such as a Magnetic Resonance Imaging (MRI) apparatus, a CT apparatus, and the like, due to differences of animals and individuals, and the like (similar to the reason of the first aspect). Particularly, for some precise experimental operations such as microinjection and the like, the related organ tissues need to be precisely positioned so as to facilitate corresponding subsequent operations. Due to the requirements of the structure and the working characteristics of the conventional nuclear magnetic resonance apparatus, the conventional CT apparatus and the like, the "visualization" operation is not allowed to be performed while scanning, so that the accurate positioning of organs/tissues in the animal body becomes a general problem.

Disclosure of Invention

In view of the above disadvantages, the technical problems to be solved by the present invention are to provide a method and a device for accurately positioning internal organs, respectively, to accurately position organs and tissues in an animal body, and to further provide accurate position information; and related applications.

A monkey brain organ positioning method is used for accurately positioning the position of an organ in the monkey brain, and comprises the following steps,

setting a reference point, and placing a cod-liver oil particle at a head bone seam point of the macaque as the reference point;

fixing, namely fixing the macaque in the magnetic resonance compatible brain positioning instrument through an ear stick and a tongue plate;

determining a scanning baseline, and respectively sticking three cod-liver oil particles on the horizontal position of the positioning instrument for confirming the horizontal plane during scanning and the scanning baseline; the three cod liver oil particles are distributed in the horizontal position in a right triangle shape;

scanning, wear with nuclear magnetic resonance appearance the head the locater the kiwi fruit scans, obtains magnetic resonance and shows the looks, through show looks, based on the liver oil granule is confirmed benchmark and/or scanning baseline determine the target position coordinate value of kiwi fruit brain tissue.

Or the like, or, alternatively,

determining a scanning baseline, and respectively sticking three cod-liver oil particles on the horizontal position of the positioning instrument for confirming the horizontal plane during scanning and the scanning baseline; the three cod liver oil particles are distributed in the horizontal position in a right triangle shape;

fixing, namely fixing the macaque in the magnetic resonance compatible brain positioning instrument through an ear stick and a tongue plate;

setting a reference point, and placing a cod-liver oil particle at a head bone seam point of the macaque as the reference point;

scanning, wear with nuclear magnetic resonance appearance the head the locater the kiwi fruit scans, obtains magnetic resonance and shows the looks, through show looks, based on the liver oil granule is confirmed benchmark and/or scanning baseline determine the target position coordinate value of kiwi fruit brain tissue.

Or the like, or, alternatively,

fixing, namely fixing the macaque in the magnetic resonance compatible brain positioning instrument through an ear stick and a tongue plate;

determining a scanning baseline, and respectively sticking three cod-liver oil particles on the horizontal position of the positioning instrument for confirming the horizontal plane during scanning and the scanning baseline; the three cod liver oil particles are distributed in the horizontal position in a right triangle shape;

setting a reference point, and placing a cod-liver oil particle at a head bone seam point of the macaque as the reference point;

scanning, wear with nuclear magnetic resonance appearance the head the locater the kiwi fruit scans, obtains magnetic resonance and shows the looks, through show looks, based on the liver oil granule is confirmed benchmark and/or scanning baseline determine the target position coordinate value of kiwi fruit brain tissue.

Or the like, or, alternatively,

fixing, namely fixing the macaque in the magnetic resonance compatible brain positioning instrument through an ear stick and a tongue plate;

setting a reference point, and placing a cod-liver oil particle at a head bone seam point of the macaque as the reference point;

determining a scanning baseline, and respectively sticking three cod-liver oil particles on the horizontal position of the positioning instrument for confirming the horizontal plane during scanning and the scanning baseline; the three cod liver oil particles are distributed in the horizontal position in a right triangle shape;

scanning, wear with nuclear magnetic resonance appearance the head the locater the kiwi fruit scans, obtains magnetic resonance and shows the looks, through show looks, based on the liver oil granule is confirmed benchmark and/or scanning baseline determine the target position coordinate value of kiwi fruit brain tissue.

Preferably, the step of setting a reference point includes,

the method is characterized by comprising the following steps of creating a place 3cm behind an eyebrow arch of a macaque, taking the place as a coordinate needle insertion point, and taking an iodophor disinfection range from 3cm to 5cm, taking the point as a center to make a coronal incision of about 4cm, stripping periosteum and fully exposing a top bone and a coordinate point, drilling a small hole at the position of a bone suture point of a head bone of the macaque, and placing cod-liver oil particles in the small hole as a datum point.

Preferably, the method further comprises, before the step of creating,

anesthesia, the method: whole hemp; a channel: a channel is established by the venous indwelling needle, and a tube is sealed by normal saline; dosage of anesthetic: ketamine 2mg, push slowly; thereafter 1.5mg ketamine was administered to maintain the ciliary reflex or the caudal and limb reflex status until the end of the experiment;

shaving hair, removing head hair, and exposing whole craniocerebral cortex.

Preferably, the reference point is used as a reference point for nuclear magnetic resonance scanner scanning, the coordinate is (0, 0, 0), and the three-dimensional coordinate parameter of the organ is determined based on the precise point coordinate.

Preferably, when the cod liver oil particles are scanned by the nuclear magnetic resonance spectrometer, the appearance phase is white imaging, and the reference point coordinates in the appearance phase are determined according to the white imaging at the precise point.

Preferably, the organ is a paraventricular nucleus.

Preferably, said scanning step comprises, in particular,

scanning by using a 3.0T magnetic resonance head coil to obtain lamellar axis T1 and T2 images, imaging at a vector position T1, and scanning data are as follows: TE100-120, TR 3800-6000; the layer thickness is 2 mm; interlayer spacing 0; FOV =280 × 280 mm; determining the accurate positions of paraventricular nuclei at two sides by referring to the magnetic resonance imaging and contrast map of the brain of the macaque;

and determining a coordinate needle insertion point, measuring and calculating coordinate values of target points, and selecting 6 target points in front of and behind the target points, at the left and right sides and at the upper and lower sides by 1mm, so as to complete accurate positioning of the paraventricular nucleus.

A monkey brain organ positioning system is used for accurately positioning internal organs of a monkey brain, and comprises,

the monkey brain locator is a magnetic resonance compatible brain locator, comprises an ear bar and a tongue plate, is used for fixing the head of a macaque, and is placed into a nuclear magnetic resonance instrument for scanning;

the cod-liver oil particles are used as positioning marks, and one cod-liver oil particle is placed at a head bone seam point of the macaque as a reference point; respectively sticking three cod liver oil particles on the horizontal position of the positioning instrument, and determining the horizontal plane and the scanning baseline during scanning, wherein the three cod liver oil particles are distributed in a right-angled triangle in the horizontal position;

and the nuclear magnetic resonance instrument is used for scanning the head of the macaque and the positioning instrument by using the nuclear magnetic resonance instrument to obtain a magnetic resonance phase display, and the target position coordinate value of the brain tissue of the macaque is determined based on the datum point and the scanning baseline determined by the cod-liver oil particles through the phase display.

A positioning method for accurately positioning a target in an animal body comprises the following steps,

setting a positioning mark, and setting the positioning mark which does not move relative to the limb where the target in the animal body is located outside the animal body;

scanning, namely scanning to obtain a target in the animal body and a phase displaying of the positioning identifier, and accurately positioning the target in the animal body based on the position relation between the positioning identifier and the target in the phase displaying.

Preferably, further comprising, fixing, said animal.

Preferably, the animal is fixed by using an animal positioning instrument.

Preferably, the localized marking is a particulate matter.

Preferably, the positioning identifier includes a first positioning identifier fixed on the horizontal position of the positioning instrument and used for confirming the horizontal plane and the scanning baseline during scanning; the horizontal position has no relative displacement with the animal limb.

Preferably, the first positioning marks are three particles which are distributed in a right-angled triangle in the horizontal position.

Preferably, the positioning mark further comprises a second positioning mark arranged on the body surface of the animal body and used as a reference point; the second positioning mark is a particle object which is fixed at the body surface position; and determining a target position coordinate value in the animal body based on the datum point determined by the positioning mark and the scanning baseline.

Preferably, the positioning mark is selected according to imaging characteristics of a scanning device used for the scanning, and the positioning mark and the animal body present different imaging characteristics in a scanning phase of the scanning device.

Preferably, the scanning device is a nuclear magnetic resonance apparatus and the location markers are fat particles.

Preferably, the scanning device is a CT and the location markers are metal particles.

