A promoter of lgals3bpb gene and its application in constructing zebrafish model

Abstract

The present invention relates to the field of nerve cell labeling methods and related animal model technologies, and specifically to a promoter of a lgals3bpb gene and its application in constructing a zebrafish model. The application of the promoter of the lgals3bpb gene in constructing a zebrafish model comprises the following steps performed in sequence: obtaining the promoter of the lgals3bpb gene through a PCR reaction; constructing an expression vector regulated by the promoter of the lgals3bpb gene; transferring the expression vector into zebrafish fertilized eggs, and obtaining a stable genetically modified zebrafish strain through culture, screening and multi-generation hybridization. This technical solution can solve the technical problem of the prior art due to the lack of a zebrafish animal model capable of locating and operating microglia, and creates conditions for studying microglia, conducting disease mechanism research, drug screening and drug efficacy testing, and has good application prospects.

Description

Technical Field

[0001] This invention relates to the field of neural cell labeling methods and related animal model technology, specifically to a promoter of the lgals3bpb gene and its application in constructing a zebrafish model. Background Technology

[0002] Zebrafish (Danio rerio) are a common tropical fish. Cell labeling, tissue transplantation, mutation, haploid breeding, transgenic, and gene activity inhibition techniques for zebrafish are well-established. With thousands of zebrafish embryonic mutants, they are an excellent resource for studying the molecular mechanisms of embryonic development, and some can even serve as models for human diseases. Because zebrafish genes share 87% similarity with human genes, results from drug experiments on zebrafish are often applicable to humans. Zebrafish embryos are transparent, making it easy for biologists to observe the effects of drugs on their internal organs. Female zebrafish can lay up to 200 eggs, and the embryos develop within 24 hours, allowing biologists to conduct different experiments on the same generation of fish, thus studying pathological evolution and identifying disease causes.

[0003] Microglia are tissue-resident macrophages in the central nervous system, whose functions include clearing cellular debris, transmitting signals to neurons and astrocytes to mediate synaptic connections, and maintaining overall nervous system homeostasis. A comprehensive study of microglia to understand their developmental mechanisms is essential for research on related diseases. Due to the aforementioned advantages of zebrafish models, an increasing number of researchers are using them to study microglia. Using zebrafish as an animal model for experimental research on microglia first requires the ability to specifically label and manipulate microglia in vivo, which places high demands on research methods and techniques. Although existing studies have revealed some genes specifically expressed in microglia, theoretically, the promoters of these genes could be used to control the specific effects of effector genes in microglia.

[0004] Currently, only two genes, apoeb and ccl34b.1, are used to label microglia. Transgenic lines based on these two genes are obtained using BAC (bacterial artificial chromosome)-mediated transgenic technology. However, reliable promoter sequences for microglia labeling have not yet been determined, limiting the scope of research and hindering its application. The varying lengths and distributions of promoters among different genes make it difficult to effectively capture ideal promoter sequences from a large pool of microglia-specific expression genes. Furthermore, even after promoter capture, it may not effectively initiate the expression of effector genes in zebrafish models in some cases.

[0005] In summary, there is an urgent need to construct a zebrafish animal model capable of locating and manipulating microglia to meet the needs of disease mechanism research, drug screening, and efficacy testing. Summary of the Invention

[0006] The present invention aims to provide an application of the promoter of the lgals3bpb gene in the construction of a zebrafish model, in order to solve the technical problem in the prior art that there is a lack of zebrafish animal models that can locate and manipulate microglia.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] The application of a promoter for the lgals3bpb gene in constructing a zebrafish model includes the following steps performed sequentially:

[0009] S1: The lgals3bpb gene promoter was obtained by PCR; the nucleotide sequence of the lgals3bpb gene promoter is shown in SEQ ID NO.1;

[0010] S2: The lgals3bpb gene promoter is integrated into a vector to obtain an expression vector; the lgals3bpb gene promoter is used to control the expression of the fluorescent protein gene;

[0011] S3: The expression vector is transferred into zebrafish fertilized eggs, and after cultivation and screening, F0 generation zebrafish are obtained; F0 generation zebrafish are crossbred with wild-type zebrafish for multiple generations to obtain a stable genetically inherited transgenic zebrafish strain.

