Preparation method and application of neural stem cells capable of being used for in-vivo visual neural circuit connection

Abstract

The invention discloses a preparation method and application of neural stem cells capable of being used for in-vivo visual neural circuit connection. The method comprises the following steps: constructing a tool virus vector which is driven by a broad-spectrum promoter EF1 alpha, is optimized by a mammal codon and is expressed by a fusion reporter gene of wheat germ lectin and tyrosinase, and transfecting the vector into neural stem cells. TYR catalyzes tyrosine in cells to synthesize melanin through a multi-step reaction, and the in-vivo visualization of a brain region connected with a neural circuit formed by transplanted neural stem cells can be realized by utilizing a nuclide labeling probe of targeted melanin. According to the method, the neural stem cells for in-vivo tracing of neural circuit connection of the neural stem cells are established for the first time, and when and where the neural stem cells are dynamically and visually traced to establish connection with the host neural network in a living body; and a powerful research tool is provided for exploring a specific brain region in which the neural stem cells form loop connection in vivo and an action mechanism of the neural stem cells in neural function repair.

Description

Technical Field

[0001] This invention belongs to the field of nuclear medicine molecular imaging, specifically relating to a method for preparing neural stem cells that can be used for in vivo visualization of neural circuit connections and its application. Background Technology

[0002] Neural stem cell transplantation therapy represents a major breakthrough in medicine and has become a potential new strategy for treating many central nervous system diseases, such as Parkinson's disease, Alzheimer's disease, and ischemic stroke. The differentiation of neural stem cells into specific nerve cells and their integration into and repair of neural circuits after transplantation into the brain is a crucial mechanism in cell transplantation therapy. However, our understanding of the dynamic changes in neural circuits of transplanted neural stem cells in the brain remains insufficient. The key reason for this is the lack of methods for in vivo visualization of neural stem cell neural circuits. Currently, there are many non-invasive imaging methods for tracking the location of stem cells in vivo, such as radionuclide imaging after labeling stem cells with radiolabeled compounds, optical imaging after labeling stem cells with fluorescent dyes, magnetic particle imaging after magnetic labeling, and ultrasound imaging after ultrasound contrast agent labeling. However, most of these methods can only present information on the location and quantity of stem cells in vivo, and cannot dynamically track the formation and connection of neural circuits within the brain regions, which are essential for stem cells to exert their biological functions in vivo. Therefore, in vivo visualization of neural stem cell neural circuits is crucial for exploring the in vivo therapeutic mechanisms of neural stem cell transplantation and for promoting the further development of cell therapies for nervous system diseases.

[0003] Positron emission tomography (PET) is a representative nuclear medicine molecular imaging technique. Compared with other imaging methods, PET imaging has advantages in the spatiotemporal dynamic visualization and quantitative tracking of biological processes in vivo at the cellular and molecular levels. PET reporter gene imaging technology involves transfecting exogenous reporter genes (encoding a specific enzyme, transporter, or receptor) along with the target gene into target cells, allowing both to be expressed simultaneously within the cell. Subsequently, a radioactive probe specifically identifies and binds to the reporter gene product, thereby achieving the visual evaluation of the target gene. However, due to limitations such as the blood-brain barrier and natural expression in the brain, PET reporter gene methods that can enter the nervous system are still relatively lacking. Tyrosinase (TYR) is a rate-limiting enzyme in melanin synthesis. In previous studies, TYR has been widely used as a reporter gene in various fields such as stem cell transplantation tracking after myocardial infarction and tumor treatment monitoring. Furthermore, melanin has low natural expression in the nervous system, giving it a unique advantage for brain tracking. Our team previously used a probe targeting melanin, fluorine […]. 18 F]-pyridineamide-benzamide ( 18The F-FPABZA gene successfully validated the potential of TYR as a PET reporter gene for the central nervous system.

[0004] Wheat germ agglutinin (WGA) is a plant lectin protein extracted from wheat germ. It binds to glycosyl groups with high affinity and specificity, and is actively taken up by neuronal synapses or cell bodies via receptor-mediated endocytosis, then transported anterogradely and retrogradely along axons. When WGA is injected into specific brain regions, it is taken up by local neurons and transmitted along their axons, clearly outlining the origin, termination, and course of neural pathways. Mammalian codon-optimized wheat germ agglutinin (mWGA) can increase WGA expression in mammalian neurons, facilitating its application in mammals. Combining mWGA with PET reporter genes holds promise for enabling in vivo neuronal tracking and visualization of neural circuits.