A positioning system for accurately positioning a target in an animal body, comprising,

the positioning mark is used for determining accurate scanning, is arranged outside the animal body and does not move relative to the limb where the target in the animal body is located;

and the scanning equipment is used for scanning the animal body and the positioning identifier to obtain a scanning display phase, and accurately positioning the target in the animal body based on the position relation between the positioning identifier and the target in the display phase.

Preferably, the animal positioning device is used for fixing the animal.

The use of the method as described above for the precise injection of a medicament into a monkey/animal.

Use of the system as described above for the precise injection of a medicament into a monkey/animal.

System as described before for accurate injection of medicament into monkeys/animals.

The method is applied to the construction of an engineering monkey model/animal model.

The application of the system in engineering monkey model/animal model construction.

The system as described above was used for engineering monkey model/animal model construction.

Through the technical scheme of the invention, the brain tissues of the macaque and the internal organs, tissues and the like of other animals can be accurately positioned based on the reference points and the scanning baselines determined by the plurality of cod-liver oil particles, and the positioning precision can exceed millimeter (mm) level. Meanwhile, the technical scheme can not cause great damage to animals such as macaques and the like, even living bodies of the animals, and even accurately position internal tissues under the condition of no damage, and has the characteristics of low biological risk, high safety, strong controllability and the like. Specifically, since the cod liver oil particles (or other fat particles) are fat components, they will form different imaging characteristics from monkey brain tissue (including skull, skin, etc. in close contact therewith) in magnetic resonance imaging (imaging), and the cod liver oil is white imaging, and monkey brain tissue, etc. is gray images of different levels, even black (as shown in fig. 8), which is sufficient to realize effective differentiation, so as to be used as a reference point for positioning reference. Meanwhile, the cod-liver oil particles adhered to the positioning instrument have imaging characteristics different from those of the positioning instrument in nuclear magnetic resonance imaging, so that the scanning baseline is positioned and determined. In addition, the distribution is in a right triangle, and only when the distribution of the three white dots in the phase development presents a right triangle, the scanning direction is vertical to the horizontal plane (scanning base line); when the three white points are in a straight line, the scanning direction is parallel to the horizontal plane (scanning baseline); meanwhile, the right triangles are distributed and can also be used as a reference system for subsequent positioning coordinates. Similarly, of course, other materials may be selected as the positioning reference point according to different scenes and scanning devices, such as CT, and metal particles may be used to achieve the same or similar technical effects.

Drawings

In order to more clearly describe the related technical solutions according to the present invention, the drawings related thereto are briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained based on these drawings without creative efforts.

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

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

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

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

FIG. 5 is a first fluorescent picture of paraventricular nuclei of macaques after virus injection (background is nuclear marker Hoechst staining);

FIG. 6 is a second fluorescent picture of paraventricular nuclei of macaques after virus injection (background is nuclear marker Hoechst staining);

FIG. 7 is a photograph of the fluorescence of the virus expressed in the brain of a mouse;

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

FIG. 9 is a graph showing the change of weight gain of experimental macaques due to the paraventricular overexpression of hNPY;

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

FIG. 11 shows the effect of nuclear overexpression of human NPY in the paraventricular nucleus of macaque on body temperature and other metabolic indicators of experimental macaque;

fig. 12 shows NPY levels, AgRP and leptin changes in cynomolgus monkey blood after virus injection.

Description of reference numerals: in the figure, brain 1, paraventricular nucleus 2, injection site 3, syringe 4, carrier injection needle point 31, auxiliary injection needle point 32.

Detailed Description

In order to facilitate further understanding of the present invention and to clearly understand technical solutions described in the present application and to fully disclose relevant technical contents of the present invention, embodiments of the present invention are described in detail below with reference to the accompanying drawings.

All other embodiments, which can be obtained by a person skilled in the art based on the embodiments of the present invention without inventive efforts, and/or even if the sequence of the individual steps is changed and various corresponding changes and modifications are made according to the present invention without departing from the spirit and the essence thereof, the corresponding changes and modifications shall fall within the protection scope of the present invention.

The invention relates to the following definition or supplementary explanation of related terms:

NPY: namely Neuropeptide Y, known collectively as Neuropeptide tyrosine, Neuropeptide Y, abbreviated NPY. NPY is a polypeptide consisting of 36 amino acid residues, belongs to the 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 from "NPY gene", but in the present application, for the sake of simplicity of description, the gene name of "NPY gene" is represented by NPY under different specific descriptions.

hNPY: namely human-derived NPY, known collectively as human-derived Neuropeptide tyrosine (also used in "human-derived Neuropeptide Y"), human Neuropeptide Y, english, abbreviated hNPY. In this document, NPY is abbreviated as hNPY unless otherwise specified. Of course, other NPY can be used to achieve the same or similar effect based on the corresponding biological properties. However, in the present invention, as a preferable embodiment, in order to obtain a clinically (human) relevant anthropomorphic animal model, human-derived NPY is selected; of course, the required NPY can be selected based on other requirements. 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 under different specific descriptions for the sake of simplicity of description.

AgRP: it is called guinea pig-related protein (also using "Agouti-related protein"), AgRP in English. The AgRP is a protein synthesized based on an "AgRP gene", but in the present application, the gene name of the "AgRP gene" is represented by AgRP in the case of different specific descriptions for the sake of simplicity of description.

hAGRP: namely, human AgRP, which is called human guinea pig-related protein (also used in "human Agouti-related protein"), and human Agouti-related protein, abbreviated as hAGRP. In this document, unless otherwise specified, the AgRP is an abbreviation for hAgRP. Similar to the selection of hNPY in the invention, other AgRPs can be applied to realize the technical scheme of the experiment, and the same or similar effect can be achieved. However, in the present invention, as a preferable scheme, in order to obtain a clinically (human) relevant anthropomorphic animal model, human-derived AgRP is selected; of course, the required AgRP can be selected based on other requirements. The "hAgRP gene" is a gene name of the "hAgRP gene" which is expressed by hAgRP in the present application, for the sake of simplicity of description.

PVN: known as the hypothalamic Paraventricular nucleus, the English Pararentrical nucleus of the hypothalamus, abbreviated PVN, also abbreviated PVH, but PVN is more commonly used and is abbreviated PVN in the present application.

Hemagglutinin, abbreviated HA.

FLAG-tag, i.e. FLAG label, corresponding to a peptide segment DYKDDDDK (peptide sequence DYKDDDDK) of 8 amino acids, which is used for marking target protein. In the research of protein expression and location, a target gene to be researched and a FLAG-tag gene sequence can be connected through a genetic engineering technical means, and can be connected to the C end or the N end of the target protein, then the integrated gene is transferred into a cell or an embryonic stem cell or a fertilized egg, the subsequent detection is mainly realized through the specific combination of an immune determinant formed by the FLAG-tag peptide chain and a monoclonal antibody thereof, and the detection means comprises immunofluorescence (immunofluorescence), Western Blotting (Blotting) and the like.

Animal models (i.e., animal disease models) are used primarily for experimental physiology, experimental pathology, and experimental therapeutics (including new drug screening) studies. The development of human diseases is very complex, people themselves are taken as experimental objects to deeply discuss disease occurrence mechanisms and promote the slow development of medicine, and the clinically accumulated experience has limitations in time and space and limits in practicalities and methods. By means of indirect research of the animal model, factors which are impossible or difficult to eliminate under natural conditions can be changed consciously, so that the experimental result of the model can be observed more accurately and compared with human diseases for research, the occurrence and development rules of the human diseases can be known more conveniently and more effectively, and prevention and treatment measures can be researched.

Animal models of human disease (animal models of human disease) refer to animals with simulated manifestations of human disease that have been established in various medical science studies.

Chimeric animal models (i.e., chimeric gene animal models, also referred to as genetic chimeric animal models, and the former is used herein collectively) are non-germline animal models that are created by purposefully engineering genes (genes in somatic cells) of local tissues or organs of animals to induce phenotype generation, and thus form relevant pathological models (animal models). The chimeric animal model is a continuation and complement of the stably inherited animal model. The genetic modification technology 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. In addition, the invention 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, which refers to the actions and activities performed by the animal, including the position movement of the animal, limb movement, eating, puppet, fighting, body color change, and the like; is the sum of all reactions adapting to the change of in vivo and in vitro environments in order to meet the basic survival and reproduction requirements of animals. All behaviors of animals depend essentially on three factors: external and internal stimulation, the functional status of the sensory organs and nervous system of animals, and the structural and physiological properties of animal bodies.