[0012] This protocol also provides a promoter for the lgals3bpb gene, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0013] The principles and advantages of this scheme are:

[0014] In this technical solution, the promoter fragment (4141bp) of the lgals3bpb gene is obtained by DNA polymerase chain reaction; then the expression vector is constructed, transgenic into zebrafish fertilized eggs, and finally, after multiple generations of hybridization, a stable transgenic fish line is obtained.

[0015] In microglia, several genes are enriched for expression, such as the apoeb gene (apolipoprotein E) and the ccl34b.1 gene (chemokine (CC motif) ligand 34b, duplicate 1). These two genes are specifically expressed in microglia and are considered by researchers to be microglia marker genes. Through sequencing and studying gene expression patterns in microglia, the inventors discovered that several genes, including the lgals3bpb gene, are enriched for expression in microglia. Further research revealed that the expression pattern of the lgals3bpb gene overlaps with the two known marker genes in microglia. Utilizing these findings, the inventors further investigated the expression regulation of these enriched genes in microglia, cloning and analyzing their promoter sequences. The study found that although a large number of candidate genes exist, the promoter sequences of these candidate genes related to microglia development are extremely difficult to clone. While some highly expressed genes can be cloned using technical means, linking these sequences to fluorescent protein genes fails to initiate fluorescent protein expression in zebrafish. Through extensive screening, the inventors obtained the promoter of the lgals3bpb gene, constructed an expression vector using this promoter, and then built a transgenic zebrafish model. The lgals3bpb gene promoter can initiate the specific expression of the fluorescent protein gene in microglia of the zebrafish model, thereby enabling microglia labeling and observation. Replacing the fluorescent protein with other effector genes can also achieve the same effector gene function. In the existing technology, no research has been reported on using the lgals3bpb gene or its promoter to label microglia, nor has a manipulable promoter sequence for specific labeling of zebrafish microglia been reported. Transgenic vectors containing the lgals3bpb gene and fluorescent protein gene are introduced into zebrafish. Zebrafish transgenic lines constructed using the lgals3bpb gene promoter are excellent zebrafish animal models for studying microglia. They can be used to study the mechanism of microglia in neurodegenerative diseases, as well as for drug screening and efficacy testing.

[0016] Furthermore, in S1, the primers for the PCR reaction include:

[0017] F1-lgals3bpb:5'-tcgaattcctgcagcccgggTGTTGCTCTGAAGTCAGAGG-3';

[0018] R1-lgals3bpb:5'-GCTGTTTGTACGTCCAAGTACCATTCATACTATTCACATTT-3';

[0019] F2-lgals3bpb:5'-GTACTTGGACGTACAACAGC-3';

[0020] R2-lgals3bpb: 5'-tcctcgcccttgctcaccatGATGTCAGAAACAGCAACGA-3'.

[0021] The above primers can be used to achieve effective amplification of the target gene.

[0022] Furthermore, in S3, the method for obtaining F0 generation zebrafish is as follows: the expression vector and transposase are simultaneously microinjected into zebrafish fertilized eggs; 3 days after fertilization, fertilized eggs expressing the fluorescent protein are selected; and then cultured until sexual maturity to obtain F0 generation zebrafish. Through transgenic methods, the target gene is integrated into the zebrafish genome, and transgenic zebrafish with the target gene are obtained through selection.

[0023] Furthermore, in S3, the DNA template sequence of the transposase is shown in SEQ ID NO.9. A transposon is a sequence capable of changing its position, containing an intermediate sequence and terminal repeat sequences; the intermediate sequence can express the transposase. The transposase in this scheme is a tool to improve plasmid insertion efficiency. If not used, insertion of exogenous genes into the zebrafish genome can still occur, but the efficiency will be reduced.

[0024] Furthermore, in S3, the culture temperature was 28.5℃. This culture temperature represents the optimal temperature for zebrafish culture.