[0005] This discovery provides a PET reporter gene-modified neural stem cell that can be used to analyze the dynamic process of neural stem cell therapy repairing neural circuits in disease states. First, this invention constructs a viral vector, mWGA-TYR, for in vitro neural stem cell transfection. After transplantation into pathologically damaged brain regions, the neural stem cells develop, differentiate, and mature within the brain, forming connections with the host neural circuits. Utilizing the transsynaptic transmission properties of mWGA, TYR can be transmitted to other neurons that have formed neural circuit connections with the transplanted cells, thereby marking the extent and timing of neural circuit connections. This technology enables dynamic and visual tracking in vivo of when and where neural stem cells establish connections with the host neural network. Currently available neural circuit tracking viruses, WGA alone, or TYR PET imaging techniques alone cannot achieve the same level of in vivo, dynamic, and spatiotemporal resolution. Summary of the Invention

[0006] The purpose of this invention is to address the shortcomings of existing technologies by proposing a method for preparing neural stem cells that can be used for in vivo visualization of neural circuit connections and its application.

[0007] The objective of this invention is achieved through the following technical solution: a method for preparing neural stem cells that can be used for in vivo visualization of neural circuit connections, characterized by the following steps: constructing a tool viral vector for expressing a reporter gene fused with wheat germ lectin and tyrosinase; transfecting the tool viral vector into neural stem cells in vitro using the broad-spectrum promoter EF1α; delivering tyrosinase to neural cells that form synaptic connections with the neural stem cells and their progeny cells using wheat germ lectin; the reporter gene is continuously expressed in the neural stem cells and their progeny cells without signal dilution during cell division; the tyrosinase catalyzes the synthesis of melanin from tyrosine in the cells; and performing in vivo dynamic imaging using a PET probe targeting melanin, thereby achieving dynamic and visual tracking in vivo of when and where neural stem cells establish connections with the host neural network, thus enabling long-term in vivo visualization of brain regions where neural stem cells and their progeny cells form neural circuit connections.

[0008] As one implementation, the viral vector for the tool is a lentivirus.

[0009] As one implementation, the expression of the reporter gene in cells is driven by the broad-spectrum promoter EF1α.

[0010] As one embodiment, the gene sequence of the tyrosinase is shown in SEQ ID NO.1.

[0011] As one implementation, the gene sequence of the mammalian codon-optimized wheat germ lectin is shown in SEQ ID NO.2.

[0012] As one embodiment, the amino acid sequence of the tyrosinase is shown in SEQ ID NO.3.

[0013] As one embodiment, the amino acid sequence of the mammalian codon-optimized wheat germ lectin is shown in SEQ ID NO.4.

[0014] As one implementation, the tool viral vector expresses fluorescent protein genes, including but not limited to GFP, RFP, BFP, EGFP, mCherry, mStrawberry, mApple, mRuby, or EosFP.

[0015] As one implementation, the viral vector of the tool carries a blastidin S (BSD) resistance gene, driven solely by a constitutive promoter, including but not limited to CMV promoter, EF1α promoter, EFS promoter, CAG promoter, Cbh promoter, SFFV promoter, SV40 promoter, Ubc promoter, Ubi promoter, hPGK promoter, and β-actin promoter, to facilitate the selection of successfully transfected cells by blastidin screening after cell transfection in in vitro experiments.

[0016] As one implementation, the PET molecular imaging probe is 18 F-FPABZA.

[0017] On the other hand, the present invention also provides the use of neural stem cells in the preparation of tracers for in vivo tracing of neural circuit connections.

[0018] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention uses the broad-spectrum promoter EF1α, and the mWGA-TYR reporter gene can be integrated into the genome of neural stem cells through a tool viral vector. The reporter gene can be continuously expressed in neural stem cells and their progeny cells without signal dilution during division, providing a novel in vivo evaluation technology for long-term tracking of neural circuits in neural stem cells and their progeny cells.

[0019] 2. The tracer PET molecular imaging probe provided by this invention 18 F-FPABZA can directly image TYR product melanin, enabling in vivo visualization of neural circuits in the brain. The probe has excellent blood-brain barrier penetration ability and does not require additional physical or chemical operations (such as ultrasound microbubbles or small molecule drugs) to open the blood-brain barrier.