The animal behavior model is an animal model established based on animal behaviors, and comprises the steps of developing the animal behaviors according to the needs of a model builder through various external and internal stimuli (such as physical stimuli, chemical stimuli, biological stimuli and the like), intervention and the like, and influencing the physiology and the like of animals based on the change of the behaviors, so that an animal disease model meeting the requirements of the model builder is established. The invention discloses a method for realizing the 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 the needs.

Over-expression of the gene: gene overexpression is achieved by using transgenic technology, so that a specific gene is expressed beyond the original level. The basic principle of gene overexpression is to obtain a target gene plasmid containing an upstream regulatory element by an artificial construction mode, transduce the plasmid into cells or tissues and organs by transgenic operation, realize mass transcription and translation of a target gene, and further realize overexpression of a gene product. Gene overexpression does not involve gene editing, and is achieved by randomly inserting a gene (target gene) carried by a vector into a chromosome, and separately transcribing the target gene fragment independently of the original gene sequence, so that the expression of the target gene in a cell exceeds a normal level, but the fragment is free from the original DNA. For example, the target gene hNPY gene in the application is introduced into the macaque in a large amount, so that the target gene is transcribed beyond the original level of the target gene, 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 also disclosed, and the relevant application is realized based on the animal model and the method. Since hyperphagia is the main cause among all the factors responsible for the obesity epidemic, the inventors have found in their studies that the hypothalamus is the main area in the brain that controls feeding, and is involved in sensing nutrient and energy levels, regulating food intake and energy expenditure, and thus maintaining a normal metabolic balance. Multiple nuclei in the hypothalamus, such as the arcuate nucleus, paraventricular nucleus, ventral medial nucleus, and the lateral hypothalamic region, are involved in regulating the balance of energy metabolism. The neurons in the nerve nuclei carry out feeding regulation through different chemical signal molecules, such as transmitters, cytokines, neuropeptides and the like, but how to establish animal models with stable characters in batch by adopting a controllable and effective technical scheme has a plurality of difficulties and obstacles, and the existing research and exploration cannot be realized by adopting a simple scheme for conversion. Through repeated theoretical analysis and experiments, the inventor of the invention finds that because NPY in the hypothalamus mainly comes from neurons in the arcuate nucleus, axons of NPY neurons directly project to paraventricular nucleus, and the paraventricular nucleus is the main target area of NPY neurons, a nerve signal path from the arcuate nucleus to the paraventricular nucleus plays an important role in regulating and controlling feeding behaviors. Based on the findings and related researches and experiments, the over-expression of NPY is realized in the hypothalamus by a (transgenic) technology, a corresponding animal model is constructed, particularly a chimeric gene animal model is constructed, and a non-human primate chimeric gene animal model is constructed based on a non-human primate-macaque, particularly a chimeric gene engineering monkey model; based on the technical scheme, the method can also be used for constructing animal behavior models.

In the aspect of specific implementation of the technical scheme, as a preferred technical scheme, the nuclear magnetic resonance scanning technology is utilized to accurately inject the hNPY lentiviruses (Lenti-hNPY) respectively containing specific expression into bilateral paraventricular nuclei of young experimental macaques. The experimental result shows that the body weight and the feeding behavior of the hNPY overexpression macaque are remarkably increased (the eating behavior is changed, the food intake is promoted to be increased, and the body weight is increased). Obviously, the non-genetic chimeric gene engineering macaque model can be used for obesity research and exploring a new clinical treatment strategy aiming at obesity as a clinical transformation obesity model; of course, further conversion to a diabetes model is also possible.

In addition, as one of the invention points of the invention, the invention provides a chimeric gene animal model, optimizes the gene sequence of the used vector, particularly optimizes the used target gene hNPY and the like (see a Lenti-hNPY sequence table specifically) so as to improve the construction effect of the chimeric gene animal model, and particularly has a remarkable effect on the construction of a chimeric gene engineering monkey model.

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

the inventor of the invention finds that in the process of realizing the invention, in order to solve the problem that the occurrence rate of obesity is gradually increased to influence the public health of people of all ages, new obesity treatment means need to be continuously developed, relevant pathogenic mechanisms need to be researched, and the like, and the appropriate animal model needs to be utilized for preclinical research. Animal models of dogs, pigs, mice and rats have been used extensively to study obesity, and the health problems associated with obesity. In order to construct a corresponding animal model, there are feeding-induced animal models and genetic engineering models in the prior art. The research of rodent model greatly promotes the exploration of the causes of human obesity. However, the discovery of rodent experiments often fails in clinical transformation, and how to realize a more effective preclinical animal research model is a technical problem to be solved currently. Because of genetic and physiological similarities to humans, a non-human primate model would be a better model to carry out valuable transformation medicine and would be a bridge linking rodent experiments with human clinics. The invention provides the construction of an animal model by the overexpression of human NPY/AgRP mediated by lentivirus in paraventricular nucleus, in particular to the construction of the animal model of macaque by the overexpression in paraventricular nucleus 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 realized by the following examples.

As shown in fig. 1, according to the requirement of animal model construction target, animal screening and vector construction are performed, the vector is introduced into animals and animal care, the target gene in the vector realizes gene overexpression transcription in animal bodies (cells), so that animals form a character meeting the animal model construction target, and the animal model character meeting the construction template becomes a corresponding animal model by inspecting the animal model character. The concrete description is as follows:

in the application, in order to construct a chimeric gene engineering monkey model for regulating and controlling feeding behaviors, macaques are taken as objects, hNPY is selected as a target gene, and the objective of the invention is realized by performing corresponding gene modification on paraventricular nucleus of the macaques through a transgenic technology, namely gene overexpression.

First, animal selection

With macaques, it is of course possible to select other types of animals as desired, and in the present invention, in order to achieve genetic and physiological similarities to humans, it would be better to select a non-human primate model, which would be a valuable transformation medical model to better simulate the relevant pathological models of humans. In the present invention, it is considered that spontaneous obesity of monkeys is gradually generated and developed in adult period. In order to investigate whether genetic engineering of young macaques can cause obesity in experimental animals, the inventors of the present invention selected male macaques of about 1 year old and half year old for experiments (macaques of other age groups may be selected, and preferably macaques of 1-2 years old). In order to facilitate the control of the experiment, male macaques of 1 year old are selected in the experimental process of the invention. Of course, the selection of other primates or macaques or primates of other ages can also practice the present invention. Based on the technical idea of the invention, different selection standards and conditions can be adopted according to different applications.

In the verification experiment of the invention, 24 male macaques aged 1 year are adopted as experimental objects, wherein 8 male macaques are injected with control viruses (used as a control group), 8 male macaques are injected with NPY gene overexpression viruses Lenti-hNPY (used for constructing the mosaic based animal model), and 8 male macaques are injected with AgRP gene overexpression viruses Lenti-hAGRP (used for constructing the mosaic based animal model). The subsequent operating conditions and procedures (including the dosage of various required instruments, medicines, reagents, etc. determined based on the types and quantities of the aforesaid experimental objects, which can be adjusted and changed according to different objects) are all explained based on this, however, the selection of the experimental objects does not represent the limitation of the technical solution of the present invention, especially does not represent the limitation of the application scope and protection scope of the present invention, and is only convenient for scientific and objective comparison and experiment to illustrate the scientificity, feasibility, practicability and corresponding technical effects of the present invention.

Secondly, vector construction (virus preparation).

In specific applications, as a preferred scheme, the Lenti virus-based construction vector is used for determining gene overexpression (of course, other corresponding virus-based construction vectors can be selected according to application requirements). Meanwhile, as the organ positioned by the invention is the brain, subtype virus with stronger infectivity on cerebral neurons is used. As shown in fig. 3, in the experiments to verify the present invention, the viral vector names used were as follows:

lenti viral vectors overexpressing genes of interest: Lenti-hsyn-NPY-2A-mcherry (abbreviated as Lenti-hNPY) (as shown in FIG. 3 (a)); Lenti-hsyn-AgRP-2A-mcherry (Lenti-hAGRP for short); the viral vector sequences are shown in the corresponding sequence listing attached to this application. Unless otherwise specified, the vectors or viruses described herein are Lenti viral vectors that overexpress the gene of interest.