[0025] Furthermore, in S3, F0 generation zebrafish are first crossed with wild-type zebrafish, and offspring expressing fluorescent proteins are selected to obtain F1 generation zebrafish; then F1 generation zebrafish are crossed with wild-type zebrafish to obtain a transgenic zebrafish strain with stable inheritance.

[0026] Furthermore, in S1, the PCR reaction system includes: 18 μL water, 25 μL buffer, 0.5 μL DNA polymerase, 1.5 μL template, 2.5 μL upstream primer, and 2.5 μL downstream primer. Using this reaction system, efficient amplification of the target DNA fragment can be achieved.

[0027] Furthermore, in S2, the sequence of the fluorescent protein gene is shown in SEQ ID NO.6. Green fluorescent protein is a commonly used marker protein in the art, regulated by the lgals3bpb gene promoter, which enables microglia to produce fluorescent signals to distinguish them from other cell types, facilitating the observation of microglia.

[0028] Furthermore, the promoter of the lgals3bpb gene is used to control the expression of fluorescent proteins in zebrafish microglia.

[0029] This approach provides an operable promoter sequence for labeling microglia, enabling specific manipulation of microglia. For example, carrying a nitroreductase gene after the promoter can specifically kill microglia without harming other macrophages; or the promoter can be used to specifically add or knock out a gene in microglia or Langerhans cells; or fluorescent proteins can be integrated after the promoter to locate microglia.

[0030] In summary, through single-cell sequencing analysis of immune cells in zebrafish brains, the inventors discovered that brain immune cells can be divided into multiple groups. They screened out several genes with expression patterns similar to the apoeb and ccl34b.1 genes (currently recognized microglia marker genes) and used these genes as candidate promoters for cloning. Furthermore, although the apoeb and ccl34b.1 genes are highly expressed in microglia, their expression is not concentrated in brain immune cells. For example, the apoeb gene also has some expression in neurons. Therefore, screening for genes that can specifically express in microglia is essential.

[0031] Due to the complexity of core promoter sequences with cell-specific expression, the inventors obtained only the sequences upstream of the start codon of some candidate genes through numerous trials. These obtained sequences were integrated into expression plasmids and transgenic into zebrafish. It was found that not all cloned sequences upstream of the start codon possessed the activity to initiate effector gene expression. Ultimately, only the sequences upstream of the start codon of the cloned *apoc1* gene and *lgals3bpb* gene were found to initiate effector gene expression, thus obtaining the core promoter sequences. Furthermore, in further experiments, we discovered that the promoters of the *apoc1* and *lgals3bpb* genes can specifically initiate effector gene expression in microglia, but not in neurons or other immune cells. This makes it possible to specifically observe and manipulate microglia. The discovery of the expression patterns of the apoc1 and lgals3bpb genes, as well as the promoters of the apoc1 and lgals3bpb genes, yielded unexpected technical results, overcoming the technical problem of the lack of animal models capable of locating and manipulating microglia in existing technologies.

[0032] In addition, transgenic lines with promoter markers of the lgals3bpb gene showed stronger fluorescence than those with promoter markers of the apoc1 gene. Furthermore, the range of promoter markers of the apoc1 gene is larger than that of the lgals3bpb gene promoter, while the lgals3bpb gene promoter has relatively higher specificity. Attached Figure Description

[0033] Figure 1 This is an electrophoresis diagram of the lgals3bpb gene promoter segment cloning in Example 1.

[0034] Figure 2 This is a schematic diagram illustrating the construction process of the pT2AL-cryaa-lgals3bpb-eGFP plasmid in Example 1.

[0035] Figure 3 This is a schematic diagram of the pT2AL-cryaa-lgals3bpb-eGFP plasmid from Example 1.

[0036] Figure 4 This is a colony PCR electrophoresis image of Example 1.

[0037] Figure 5 This is a schematic diagram of the construction and screening of transgenic zebrafish in Example 1.

[0038] Figure 6The results of the observation of cryaa-cerulean labeling in 3dpf of Example 1 are shown.