[0020] 3. The tracing method provided by the present invention is not limited by the depth of neural stem cell transplantation in the brain, and does not require invasive imaging by inserting optical fibers or lenses into the brain. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1A schematic diagram illustrating the in vivo imaging neural circuit of neural stem cells modified with the mWGA-TYR PET reporter gene. (a) shows the rLV-EF1α-mWGA-TYR-CMV-EGFP-P2A-BSD-WPRE plasmid sequence; (b) lentivirus infection of neural stem cells, with mWGA transmitting TYR to other neurons forming neural circuit connections; (c) PET reporter gene-modified neural stem cells are transplanted into the brain damaged by ischemic stroke and form neural circuit connections in vivo, transmitting TYR to host neurons via mWGA. This process can be dynamically tracked in vivo using PET imaging.

[0023] Figure 2 A schematic diagram of the in vivo visualization of neural circuits formed by transplantation of mWGA-TYR PET reporter gene-modified neural stem cells into the brain of an ischemic stroke patient. (a) mWGA-TYR PET reporter gene-modified neural stem cells can develop into mature neurons in the brain; (b) 2 weeks after in vivo transplantation of neural stem cells. 18 F-FPABZA PET imaging showed signals in the injection site and distal brain regions; (c) In vitro immunofluorescence staining verified the in vivo imaging results, showing signals of GFP, WGA, and TYR in the injection site and distal brain regions. Detailed Implementation

[0024] The present invention will be further illustrated below with reference to specific embodiments. These embodiments are for illustrative purposes only and should not be construed as limiting the invention. Those skilled in the art will understand that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the invention. The scope of the invention is defined by the claims and their equivalents. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or according to the manufacturer's recommendations.

[0025] This invention utilizes a viral vector to transfect neural stem cells into expressing tyrosinase (catalyzing melanin synthesis) under neural circuit conditions. The gene sequence of tyrosinase is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.3. After transplantation into a nude mouse model of cerebral infarction, the neural stem cells survive and differentiate into neurons, forming synaptic connections with peripheral or distant neurons. Driven by the EF1α promoter, neurons derived from neural stem cells synchronously express tyrosinase and catalyze the production of melanin. 18 F-FPABZA PET combined with melanin can enable in vivo visualization of neural circuits from neural stem cells. A specific operational example is shown below: 1. Lentiviral particle production and packaging In this invention, the EF1α-mWGA-TYR-CMV-EGFP-P2A-BSD-WPRE reporter gene was first cloned into a tool viral vector plasmid. The gene sequence of the mammalian codon-optimized wheat germ lectin mWGA is shown in SEQ ID NO.2, and the amino acid sequence of the mammalian codon-optimized wheat germ lectin is shown in SEQ ID NO.4. Then, 10.5 μg of recombinant plasmid was mixed with 3 μg of Rev (helper plasmid), 5 μg of Gag-Pol (packaging plasmid), and 1.5 μg of VSV-G (enveloping plasmid) and dissolved in Opti-MEM to prepare solution A. The amount of PEI (1 μg / μL) per culture dish was 2-3 times the mass of the plasmid, and the mixture was dissolved in 250 μL of Opti-MEM to prepare solution B. After mixing solutions A and B, the mixture was transfected into HEK 293T cells. The viral supernatant produced after transfection was collected, and finally, high-titer lentivirus was obtained through concentration and purification steps.

[0026] 2. Neural stem cell reporter gene transfection and intracranial transplantation in a nude mouse model of cerebral infarction This invention involves in vitro infection of neural stem cells with lentivirus. After 3 days, the cells are screened using 20 μg / mL blastomycin. The neural stem cells are then transplanted into the peri-infarction area of ​​a nude mouse model of cerebral infarction. Two weeks after transplantation, the in vivo visualization of neural circuits of the neural stem cells is evaluated using PET.

[0027] 3. 18 F-FPABZA PET Imaging This invention involves injecting 9.25 MBq into the tail vein of nude mice. 18 After injection of F-FPABZA imaging agent, nude mice were returned to their cages and allowed to move freely. Sixty minutes later, the mice were anesthetized for further processing. 18 F-FPABZA PET static scan for 10 minutes.