Control viral vectors: lenti-hsyn-mcherry (shown in FIG. 3 (b)) uses the same lentiviruses as Lenti-hNPY and Lenti-hAGRP described above, and is different in that the target genes hNPY and hAGRP are not included. In practical applications, the control viral vector is not of practical significance, and in the present application, the injection of the vector is aimed to be compared with the macaques injected with the aforementioned viral vectors Lenti-hNPY and Lenti-hAGRP, so as to test the practical technical effects of the present invention.

Description of the role of elements in viral vectors:

lenti is a name prefix used for distinguishing virus types, such as Lenti (namely Lenti virus); hsyn is the name of the promoter driving expression of different genes; NPY, AgRP and the like are target genes or sgRNA names; 2A is a connecting sequence between two genes; mCherry is a fluorescent protein gene, and forms a fluorescent detection mark for indicating the expression position of the virus on the body; flag, HA is usually expressed as a fusion with the gene of interest, and is a tag sequence for detecting the gene of interest. It should be noted that, because of the problem of writing habit, the same meaning is given by case in this application for each element of the carrier and unless otherwise specified except for the unit of measure (international unit).

Lentivirus (generally referred to as lentii) is a widely used transgenic vector, which can effectively transfer exogenous genetic materials to both dividing and non-dividing mammalian cells and finally integrate into the cell genome. The Lenti lentivirus is used as a vector, and a target gene is transferred to a somatic gene to be modified. FIG. 3 (a) schematically shows the structure of the Lenti-NPY lentiviral expression vector for the corresponding vector (only relevant elements for the specific part are shown, no other elements are shown, the complete structure is in a closed-loop plasmid.)

Further, for safety and effectiveness verification, the virus should pass through a mouse in-vivo expression test experiment before use, and the specific test experiment mode is the prior known technical scheme. FIG. 7 shows fluorescence images of the expression of the above virus (Lenti-hNPY viral vector) in mouse brain. The picture shows that the human NPY lentivirus has high expression in the brain of a mouse, and the virus can achieve the aim of gene modification and has safety.

As shown in FIG. 3 (a), a first constructed Synapsin (Chinese name: neuronal Synapsin, abbreviated as Syn) is a promoter-driven FLAG-labeled hNPY lentiviral expression vector and an HA-labeled hAGRP lentiviral expression vector (FLAG and HA are two commonly used labels, and different labels are used for hNPY and hAGRP in the experiment of the invention in order to distinguish NPY target proteins from AgRP target proteins in the future experiment, so that over-expression of hNPY and hAGRP can be distinguished, other labels can be selected in specific application or exchanged, namely the FLAG and the HA can be selected according to needs. In order to examine the expression efficiency of the vector, the expression efficiency was examined by injecting two lentiviral vectors into the brain of a mouse, respectively (this is not an essential step and can be omitted in specific applications). Three weeks after microinjection, the inventors of the present invention obtained frozen sections of mouse brains and detected mCherry red fluorescence signals co-expressed with NPY or AgRP. The results showed that both lentiviruses produced a large amount of red fluorescence signal in the mouse hippocampal nerve dentate gyrus region (as shown in FIG. 7), and that the Lenti-hNPY virus showed high levels of hNPY expression 4-6 weeks after mouse hypothalamic injection. Obviously, these two lentiviruses can be used for the next experimental procedure of brain injection in macaques. Preferably, the invention uses hSynapsin (human Synapsin) -promoter, which is named as human neuron synapsin promoter in Chinese, abbreviated as hSYN (both upper and lower cases). Specifically, in the characteristic partial structure of the vector shown in FIGS. 3 (a), (b), LTR (abbreviation of Long Terminal Repeat, Chinese Long Terminal Repeat, which is a Long Terminal Repeat present on both flanks of LTR retrotransposons (LTRs) and is a part of a plasmid) is a fixed sequence in a viral vector; hSynapsin is a promoter; hNPY is a target gene needing overexpression; 2a is a coupling fetus for co-expression of mcherry; mcherry is a red fluorescent protein used to indicate viral expression; WPRE is a transcription enhancing element. FIGS. 3 (a) and (b) show the structure of a vector expressed in the form of a linear plasmid, in which LTRs at both ends of the linear plasmid are linked by a closed-loop plasmid.

The expression of each gene is controlled by its own associated regulatory elements, common ones including: 1, promoter (promoter), the promoter in the invention is hSynapsin (i.e., human Synapsin, abbreviated as hSyn); enhancer (enhancer) (not shown).

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

By analyzing brain slices of one euthanized cynomolgus monkey, the inventors of the present invention found that the expression of AgRP lentivirus could be detected in the paraventricular nucleus region with high expression efficiency (as shown in fig. 5 and 6). Fig. 5 shows fluorescence images of paraventricular nuclei of macaque after virus injection (background is cell nucleus marker Hoechst staining), wherein the left side of the image is a macaque brain structure schematic diagram, and the right 4 images are sequential slice fluorescence images of the PVN part of macaque. For experimental reasons (to reduce the cost of the experiment and to manage the surviving macaques, only the Lenti-hAGRP vector was sectioned to examine the experimental status), in fact, the Lenti-hNPY vector also 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 10⁹Mu/l.

The vector and the vector reagent containing the vector can be prepared according to the conventional standard procedures, and the introduced vector is injected into the paraventricular nucleus 2 of the macaque in a carrier reagent mode. The reagents used may be any conventionally known reagents.

Third, introduction of vector

The vector is introduced into the body of the macaque, particularly in the appropriate location of the brain. The introduction can be realized by adopting the prior art means such as direct injection, microinjection, introduction by an intervention mode and the like, and can also adopt the novel introduction means appearing in the future. In the present invention, the following precise positioning introduction scheme is preferably adopted, specifically including:

the required instruments are as follows: nuclear magnetic resonance apparatus (MRI for english abbreviation, such as 3.0T, also can use other specifications), brain locator for monkey, brain locator operating arm, micro-injection pump, cranial drill, operating lamp, surgical instrument, special transport vehicle (platform) for macaque, etc.

Reagents, drugs and disinfectants: vectors obtained as described above, such as Lenti-hNPY, Lenti-hAGRP overexpression virus, PBS, saline, ketamine 0.1 g/log, midazolam 10 mg/log, nicotemam 0.375 mg/log, cod liver oil phi 5mm, saline 0.9% (250 ml), iodophor disinfectant 500 ml/bottle, veterinary antibiotics. The above-mentioned other drugs, besides the related vector virus, can be selected and matched as required.

Consumable material: the surgical operation device comprises an operation coat, an operation cap, a plurality of masks, ten pairs of sterile operation gloves, 24 skin preparation bags, 24 debridement suture bags, 24 gelatin hemostatic sponge bags, 24 seven-gauge indwelling needles and empty needles (1 ml × 24 tools, 2ml × 24 tools and 20ml × 24 tools).

Step (as shown in figure 1 and combined with figure 2):

1. anaesthesia

1) The method comprises the following steps: general anesthesia, channel: a channel is established by the venous indwelling needle, a normal saline tube is sealed, and the dosage of anesthetic is as follows: ketamine 2mg was pushed slowly. Thereafter 1.5mg ketamine was administered to maintain the ciliary reflex or the caudal and limb reflex status until the end of the experiment. (the initial ketamine dose can be increased).

2) The hair of the head is removed, and the whole craniocerebral cortex is exposed.

3) Opening a wound: taking the position 3cm behind the eyebrow arch as a coordinate needle insertion point (coordinate point), the iodine disinfection range is 3cm to 5cm, taking the point as the center to make a coronal incision about 4cm, stripping periosteum to fully expose the apical bone and the position of the coordinate point. A small hole is drilled at the position of the seam of the head bone of the macaque for placing a cod liver oil particle as the following MRI scanning base point (the original point: 0, 0, 0). The cod-liver oil particles aim at forming imaging characteristics (the cod-liver oil is white imaging) different from monkey brain tissues on a Magnetic Resonance Imaging (MRI) by using grease so as to be conveniently used as reference points for positioning, of course, other materials can be selected as positioning reference points according to different scenes and scanning equipment, and metal particles and the like are adopted, such as CT and the like. Figure 8 shows a picture of a part of the experimental macaque nmr scan. The original scan picture of experimental macaque number C1406067 was lost. The white bright spots at the top of the head of the experimental macaque 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 convenient to display, and subsequent marks are artificially added and used for determining the three-dimensional coordinate parameters of the paraventricular nucleus of the experimental animal.

4) Fixing: the macaque is fixed in the magnetic resonance compatible brain locator through the ear stick and the tongue plate.