[0039] Figure 7 The image shows a fluorescence micrograph of the whole-body eGFP positive signal detection of zebrafish Tg(lgals3bpb:eGFP) 3 days after fertilization, as described in Example 1.

[0040] Figure 8 The results show the colocalization of eGFP in transgenic zebrafish and panmicroglia in the midbrain three days after fertilization in Example 1.

[0041] Figure 9 The results show the colocalization of eGFP with panmicroglia in the midbrain at 12 months of development in the transgenic line of Example 1.

[0042] Figure 10 The results show the expression patterns of the lgals3bpb gene and known marker genes in microglia, as described in Example 2.

[0043] Figure 11 This is the expression of green fluorescent protein regulated by the Apoeb gene promoter in the prior art study of Example 2.

[0044] Figure 12 The image shows a fluorescence micrograph of the zebrafish Tg(coro1a:DsRed;vmp1-eGFP) strain from Example 2.

[0045] Figure 13 The image shows a fluorescence micrograph of the zebrafish Tg(coro1a:DsRed; havcr1-eGFP) strain from Example 2.

[0046] Figure 14 The image shows a fluorescence micrograph of the whole-body eGFP positive signal detection of zebrafish Tg(apoc1:eGFP) 3 days after fertilization, as described in Example 3. Detailed Implementation

[0047] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. Unless otherwise specified, the technical means used in the following embodiments and experimental examples are conventional means well known to those skilled in the art, and the materials and reagents used can all be obtained commercially.

[0048] Example 1: Construction of a zebrafish animal model

[0049] S1: Cloning of the lgals3bpb gene promoter

[0050] The promoter of the lgals3bpb gene (SEQ ID NO.1, 4141bp) was amplified by PCR using the following primers:

[0051] F1-lgals3bpb: 5'-tcgaattcctgcagcccgggTGTTGCTCTGAAGTCAGAGG-3', SEQ IDNO.2;

[0052] R1-lgals3bpb: 5'-GCTGTTGTACGTCCAAGTACCATTCATACTATTCACATTT-3', SEQ IDNO.3;

[0053] F2-lgals3bpb: 5'-GTACTTGGACGTACAACAGC-3', SEQ ID NO.4;

[0054] R2-lgals3bpb: 5'-tcctcgcccttgctcaccatGATGTCAGAAACAGCAACGA-3', SEQ ID NO.5.

[0055] The PCR system (using the PCR kit from Yisheng Biotechnology (Shanghai) Co., Ltd.) is as follows:

[0056] Water: 18 μL; 2×heiff buffer: 25 μL; heiff enzyme: 0.5 μL; genomic template (zebrafish genomic DNA): 1.5 μL; F-terminal primer (10 μM): 2.5 μL; R-terminal primer (10 μM): 2.5 μL.

[0057] The PCR procedure is as follows:

[0058] 98℃, 3 min, ×1;

[0059] (98℃, 10s)→(60℃, 20s)→(72℃, 2min), ×40;

[0060] 72℃, 5min, ×1.

[0061] The purified and enriched promoter amplification product of the lgals3bpb gene was obtained using conventional agarose gel electrophoresis and gel extraction techniques. The product was then sent to a third-party company for sequencing to confirm that the recovered product was the target sequence. See the electrophoresis images of the two DNA fragments below. Figure 1 .

[0062] The following primers were used to amplify the eGFP fluorescent protein (SEQ ID NO.6) by PCR:

[0063] F1-eGFP: 5'-atggtgagcaagggcgagga-3', SEQ ID NO.7;

[0064] R1-eGFP: 5'-tggatcatcatcgataggcctttacttgtacagctcgtcca-3', SEQ ID NO. 8.

[0065] Water: 18 μL; 2×heiff buffer: 25 μL; heiff enzyme: 0.5 μL; plasmid template: 1.5 μL; F-terminal primer: 2.5 μL; R-terminal primer: 2.5 μL.

[0066] The PCR procedure is as follows:

[0067] 98℃, 3 min, ×1;

[0068] (98℃, 10s)→(60℃, 20s)→(72℃, 2min), ×40;

[0069] 72℃, 5min, ×1.