[0028] 4. In vitro immunofluorescence staining of stem cell transplanted nude mouse brain slices In this invention, brain tissue was harvested from nude mice two weeks after neural stem cell transplantation via cardiac perfusion. The brain tissue was fixed, dehydrated, and then frozen sectioned. The brain slices were then subjected to WGA, TYR, GFP, and MAP2 immunofluorescence staining.

[0029] like Figure 1 As shown, in this invention, EF1α-mWGA-TYR-CMV-EGFP-P2A-BSD-WPRE lentivirus infects neural stem cells. These neural stem cells gradually mature into neurons, forming synaptic connections with surrounding or distant neurons. Driven by the EF1α promoter, they continuously express mWGA and TYR. mWGA carries TYR across the synapse and transmits it to other neurons that form neural circuits with it. TYR can catalyze the production of melanin, through…18 F-FPABZA PET combined with melanin enables in vivo visualization of neural circuits in neural stem cells. EGFP, driven by the CMV promoter, is expressed only in the initially infected neural cells and cannot be transmitted to the next neuron, thus identifying the initially virus-infected cells and the cells that form connections with them. Using BSD to screen lentivirus-infected neural stem cells, neural stem cells successfully expressing the PET reporter gene were transplanted into the brain damaged by ischemic stroke, forming neural circuit connections in vivo. This process can be dynamically tracked in vivo using PET imaging.

[0030] See the following embodiments for details: Example 1: Construction of a lentiviral vector plasmid for overexpression of tyrosinase reporter gene 1. Synthesized gene: The mWGA-TYR reporter gene was synthesized using a DNA synthesizer. 2. Amplification of the reporter gene: The mWGA-TYR reporter gene was amplified in large quantities by polymerase chain reaction (PCR). The primers at the 5' and 3' ends contained KpnI and BamHI restriction endonuclease sequences, respectively. The product gene was purified using a gel extraction kit.

[0031] 3. The PCR reaction system is as follows: 4. The PCR reaction procedure is as follows: 5. Insert the gene fragment digested with BamHI and KpnI into the multiple cloning site of the prepared vector and ligate it using DNA ligase. The vector sequence is EF1α-mWGA-TYR-CMV-EGFP-P2A-BSD-WPRE.

[0032] 6. After screening for correct recombinant plasmids using PCR and enzyme digestion methods, and confirming the correct vector construction through sequencing, large-scale amplification of the recombinant plasmid (EF1α-mWGA-TYR-CMV-EGFP-P2A-BSD-WPRE) begins.

[0033] 7. HEK-293T cell preparation: Seed 4 million HEK-293T cells in a 10cm culture dish, culture overnight to restore cell state, and replace with fresh complete culture medium before transfection.

[0034] 8. HEK-293T Transfection: Mix 0.5 μg of recombinant plasmid with 3 μg of Rev (helper plasmid), 5 μg of Gag-Pol (packaging plasmid), and 1.5 μg of VSV-G (enveloping plasmid) in Opti-MEM to prepare solution A. Dissolve PEI (1 μg / μL) at 2-3 times the mass of the plasmid in 250 μL of Opti-MEM to prepare solution B. Mix thoroughly by pipetting. Let solutions A and B stand at room temperature for 5 minutes. Add Opti-MEM containing PEI dropwise to the Opti-MEM containing the mixed plasmid one drop at a time and let stand for 15-20 minutes. Add 500 μL of the mixture (250 μL PEI + 250 μL mixed plasmid) to each dish. Change the solution after 4-6 hours.

[0035] 9. Virus particle acquisition: 48 hours after transfection, aspirate the supernatant into a new centrifuge tube, centrifuge at 3000 rpm for 10 minutes at 4°C to remove cell debris, aspirate the supernatant, filter through a 0.45 μm filter, and transfer to a new centrifuge tube. Add 1 / 4 volume of 5×PEG8000 to the virus solution and immediately invert to mix. Place on ice, invert 5 times every 30 minutes, for a total of 5 times, then incubate overnight at 4°C (a clear white precipitate indicates the presence of virus).

[0036] Example 2: Intracerebral transplantation of neural stem cell reporter gene transfection into a nude mouse model of cerebral infarction 1. Neural stem cell transfection: Seed neural stem cells at a density of 500,000 per well in 24-well plates and culture until the cell density is about 70%. Add a certain volume of lentivirus at a multiplicity of infection of 10 according to the lentivirus titer. Replace the complete culture medium about 8 hours after infection. If changes in cell morphology are observed during the process, the complete culture medium can be replaced earlier.