5) MRI scan and target calculation: the macaque wears the locater to carry out magnetic resonance scanning. Scanning by using a 3.0T magnetic resonance head coil to obtain lamellar axis T1 and T2 images, imaging at a vector position T1, and scanning data are as follows: TE100-120, TR 3800-6000; the layer thickness is 2 mm; interlayer spacing 0; FOV =280 × 280 mm; a small hole is arranged at the position of the head bone seam of the macaque, and one cod-liver oil particle is placed as a base point for confirming the coordinate of the injection part. Three cod-liver oil particles are adhered to the horizontal position of the positioning instrument, are placed in a right triangle and are used for confirming the horizontal plane during scanning and a scanning base line, and the scanning range is from the skull base to the skull top and comprises all magnetic resonance positioning points. Bilateral paraventricular nuclei were determined by reference to cynomolgus magnetic resonance imaging (i.e., imaging) and control maps. And determining a coordinate needle insertion point, measuring and calculating the coordinate value of a target point, and selecting 6 target points in front of and behind the target point, at the left and right sides and at the upper and lower sides by 1mm, thereby completing accurate positioning of the paraventricular nucleus of the hypothalamus. The whole process is about 1-1.5hr after modification.

The purpose of this step is to determine the exact injection site for each individual, because the exact location of the corresponding organ and operation site for each individual is different due to individual differences. Other measurement modes or direct operation can be adopted for other objects and other parts which can be visually displayed.

Simultaneously, above-mentioned 1) -5) aim at be convenient for the kiwi fruit fixed, carry out the accurate location and the operation of injection position, technical scheme also can realize with other modes, also can replace or omit as required.

6) Opening a hole: carefully taking the macaque strip locator out of the magnetic resonance apparatus, and paying attention to ensure that the skull does not displace in the locator in the process. A bone window of about 6mm to 8mm in size centered on the injection origin was opened to fully expose the meninges. The temperature reduction is noticed by normal saline in the process of opening the hole, the skull drilling force is reduced when the skull is close to the periosteum, and the skull and the meninges are prevented from being penetrated instantly to cause cortical injury. Finally, the thin layer of the left skull is removed by a needle or forceps, and the meninges are exposed.

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

7) And (3) injecting a reagent: the mechanical arm, the micro-injection pump and the injection needle are assembled.

One of the experimental macaques is used as follows:

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 anteroposterior and depth distances between the paraventricular nucleus converted from the previous magnetic resonance scanning and the origin on the coordinates of the origin. The movement of the positioning arm and the needle inserting operation are carried out according to the coordinates, if a blood vessel is obvious right below the needle inserting point, the positioning arm is moved forward or backward a certain distance to avoid the blood vessel and then the needle is inserted, and the distance is recorded.

Injection time per site: 2 min/mul virus volume (i.e. vector reagent), needle retention for 7-8min per site. The slow extraction of the syringe takes 3-4 min. The concentration of vector particles in the injected viral (vector) reagent was 10⁹μ l, such that the number of vector particles entering paraventricular nucleus 2 is significantly greater than the cell number of paraventricular nucleus 2 and more than 2 times the cell number to ensure the achievement of overexpression.

For the convenience of understanding, the following references are given by way of example, and the specific coordinate values shown below are only illustrative, and in the specific implementation, the relevant data needs to be checked and recorded according to specific situations.

In-situ coordinates: x =7.08cm, Y = -2.19cm, Z =6.32 cm;

and (3) right: x =1mm, Y = -2.19cm, Z =6.32 cm;

left: x =1mm, Y = -2.19cm, Z =6.32cm (30 cm full scale)

As shown in fig. 4, it is a schematic diagram that the whole cynomolgus monkey brain divides the monkey brain into four parts, upper, lower, left and right, with sagittal plane and coronal plane respectively (the dotted line in the figure is the information boundary of the four parts): the carrier injection needle-feeding points 31 are located at the center, and the auxiliary injection needle-feeding points 32 located around the modified points include the upper left (point a), the lower right (point c), the upper right (point d), and the lower left (point b).

Carrier injection needle entry point 31: injecting 2 mul of carrier;

and a point: injecting 2 mul of purple staining agent;

X=7.38 cm，Y=0.29cm，Z=3.32cm；

correcting an original point: x =7.08cm, Y = -2.19cm, Z =6.68 cm.

b, point: injecting 5 mul of grease;

X=7.08-0.1=6.98cm；

Y=-2.19cm，Z=6.68-2.8=3.88cm。

and c, point: injecting 2 mul of green staining agent;

correcting an original point: x =7.08cm, Y = -2.19cm, Z =5.85cm

C point coordinate: x =7.38cm, Y =0.09cm, Z =5.85-3.0=2.85cm

And d, point: injection regulation and control virus 2 mul

X =6.98cm, Y = -2.19cm, Z = subcutaneous 0.4 cm.

8) Post-injection magnetic resonance imaging scans (derived imaging data maps for subsequent analysis to verify experimental results).

9) Suturing an incision

Sterilizing the wound surface and the periphery of the wound surface by 3-5 cm, filling the open bone wound part with gelatin sponge, and suturing the scalp. Cleaning the suture wound surface, and supplementing physiological saline and antibiotics in time.

Of particular note, the diets of the macaques prior to anesthesia were fasted for 8 hours and prohibited for 6 hours; during anesthesia, the breathing and the heart rate of the macaque are closely observed, and a person who is specially used for observing whether the macaque is abnormally rescued immediately; stopping bleeding at the incision and preventing outflow of intracranial contents due to pressure or posture change when the incision is opened; the vital signs of the macaques are closely observed in the process of transporting the macaques; the whole experiment process pays attention to the heat preservation of the macaques and the constancy of the external temperature; the operation is rapid and accurate, the progress of each step of the experiment is not delayed, and the fixed reading and the medication of each operation are skillfully carried out; surely, do not mistakenly put the iodophor disinfectant into the cranium; recording in time and recording of magnetic resonance imaging in the scanning process; nursing records of nursing specially-assigned persons after operation; especially to avoid postoperative infections, and to observe later the associated behavior by specific personnel.

In practical application, besides the above manner, direct positioning injection can be adopted without creating skull, such as positioning high-speed steel needle, high-speed insertion under the conditions of disinfection and anesthesia, virus injection, etc., or other existing or new technologies for relevant operations.

It should be noted that, although there is a technical scheme for introducing the virus by microinjection that a local part of the head of the macaque is subjected to traumatic treatment by using medical equipment, instruments and medicines, in order to accurately introduce the vector into a proper position (such as paraventricular nucleus) of the brain of the macaque while minimizing the damage to the macaque and minimizing the influence of other medicines and the like on the macaque, the treatment does not relate to the diagnosis and treatment of diseases.

And fourthly, animal nursing (nursing and feeding).

As shown in fig. 2, for the carrier introduction implemented by the above-mentioned incision wound manner, the macaque after the carrier introduction is sent into the observation room, and after the macaque is fully revived and the vital signs are normal and stable, and the wound has no bleeding, the antibiotic treatment is started, and preferably, the injection can be performed, and the specific injection measurement can be implemented according to the scheme disclosed in the prior art. In the healing process of the macaque wound, the macaque wound is treated correspondingly by adopting the prior art scheme, and medicines can be used as required to promote the healing of the macaque wound. Subsequently, the macaque is fed by adopting the existing common feeding mode, the vital sign change condition of the macaque is noticed, and the corresponding observation record is made. The macaque can be recovered basically and completely after two weeks of careful attention. After the carrier injection, the state of the macaques must be monitored in real time (BW monitoring) during the period of macaque resuscitation and wound healing, and after about two weeks (day 14) after the carrier injection, the macaques can be managed and raised in a conventional manner.

For macaques which are introduced with the vector in other modes, the macaques should be sent into an observation room to be observed correspondingly until the vital signs are normal and stable.