[0070] The purified and enriched eGFP fluorescent protein gene amplification product was obtained using conventional agarose gel electrophoresis and gel extraction techniques. The product was then sent to a third-party company for sequencing to confirm that the recovered product was the target sequence.

[0071] S2: Construction of expression vector

[0072] The promoter of the lgals3bpb gene, the eGFP fluorescent protein sequence, and the pTAL2-cryaa vector (catalog number: #31823; see reference: Darius Balciunas, Harnessing a high cargo-capacity transposon for genetic applications in vertebrates, PLoS Genet. 2006 Nov; 2(11):e169. doi:10.1371 / journal.pgen.0020169) were fused via homologous recombination to obtain the pT2AL-cryaa-lgals3bpb-eGFP plasmid. The general process is described in [link to relevant documentation]. Figure 2 The specific process is as follows:

[0073] The pTAL2-cryaa vector was digested with restriction endonucleases Xma1 and Cla1. DNA polymerase chain reaction (PCR) was used to obtain lgals3bpb gene promoter amplification product 1, which was purified and recovered to obtain PCR product A. DNA polymerase chain reaction (PCR) was used to obtain lgals3bpb gene promoter amplification product 2, which was purified and recovered to obtain PCR product B. A plasmid containing the eGFP fluorescent protein sequence was amplified by DNA polymerase chain reaction (PCR) to obtain the eGFP sequence amplification product, which was purified and recovered to obtain PCR product C. Then, PCR products A, B, and C were ligated using homologous recombinase to form the pT2AL-cryaa-lgals3bpb-eGFP plasmid. A schematic diagram of this plasmid is shown below. Figure 3 .

[0074] The ligation system was as follows: 2× homologous recombinase: 10 μL; digested pTAL2-cryaa vector: 100 μg; PCR product A: 25 μg; PCR product B: 23 μg; PCR product C: 30 μg.

[0075] Connection time: 15 minutes; temperature: 50℃.

[0076] The constructed plasmid was transformed into *E. coli* DH5 competent cells using conventional techniques. Single colonies were picked and cultured in 40 ml of ampicillin-containing medium at 37°C and 180 rpm for 12 h. The plasmid was then purified using an endotoxin-free plasmid extraction kit to obtain a highly pure plasmid vector, pT2AL-cryaa-lgals3bpb-eGFP, free of *E. coli* cell bodies and other substances. Images of positive colonies selected by colony PCR are shown below. Figure 4 .

[0077] S3: Obtaining Genetically Modified Zebrafish Strains

[0078] See the flowchart for this step. Figure 5The specific process is as follows: The pT2AL-cryaa-lgals3bpb-eGFP plasmid (40 ng / μL) and transposase (100 ng / μL, Tol2 transposase mRNA, whose template DNA sequence is shown in SEQ ID NO.9) were mixed in a 1:1 volume ratio and injected into zebrafish single-cell fertilized eggs (AB strain zebrafish, injection volume 1 nL). The plasmid vector was then randomly integrated into the zebrafish genome by the action of the transposase. A transposon is a sequence that can change its position, containing an intermediate sequence and terminal repeats. The intermediate sequence can express transposase. The transposase can recognize the terminal repeats, thereby excising the transposon sequence and transferring it to other locations in the genome. The transposase in this protocol is a tool to improve the plasmid insertion efficiency. Without its use, the insertion of foreign genes into the zebrafish genome can still occur, but the efficiency will be reduced.

[0079] Two days post-microinjection of single-cell zygotes into embryos, the expression of Cryaa fluorescent protein signal in the eyes was detected under a fluorescence microscope to determine the success of the microinjection. Then, three days post-fertilization, the presence and location of eGFP expression were detected under a fluorescence microscope to determine whether the recombinant plasmid could drive eGFP expression and specifically label microglia. The results of Cryaa-cerulean labeling observation three days post-fertilization are shown below. Figure 6 (cryaa-cerulean specifically expresses blue fluorescence in the lens of zebrafish, and is commonly used to detect whether plasmids have integrated into the chromosome after injection into zebrafish). Fertilized eggs exhibiting fluorescent expression were selected and cultured at a standard temperature of 28.5℃ until sexual maturity (F0 generation; founder). The F0 generation was mated with wild-type zebrafish to obtain fluorescent offspring, designated as the F1 generation. The F1 generation was then cultured until sexual maturity and mated with wild-type zebrafish to obtain the fluorescent F2 generation. At this point, the fluorescent zebrafish Tg(lgals3bpb:eGFP) strain was successfully constructed.