[0037] 2. Establishment of a nude mouse cerebral infarction model: Nude mice weighing approximately 20g were anesthetized by intraperitoneal injection of 1.5% sodium pentobarbital (1.5g sodium pentobarbital powder dissolved in 100mL physiological saline), with an injection dose of 50mg / kg. The mice were fixed in a stereotaxic apparatus, and the anterior and posterior fontanelles were leveled. 0.1mL of 15mg / mL rose red solution was injected into the tail vein. A square area on the right side of the skull was irradiated with a 25mW laser (1.8mm-3mm to the right of the midline, 0.5mm anterior to 1.3mm posterior to the anterior fontanelle). The remaining exposed areas were covered with aluminum foil to avoid light. After irradiation for 15 minutes, the skin was disinfected and sutured. Penicillin was injected intraperitoneally for 3 days to prevent infection.

[0038] 3. Intracerebral transplantation of neural stem cells in nude mice with cerebral infarction: Two weeks after cerebral infarction modeling in nude mice, the mice were anesthetized and fixed in a stereotaxic apparatus. Three holes (evenly distributed anteriorly and posteriorly) were drilled on the left side of the infarct area. 0.33 μL of transfected neural stem cells (approximately 33,000) were aspirated using a micro-injection needle and transplanted into the peri-infarct area at a depth of 0.9 mm into the brain tissue at a rate of 0.1 μL / min. After injection, the mice were allowed to stand for 5 minutes before being slowly withdrawn. The skin of the nude mice was sutured, the area was disinfected, and penicillin was administered intraperitoneally for 3 days to prevent infection.

[0039] Example 3: Tracer Injection 18 F-FPABZA Synthesis 1. After the fluoride source in the fluoride-containing heavy oxygen water is adsorbed onto the anion exchange column (QMA column), the QMA column is eluted with 0.8 mL of eluent (15 mg K222, 3 mg potassium carbonate, 0.64 mL acetonitrile, and 0.16 mL sterile water) and the solution is transferred to a reaction flask. The liquid in the reaction flask is evaporated at 110 °C, with 1 mL of anhydrous acetonitrile added during the process, and the azeotropic evaporation is repeated three times.

[0040] 2. After complete drying, add 1 mL of DMF solution containing 7.5 mg of the precursor BrPABZA, and react in a sealed environment at 140 °C for 30 min. Dilute the reaction solution and inject it into an HPLC column for purification and separation. Wash the column with a mobile phase of acetonitrile and 0.5 / 1000 trifluoroacetic acid aqueous solution in a volume ratio of 3:7. The product peak appears at approximately 16 min and is collected.

[0041] 3. Dilute the product peak solution with 30 mL of pure water and pass it through a C18 separation column to adsorb the product onto the C18 column. Then wash the C18 column with 10 mL of pure water. Elute the product from the C18 column with 1 mL of ethanol, dilute with 10 mL of physiological saline containing 10 mg / mL ascorbic acid, and finally filter through a 0.22 μm sterile filter membrane to obtain the final product. 18 F-FPABZA injection.

[0042] Example 4: 18 F-FPABZA PET scan to trace transplanted neural stem cells 1. 18 F-FPABZA PET scan: Two weeks after neural stem cell transplantation, nude mice were anesthetized with 4% isoflurane (v / v) and injected with approximately 9.25 MBq (250 μCi) via the tail vein. 18 After F-FPABZA, the mice were returned to their cages and allowed to move freely. Sixty minutes later, they were anesthetized again with 4% isoflurane (v / v). The nude mice were then fixed in a prone position on a small animal PET scan window for static scanning over 10 minutes. Anesthesia was maintained during the scan with 1.5% isoflurane (v / v). Figure 2As shown, neural stem cells modified with the mWGA-TYR PET reporter gene can develop into mature neurons in the brain. Two weeks after in vivo transplantation of neural stem cells... 18 F-FPABZA PET imaging detected PET signals in the injection site and distal brain regions, and the in vivo imaging results were verified by in vitro immunofluorescence staining, further validating that the technology described in this patent can visualize the neural circuits of transplanted neural stem cells in vivo.

[0043] 2. 18 F-FPABZA PET Reconstruction: The initial image obtained from the scan is reconstructed using the Ordered Subset Expectation-Maximization (OSEM) method, and the reconstructed image is processed using Pmod software.