Gene overexpression transcription

After the vector is introduced into paraventricular nucleus 2 of a macaque, the injected Lenti-hNPY vector is transcribed into genes in somatic cells of the paraventricular nucleus 2 due to biological activity, so that over-expression of hNPY in the paraventricular nucleus 2 is realized, phenotype generation is induced, and a related pathological model is formed. The realization form comprises:

the hNPY is free from the original target DNA, is not randomly integrated and inserted into the chromosome DNA, is only transiently expressed in cells, and gradually diluted with the over-expressed gene fragment hNPY along with cell division, but cannot be diluted due to the non-regenerability of brain cells, so that the stability of the character of the chimeric gene animal model is ensured. Alternatively, the hNPY is free from the original target DNA, but randomly integrated into the genomic DNA, and the overexpressed gene fragment can be transferred to new cells formed by division during cell division (which also may be useful for other regenerable tissues or organs, and to some extent, maintains the stability of the chimeric gene animal model trait). Since the overexpression of the inventor is carried out in somatic cells, but not germ cells, the overexpression cannot be inherited to next generation animals, and the stability of the self genetic characteristics of the animals is not influenced; of course, other chimeric gene animal models can be constructed again for the offspring without being influenced by the chimeric gene animal model constructed in the previous generation.

Of course, random integration of over-expressed gene fragments into genomic DNA will result in loss of function or gain of the gene or element associated with the insertion site depending on the location of the insertion site and whether the insertion site is functional. However, since the principle is random, the process is usually defined as a transgenic operation, not a gene editing. In contrast, gene editing is a fixed point, and the post-editing result can be predicted before the experiment begins.

Sixthly, animal model character test

By analyzing brain slices of one euthanized cynomolgus monkey, the inventors of the present invention found that the expression of AgRP lentivirus could be detected in the paraventricular nucleus region with high expression efficiency (as shown in fig. 5 and 6). Fig. 5 shows fluorescence images of paraventricular nuclei of macaque after virus injection (background is cell nucleus marker Hoechst staining), wherein the left side of the image is a macaque brain structure schematic diagram, and the right 4 images are sequential slice fluorescence images of the PVN part of macaque. For experimental reasons (to reduce the cost of the experiment and to manage the surviving macaques, only the Lenti-hAGRP vector was sectioned to examine the experimental status), in fact, the Lenti-hNPY vector also had the same expression due to the use of the same lentivirus.

Further, the experimental results shown in FIGS. 9 to 12 are explained in detail below.

The effect on the weight of the experimental macaques on the overexpression of hNPY is as follows:

after the completion of the virus microinjection experiment, the experimental macaque was almost completely recovered after two weeks of careful attention. The inventors of the present invention weighed the weight of each experimental macaque every two weeks. Previous experimental results in adult rats showed that virus-mediated overexpression of NPY in the paraventricular nucleus would lead to weight gain. When experimental macaques were 2 years old, the weight of the NPY over-expressed macaques increased significantly compared to the control macaques. Interestingly, the experimental macaques in the NPY group showed significant weight gain at month 6 after virus injection. As shown in fig. 9, the change of weight of experimental macaque caused by the overexpression of hNPY in paraventricular nucleus of macaque is shown in the graph of weight change per month, and the weight of experimental macaque in NPY group starts to increase significantly after 6 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: mean standard error (error bars are mean s.e.m. (standard error), i.e., mean standard error, the same below).

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

FIG. 10 shows the effect of month 7 after virus injection (analyzed for the first 3 days) on feeding behavior, food intake and serum insulin in macaques, showing that both feeding time and food intake increase in experimental macaques of NPY group (a, b in the figure) at month 7 after virus injection; the motor behaviour and sleep time were not affected by the virus injection (c, d in the figure). The blood glucose concentration of experimental macaques in the NPY group did not change, while serum insulin levels increased at month 10 after virus injection; after month 12 post virus injection, serum insulin levels returned to normal (e, f in the figure). Data: mean standard error. In d of FIG. 11, the body temperature changes of the control group and the NPY experimental group continuously monitored for three days at month 7 after the virus injection are shown, and no significant difference is observed.

The influence of human-derived NPY overexpression on the feeding behavior of the experimental macaque is detected through video recording analysis and feeding monitoring. As shown in fig. 9, the weight of the cynomolgus monkeys in the NPY overexpression experimental group significantly increased 7 months after virus injection compared to the control cynomolgus monkeys; as shown in fig. 10, the feeding time and food intake of the macaques in the NPY overexpression experimental group were increased during the 3-day continuous video recording period. In addition, the exercise capacity and sleep time of the experimental macaques in the contemporary NPY overexpression group were similar to those in 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 are caused by the overexpression of NPY in the hypothalamic paraventricular nucleus, and are not related to the change in exercise capacity. The inventors of the present invention further measured the body temperature of experimental animals and found that the body temperature of experimental macaques of NPY or AgRP overexpression group was not abnormal at 7 months after virus injection, compared to macaques of control group (fig. 11 e).

The influence of hNPY over-expression on endocrine and metabolic biochemical indexes of experimental macaques is as follows:

FIG. 11 shows the effect of hNPY overexpression in cynomolgus monkey paranuclear on body temperature and other metabolic indicators in experimental macaques, wherein: and a, the concentration of triglyceride, cholesterol, high density lipoprotein, low density lipoprotein and non-esterified lipid in a control group and an NPY experimental group before virus injection. b, 10 months after virus injection, concentration of triglyceride, cholesterol, high density lipoprotein, low density lipoprotein, non-esterified lipid in control group, NPY experimental group and AgRP experimental group. c, month 12 after virus injection, concentration of triglyceride, cholesterol, high density lipoprotein, low density lipoprotein, non-esterified lipid in control group, NPY experimental group and AgRP experimental group.

The inventor of the invention analyzes partial endocrine and metabolic biochemical indexes from the collected serum of experimental macaques in the 10 th and 12 th month periods before (0 point) and after NPY or AgRP virus injection. As shown in e of fig. 10, blood glucose levels were normal in the NPY or AgRP group experimental macaques at three periods; in the 10 th month after injection, the serum insulin concentration of the experimental macaque in the NPY group is obviously higher than that of the control group; however, at month 12, as the body weight of the macaques tested in the NPY group did not change significantly in the winter, the change in serum insulin concentration was also not significant (see fig. 10 f). No significant difference was observed in all three phases from their body weights. The change in serum insulin concentration correlates with the body weight of the experimental animals. The detection results of other blood biochemical indexes show that the concentration of triglyceride, cholesterol, high-density lipoprotein, low-density lipoprotein and non-esterified lipid in the blood serum of the experimental macaque is not abnormal in the over-expression process and is not influenced by the over-expression of NPY.

The influence of the paraventricular nuclear overexpression of the lentivirus-mediated hNPY on the body weight, the feeding behavior and the endocrine metabolism index of the young macaque is detected. Overexpression of human NPY in the paranuclear region of the experimental macaque caused increases 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 paraventricular nucleus of experimental macaques, at month 19 post-viral injection, caused weight gain; this finding is different from the results of AgRP in rodent experiments. Because the expression levels of lentiviruses respectively expressing hNPY in animal bodies are close, the inventor analyzes that the regulation and control of hNPY on the body weight are realized by different mechanisms in the early development stage of the macaque, so that the hNPY can regulate and mediate the body weight change of the macaque at different stages.

The NPY group generated weight gain in experimental macaques 10 months after virus injection, accompanied by an increase in serum insulin levels. When the weight of the experimental macaques in the NPY group is reduced to be similar to that of the macaques in the control group in winter, the serum insulin level is also recovered to be normal. This finding is consistent with the known weight loss in humans being a determinant of blood insulin levels. In previous studies, the effects of NPY overexpression on blood glucose concentration and body temperature were inconsistent. The present inventors' studies were not observed before and after the virus injection.

The differences between the body temperature and the blood sugar concentration of the experimental macaques in the hNPY group and the body temperature of the control group are as follows:

the inventor of the invention does not observe the concentration abnormality of other blood biochemical indexes such as triglyceride, cholesterol, high-density lipoprotein, low-density lipoprotein, non-esterified lipid and the like in the blood serum of the experimental macaque during overexpression. This observation is consistent with the reported transient effect of long-term NPY perfusion on fatty acids.

In addition, the NPY level, AgRP and leptin change of the blood of the macaque after virus injection are shown in FIG. 12, and the meridional analysis of variance and post-hoc tests show that the NPY level, AgRP and leptin of the blood are not changed in the macaque injected with Lenti-hNPY at the 10 th, 12 th and 19 th months after virus injection (data: standard error of mean value), and it can be seen that the Lenti-hNPY injected into the paraventricular nucleus 2 only affects the gene of the local tissue of the macaque, thus forming the chimeric gene animal model.