[0080] The whole-body eGFP signal of F1 generation (3dpf) zebrafish was detected. See the experimental results below. Figure 7The study found that eGFP signaling specifically colonizes in the brain and skin. Langerhans cells, a type of tissue-resident macrophage, are present in the skin and share functional similarities with microglia. The lgals3bpb gene is involved in immune processes, hence the presence of green fluorescent protein (GFP) markers on zebrafish skin. Langerhans cells and microglia are tissue-resident macrophages belonging to different tissues. If the promoter of the lgals3bpb gene is manipulated on cells expressing this gene, in microglia, these gene manipulations primarily affect the brain microenvironment, such as neuronal development and function; in Langerhans cells, these gene manipulations primarily affect viral infection at their colonization sites and tumor-related immune processes. Therefore, the initiation of effector gene expression by the lgals3bpb gene promoter in Langerhans cells and microglia does not affect manipulation of microglia or experimental results. In addition, if we use MTZ-NTR to specifically eliminate cells containing the promoter of the lgals3bpb gene, we can control the concentration of MTZ to ensure that only microglia in the brain are eliminated without affecting Langerhans cells.

[0081] The zebrafish Tg(coro1a:DsRed) strain was crossed with the aforementioned F0 generation expressing eGFP (Tg(lgals3bpb:eGFP)) to obtain the hybrid strain Tg(coro1a:DsRed;lgals3bpb:eGPF). See also Figure 8 At 3 dpf, observations were made on coro1a-DsRed+ cells (red, left image) and lgals3bpb-eGFP+ cells (green, middle image). A high degree of overlap was found between cells labeled with the lgals3bpb gene promoter and those labeled with the Coro1a gene promoter (fused image, right image), indicating that the lgals3bpb gene promoter can initiate the specific expression of effector genes in microglia. The zebrafish Tg(coro1a:DsRed) strain was obtained as described earlier in this embodiment, except that the promoter and effector gene were replaced with the coro1a gene promoter (information on this promoter can be found in the literature Li, L., Liveimaging reveals differing roles of macrophages and neutrophils during zebrafish tail fin regeneration. 2012, Journal of Biological Chemistry 287, 25353-25360.) and DsRed fluorescent protein. Figure 8In the diagram, arrows indicate cells co-stained with DsRed and eGFP, while arrows indicate cells without co-staining (only DsRed signal). The Coro1a gene promoter is primarily used to specifically label immune cells (leukocytes), including microglia, while the lgals3bpb gene promoter primarily labels microglia. Since brain immune cells are mainly microglia, overlapping labeled cells occurred. The fluorescence image shows cells labeled only by the Coro1a gene promoter and not by the lgals3bpb gene promoter. The inventors analyzed that this is because these cells not labeled by the lgals3bpb gene promoter are not tissue-resident cells (i.e., not microglia), but rather macrophages patrolling to that location and being labeled by the Coro1a gene promoter. These experimental results further demonstrate the specificity of the lgals3bpb gene promoter labeling, marking only microglia and not other brain immune cells or neurons. We can utilize the characteristics of the lgals3bpb gene promoter to conduct microglial cell-specific research and related procedures.