[0044] Example 5: In vitro immunofluorescence staining of stem cell transplanted nude mouse brain slices 1. Nude mice were deeply anesthetized by intraperitoneal injection of 1.5% sodium pentobarbital (1.5g sodium pentobarbital powder dissolved in 100mL physiological saline). The heart was exposed, and a perfusion needle was inserted through the apex of the heart, positioned along the left ventricular outflow tract to the origin of the aortic arch. Physiological saline was rapidly perfused until no blood flowed out. Then, approximately 50mL of 4% paraformaldehyde (4g paraformaldehyde powder dissolved in 100mL 1× PBS buffer) was slowly perfused until the mouse became rigid. The intact brain tissue was carefully removed. The removed brain was placed in 4% paraformaldehyde solution and externally fixed overnight at 4°C. The solution was then replaced with 30% sucrose (30g solid sucrose dissolved in 100mL 1× PBS buffer) for dehydration. After dehydration, the brain tissue was embedded and frozen sectioned.

[0045] 2. Select brain slices and place them in a 24-well plate containing PBS. Shake rapidly for 5 minutes on a shaker, repeating 3 times. Fix with 4% paraformaldehyde at room temperature for 10 minutes, then wash with PBS for 5 minutes, repeating 3 times. Add 0.25 mL of rapid blocking buffer and block with gentle shaking on a shaker at room temperature for 1 hour, then wash with PBS 3 times, 5 minutes each time. Prepare primary antibody according to the antibody manufacturer's instructions and incubate overnight at 4°C with gentle shaking. Recover the primary antibody and wash with PBS 3 times. Prepare secondary antibody according to the primary and secondary antibody manufacturer's instructions and incubate with gentle shaking at room temperature in the dark for 1 hour. Recover the secondary antibody and wash with PBS 3 times. Add DAPI staining solution and incubate at room temperature for 10 minutes, then add 0.5 mL of PBS to each well and wash 3 times. Spread and mount the brain slices in a culture dish containing PBS. Add anti-quenching mounting medium to the brain slices, mount, and observe.

[0046] The above embodiments are used to explain and illustrate the present invention, but not to limit the present invention. Any modifications and changes made to the present invention within the spirit and scope of the claims shall fall within the protection scope of the present invention.

Claims

1. A method for preparing neural stem cells useful for in vivo visualization of neural circuit connectivity, comprising, The method comprises the following steps: Constructing a tool virus vector for expression of wheat germ agglutinin and tyrosinase fusion reporter gene, and transfecting the tool virus vector into neural stem cells in vitro driven by a broad-spectrum promoter EF1α; delivering the tyrosinase into nerve cells connected with the neural stem cells and their daughter cells by the wheat germ agglutinin, and continuously expressing the reporter gene in the neural stem cells and their daughter cells without signal dilution during division; the tyrosinase catalyzes the synthesis of melanin from tyrosine in cells, and the in vivo dynamic imaging is performed by a PET probe targeting melanin, so as to realize dynamic and visual tracing of when and where the neural stem cells connect with the host neural network, and to realize in vivo visualization of long-term tracking of brain regions where the neural stem cells and their daughter cells form neural circuit connections.

2. The method of claim 1, wherein, The tool virus vector is a lentivirus vector.

3. The method of claim 1, wherein, In the tool virus vector, the gene sequence for expressing the tyrosinase TYR comprises a sequence as shown in SEQ ID NO:

1.

4. The method of claim 1, wherein, In the tool virus vector, the gene sequence for expressing the mammalian codon-optimized wheat germ agglutinin mWGA comprises a sequence as shown in SEQ ID NO:

2.

5. The method of claim 2, wherein, The tool virus vector further comprises a blasticidin resistance gene for screening of in vitro successfully transfected cells.

6. The method of claim 1, wherein, The tool virus vector further expresses a fluorescent protein gene; the fluorescent protein is selected from any one of GFP, RFP, BFP, EGFP, mCherry, mStrawberry, mApple, mRuby or EosFP.

7. The method of claim 1, wherein, The melanin-targeting PET probe is 18 F-pyridinamide-benzamide 18 F-FPABZA.

8. Use of neural stem cells prepared by the method according to any one of claims 1-7 in preparation of a tracer for in vivo tracing of neural circuit connections.

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