In summary, experimental data of the present inventors show that overexpression of hNPY in the paraventricular nucleus of young macaques will lead to weight gain. The changes of feeding behavior, food intake and serum insulin level are positively correlated with the body weight change of the experimental macaque. In future studies, the inventors of the present invention also need to continue to investigate and analyze the long-term effects of the overexpression of human-derived NPY or AgRP in the paraventricular nucleus on experimental animals. Obviously, the non-genetic chimeric gene engineering monkey is a good transformation 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 (including the method, the animal model and the like) provided by the invention can be used for researching the pathogenic mechanism for preventing and treating diseases including obesity/nervous system diseases, diabetes and the like, clinical treatment means, verification and test of related treatment medicines and other related researches before clinic, exploring new clinical treatment strategies for diseases including obesity, diabetes and the like, and the like. And raising other animals according to specific requirements, such as increasing the weight of the test animal, promoting the feeding of the animal and the like, so as to provide the efficiency and quality of animal breeding, and because the modified gene is in a specific tissue or organ and does not relate to other tissues or organs of the animal, the safety of the transgenic technology in the food field is not concerned by the public.

Since the organ operated by the present invention is the brain. The brain nerve cells do not have the capacity of continuously dividing and regenerating, so theoretically, no new cells can replace the brain nerve cells after gene knockout is carried out in the brain, the knockout or over-expression effect is permanent, the characteristic of a relevant model cannot be lost due to the self-repairing capacity of an organism, and the relevant characters are good in stability and persistence and high in reproducibility degree. Meanwhile, the animal model obtained by the scheme has no modification or editing (for the part of the pathological model itself) on the individual gene, and no other factors are introduced, so that the influence of other uncontrollable factors is reduced, and the scientificity and accuracy of related researches and experiments are ensured. In operation, the number of the virus vectors is far larger than that of brain cells of tissues or organs at corresponding positions, so that all brain cells in corresponding regions can be ensured to be infected, and interference on the brain cells is realized. Thereby realizing the gene over-expression in the local brain area or the nerve nucleus and forming a chimeric gene animal model.

Compared with the traditional animal model capable of genetic gene editing, the chimeric animal model has the advantages of short period, high phenotype penetrance, high synchronism, high repeatability, easy large-scale batch acquisition, low cost and the like. Moreover, the technical means for realizing the method can be diversified, for example, the stem cells after the gene modification are locally injected, or the vectors (such as viruses) after the gene modification are locally injected, so that the gene modification is locally caused in an animal body, and the phenotype is induced to be generated. In addition, cell-specific diseases such as those represented by neoplastic diseases typically occur in the presence of localized genetic mutations, with diseased cells in the surrounding normal cellular microenvironment. Clearly, a systemically heritable gene mutation animal model cannot fully mimic the progression under the actual physiological state of cell-specific disease development. The chimeric animal model can truly simulate the occurrence and development of cell-specific diseases in a normal physiological microenvironment, but not the abnormal mutation background of the traditional model. More importantly, the scheme solves the restriction of high cost, long time, small quantity, high difficulty and the like in establishing the germ line genetic engineering monkey. Especially aiming at the characteristic that a plurality of human diseases are heterogeneous local lesions of organs, tissues and cells, the chimeric animal model can be established by accurately performing gene modification in related animal tissues and organ somatic cells by using related gene technology. 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 the establishment of related models can be carried out as required (by using the technical scheme, proper viral vectors and gene segments are selected, and corresponding parts, organs and tissues are operated according to the technical scheme, so that the method can be realized).

The model is mainly characterized in that: a, in adult primates; the invention relates to local gene modification, which can be single gene locus or combination of a plurality of gene loci; the model of the invention can be used for screening clinical drugs.

It should be noted that, in the present application, the priority is based on the application number 201811343685.4, which is filed by the applicant to the intellectual property office of china on 11/13/2018 and the name of the invention is 'application of animal model construction method, animal breeding method, animal model and application thereof'. In the present application, some terms and descriptions are slightly different from the above-mentioned expressions as a priority basis in order to make the related expressions clearer, more accurate and complete, but are sufficient for those skilled in the art to distinguish and enable the related contents of the present application to be supported.

In the specification 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 recited 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," "has," "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 technical solutions provided by the embodiments of the present invention are described in detail above, and the principles and embodiments of the present invention are described herein by using specific examples, and the description of the embodiments is only used to help understanding the method and the core ideas of the present invention; meanwhile, for a person skilled in the art, according to the idea of the present invention, there may be variations in the specific embodiments and the application scope, and in summary, the content of the present specification should not be construed as a limitation to the present invention.

Sequence listing

<110> Sichuan Cross vertical Biotechnology Ltd

<120> animal in-vivo target positioning method, system and application

<141> 2019-11-13

<150> 2018113436854

<151> 2018-11-13

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 7664

<212> DNA

<213> Artificial sequence Lenti-hNPY plasmid (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

Claims (10)

1. A monkey brain organ positioning method is used for accurately positioning the position of an organ in the monkey brain, and comprises the following steps,

setting a reference point, and placing a cod-liver oil particle at a head bone seam point of the macaque as the reference point;

fixing, namely fixing the macaque in the magnetic resonance compatible brain positioning instrument through an ear stick and a tongue plate;

determining a scanning baseline, and respectively sticking three cod-liver oil particles on the horizontal position of the positioning instrument for confirming the horizontal plane during scanning and the scanning baseline; the three cod liver oil particles are distributed in the horizontal position in a right triangle shape;

scanning, namely scanning the macaque wearing the locator on the head by using a nuclear magnetic resonance apparatus to obtain a magnetic resonance phase display, and determining a target position coordinate value of the brain tissue of the macaque based on the datum point and/or the scanning baseline determined by the cod-liver oil particles through the phase display; or the like, or, alternatively,

a monkey brain organ positioning method is used for accurately positioning the position of an organ in the monkey brain, and comprises the following steps,

determining a scanning baseline, and respectively sticking three cod-liver oil particles on the horizontal position of the positioning instrument for confirming the horizontal plane during scanning and the scanning baseline; the three cod liver oil particles are distributed in the horizontal position in a right triangle shape;

fixing, namely fixing the macaque in the magnetic resonance compatible brain positioning instrument through an ear stick and a tongue plate;

setting a reference point, and placing a cod-liver oil particle at a head bone seam point of the macaque as the reference point;

scanning, namely scanning the macaque wearing the locator on the head by using a nuclear magnetic resonance apparatus to obtain a magnetic resonance phase display, and determining a target position coordinate value of the brain tissue of the macaque based on the datum point and/or the scanning baseline determined by the cod-liver oil particles through the phase display; or the like, or, alternatively,

a monkey brain organ positioning method is used for accurately positioning the position of an organ in the monkey brain, and comprises the following steps,

fixing, namely fixing the macaque in the magnetic resonance compatible brain positioning instrument through an ear stick and a tongue plate;

determining a scanning baseline, and respectively sticking three cod-liver oil particles on the horizontal position of the positioning instrument for confirming the horizontal plane during scanning and the scanning baseline; the three cod liver oil particles are distributed in the horizontal position in a right triangle shape;

setting a reference point, and placing a cod-liver oil particle at a head bone seam point of the macaque as the reference point;

scanning, namely scanning the macaque wearing the locator on the head by using a nuclear magnetic resonance apparatus to obtain a magnetic resonance phase display, and determining a target position coordinate value of the brain tissue of the macaque based on the datum point and/or the scanning baseline determined by the cod-liver oil particles through the phase display; or the like, or, alternatively,

a monkey brain organ positioning method is used for accurately positioning the position of an organ in the monkey brain, and comprises the following steps,

fixing, namely fixing the macaque in the magnetic resonance compatible brain positioning instrument through an ear stick and a tongue plate;

setting a reference point, and placing a cod-liver oil particle at a head bone seam point of the macaque as the reference point;

determining a scanning baseline, and respectively sticking three cod-liver oil particles on the horizontal position of the positioning instrument for confirming the horizontal plane during scanning and the scanning baseline; the three cod liver oil particles are distributed in the horizontal position in a right triangle shape;

scanning, wear with nuclear magnetic resonance appearance the head the locater the kiwi fruit scans, obtains magnetic resonance and shows the looks, through show looks, based on the liver oil granule is confirmed benchmark and/or scanning baseline determine the target position coordinate value of kiwi fruit brain tissue.

2. The method of claim 1, wherein the setting a reference point step includes,

the method comprises the steps of creating, taking a position 3cm behind an eyebrow arch of a macaque as a coordinate needle insertion point, taking an iodophor disinfection range from 3cm to 5cm, making a coronal incision of about 4cm by taking the point as a center, stripping periosteum to fully expose a top bone and a coordinate point, drilling a small hole at the position of a skull bone suture of the macaque, and placing cod-liver oil particles in the small hole to serve as a reference point;

further, before the step of creating, further comprising,

anesthesia, the method: whole hemp; a channel: a channel is established by the venous indwelling needle, and a tube is sealed by normal saline; dosage of anesthetic: ketamine 2mg, push slowly; thereafter 1.5mg ketamine was administered to maintain the ciliary reflex or the caudal and limb reflex status until the end of the experiment;

shaving hair, removing head hair, and exposing whole craniocerebral cortex;

further, the reference point is used as a reference point for nuclear magnetic resonance scanner scanning, the coordinate is (0, 0, 0), and the three-dimensional coordinate parameter of the organ is determined based on the precise point coordinate;

further, when the cod liver oil particles are scanned by the nuclear magnetic resonance spectrometer, the appearance phase is white imaging, and the reference point coordinates in the appearance phase are determined according to the white imaging at the accurate point;

further, the organ is paraventricular nucleus;

further, the scanning step specifically includes,

scanning by using a 3.0T magnetic resonance head coil to obtain lamellar axis T1 and T2 images, imaging at a vector position T1, and scanning data are as follows: TE100-120, TR 3800-6000; the layer thickness is 2 mm; interlayer spacing 0; FOV =280 × 280 mm; determining the accurate positions of paraventricular nuclei at two sides by referring to the magnetic resonance imaging and contrast map of the brain of the macaque;

and determining a coordinate needle insertion point, measuring and calculating coordinate values of target points, and selecting 6 target points in front of and behind the target points, at the left and right sides and at the upper and lower sides by 1mm, so as to complete accurate positioning of the paraventricular nucleus.

3. A monkey brain organ positioning system is used for accurately positioning internal organs of a monkey brain, and comprises,

the monkey brain locator is a magnetic resonance compatible brain locator, comprises an ear bar and a tongue plate, is used for fixing the head of a macaque, and is placed into a nuclear magnetic resonance instrument for scanning;

the cod-liver oil particles are used as positioning marks, and one cod-liver oil particle is placed at a head bone seam point of the macaque as a reference point; respectively sticking three cod liver oil particles on the horizontal position of the positioning instrument, and determining the horizontal plane and the scanning baseline during scanning, wherein the three cod liver oil particles are distributed in a right-angled triangle in the horizontal position;

and the nuclear magnetic resonance instrument is used for scanning the head of the macaque and the positioning instrument by using the nuclear magnetic resonance instrument to obtain a magnetic resonance phase display, and the target position coordinate value of the brain tissue of the macaque is determined based on the datum point and the scanning baseline determined by the cod-liver oil particles through the phase display.

4. The system of claim 3, wherein the fiducial point determination method comprises,

anesthesia, the method: whole hemp; a channel: a channel is established by the venous indwelling needle, and a tube is sealed by normal saline; dosage of anesthetic: ketamine 2mg, push slowly; thereafter 1.5mg ketamine was administered to maintain the ciliary reflex or the caudal and limb reflex status until the end of the experiment;

shaving hair, removing head hair, and exposing whole craniocerebral cortex;

the method comprises the steps of creating a wound, taking a position 3cm behind an eyebrow arch of a macaque as a coordinate needle insertion point, taking an iodophor disinfection range from 3cm to 5cm, making a coronal incision about 4cm by taking the point as a center, stripping periosteum to fully expose a top bone and a coordinate point, drilling a small hole at a position of a skull bone suture of the macaque, and placing cod-liver oil particles in the small hole;

further, the reference point is used as a reference point for scanning by the nuclear magnetic resonance apparatus, the coordinate is (0, 0, 0), and based on the precise point coordinate, the three-dimensional coordinate parameter of the organ is determined, wherein the organ is a paraventricular nucleus;

further, when the cod liver oil particles are scanned by the nuclear magnetic resonance spectrometer, the appearance phase is white imaging, and the reference point coordinates in the appearance phase are determined according to the white imaging at the accurate point;

further, the scanning performed by the nuclear magnetic resonance apparatus specifically includes,

scanning by using a 3.0T magnetic resonance head coil to obtain lamellar axis T1 and T2 images, imaging at a vector position T1, and scanning data are as follows: TE100-120, TR 3800-6000; the layer thickness is 2 mm; interlayer spacing 0; FOV =280 × 280 mm; determining the accurate positions of paraventricular nuclei at two sides by referring to the magnetic resonance imaging and contrast map of the brain of the macaque;

and determining a coordinate needle insertion point, measuring and calculating coordinate values of target points, and selecting 6 target points in front of and behind the target points, at the left and right sides and at the upper and lower sides by 1mm, so as to complete accurate positioning of the paraventricular nucleus.

5. A positioning method for accurately positioning a target in an animal body comprises the following steps,

setting a positioning mark, and setting the positioning mark which does not move relative to the limb where the target in the animal body is located outside the animal body;

scanning, namely scanning to obtain a target in the animal body and a phase displaying of the positioning identifier, and accurately positioning the target in the animal body based on the position relation between the positioning identifier and the target in the phase displaying.

6. The method of claim 5, further comprising,

fixing, namely fixing the animal;

further, the animal is fixed by an animal positioning instrument;

further, the localized markings are particulate matter;

further, the positioning identification comprises,

the first positioning identifier is fixed on the horizontal position of the positioning instrument and used for confirming the horizontal plane and the scanning baseline during scanning; the horizontal position has no relative displacement with the animal limb;

further, the first positioning marks are three particles which are distributed in a right-angled triangle in the horizontal position;

further, the positioning identifier further comprises,

the second positioning mark is arranged on the body surface of the animal body and used as a reference point; the second positioning mark is a particle object which is fixed at the body surface position;

determining a target position coordinate value in the animal body based on the datum point determined by the positioning mark and a scanning baseline;

furthermore, the positioning mark is selected according to the imaging characteristics of the scanning equipment used for scanning, and the part of the positioning mark, which is in contact with the positioning mark on the animal body, presents different imaging characteristics in the scanning phase of the scanning equipment;

further, the scanning device is a nuclear magnetic resonance apparatus, and the positioning marks are fat particles;

further, the scanning device is a CT, and the positioning markers are metal particles.

7. A positioning system for accurately positioning a target in an animal body, comprising,

the positioning mark is used for determining accurate scanning, is arranged outside the animal body and does not move relative to the limb where the target in the animal body is located;

and the scanning equipment is used for scanning the animal body and the positioning identifier to obtain a scanning display phase, and accurately positioning the target in the animal body based on the position relation between the positioning identifier and the target in the display phase.

8. The system of claim 7, further comprising,

an animal positioning apparatus for fixing the animal;

further, the localized markings are particulate matter;

further, the positioning identification comprises,

the first positioning identifier is fixed on the horizontal position of the positioning instrument and used for confirming the horizontal plane and the scanning baseline during scanning; the horizontal position has no relative displacement with the animal limb.

9. The system of claim 8, wherein the first localized marker is three particles distributed in a right triangle within the horizontal position;

further, the positioning identifier further comprises,

the second positioning mark is arranged on the body surface of the animal body and used as a reference point; the second positioning mark is a particle object which is fixed at the body surface position;

further, determining a target position coordinate value in the animal body based on the datum point and the scanning baseline respectively determined by the positioning identifier;

furthermore, the positioning mark is selected according to the imaging characteristics of the scanning equipment used for scanning, and the part of the positioning mark, which is in contact with the positioning mark on the animal body, presents different imaging characteristics in the scanning phase of the scanning equipment;

further, the scanning device is a nuclear magnetic resonance apparatus, and the positioning marks are fat particles;

further, the scanning device is a CT, and the positioning markers are metal particles.

10. Use of a method according to any preceding claim for the precise injection of a pharmaceutical agent into a monkey/animal; or the like, or, alternatively,

use of a system according to any of the preceding claims for precise injection of a medicament in a monkey/animal; or the like, or, alternatively,

the system of any one of the preceding claims for precise injection of a medicament into a monkey/animal; or the like, or, alternatively,

use of the method of any preceding claim in the construction of an engineered monkey model/animal model; or the like, or, alternatively,

use of the system of any one of the preceding claims in the construction of an engineered monkey model/animal model; or the like, or, alternatively,

the system of any preceding claim for use in engineering monkey model/animal model construction.

Images