[0082] The colocalization results of the newly constructed transgenic line (the fluorescent zebrafish Tg(lgals3bpb:eGFP)) with panmicroglia in the midbrain at 12 months of development are shown in [reference needed]. Figure 9 (The left image shows the red Lcp1 label, the middle image shows the green eGFP label, and the right image is a fused version of the two). Anti-Lcp1 refers to an antibody against LCP1 (lymphocyte cytosolic protein 1). The promoters of the lcp1 and coro1a genes label the same type of cell; this antibody is generally used to label panmicroglia in the zebrafish brain. The above experimental results indicate that the expression of effector genes controlled by the coro1a gene promoter is concentrated in panmicroglia in the midbrain. Figure 9 In the diagram, the arrowhead indicates cells co-stained with Lcp1 and eGFP, while the arrow itself indicates cells without co-staining (only Lcp1 signal). Adult zebrafish may have macrophages from other sources replacing some microglia in the brain, resulting in cells that do not express eGFP. This further illustrates the specificity of the lgals3bpb gene promoter marker, which only marks microglia and not other immune cells or neurons.

[0083] Example 2: Study of the lgals3bpb gene and its promoter

[0084] (1) Study on the expression pattern of the lgals3bpb gene

[0085] like Figure 10 As shown, the apoeb gene (apolipoprotein E, accession number NM_131098.2) and the ccl34b.1 gene (chemokine(CC motif) ligand 34b,duplicate 1, accession number NM_001115054.2) are both marker genes for microglia reported in the prior art, and are highly expressed in microglia. For reports on the apoeb gene and ccl34b.1 gene as marker genes in microglia, please refer to existing technical literature (Francesca Peri, Live Imaging of Neuronal Degradation by Microglia Reveals aRole for v0-ATPase a1 in Phagosomal Fusion In Vivo, Cell. 2008 May 30; 133(5):916-27. doi:10.1016 / j.cell.2008.04.037; Shuting Wu, Two phenotypically and functionally distinct microglial populations in adult zebrafish, SciAdv. 2020 Nov18; 6(47):eabd1160. doi:10.1126 / sciadv.abd1160.). The inventors discovered a variety of genes enriched and expressed in microglia through gene sequencing and other methods. Expression pattern studies were conducted on these enriched genes, revealing that the expression pattern of the lgals3bpb gene (accession number: NM_212873.1, galectin 3-binding protein) highly overlapped with that of the apoeb and ccl34b.1 genes. (See [link to relevant documentation]). Figure 10 .

[0086] exist Figure 10 In the diagram, the coordinate axes represent the gradients between data points, used to simulate their distance functions in high-dimensional and low-dimensional spaces. Positive gradient values ​​indicate attraction between data points in low-dimensional space, while negative values ​​indicate repulsion. The gradient on the right, ranging from 0 to 6, represents the gene's expression level in different taxa, using color intensity to indicate expression levels. Figure 10In the image, the genes in the right-hand box have the same expression patterns as the apoeb and ccl34b.1 genes in the immune cells of the zebrafish brain, meaning they are enriched in the same cell populations, including lgals3bpb, p2ry12, vmp1, fcer1gl, havcr1, and apoc1 genes.

[0087] Literature reports (Francesca Peri, Live Imaging of Neuronal Degradation by Microglia Reveals a Role for v0-ATPase a1 in Phagosomal Fusion In Vivo, Cell. 2008 May 30; 133(5):916-27. doi:10.1016 / j.cell.2008.04.037.) that in the existing transgenic expression pattern of apoeb constructed by BAC, the effector gene (GFP) carried by apoeb is not only expressed in microglia, but can also be detected in brain neurons (immune cells in non-zebrafish brains) (see [link to article]). Figure 11 (This figure is from the aforementioned paper by Francesca Peri). Figure 11 The thin arrows refer to microglia, and the thick arrows refer to neurons. Neurons are characterized by their long, linear branches. This indicates that using the apoeb gene to label microglia lacks specificity and also marks neurons, hindering observation and manipulation of microglia. Therefore, some commonly used microglia markers are only highly expressed in microglia but also expressed to some extent in other cell types. In particular, the apoeb gene can be expressed simultaneously in both microglia and neurons, significantly interfering with microglia research. It is evident that current technology lacks genes (promoters) capable of specifically labeling microglia, and the discovery of the lgals3bpb gene expression pattern can overcome these shortcomings.

[0088] (2) Cloning of promoters of genes enriched in microglia.

[0089] Based on the genes highly expressed in microglia screened in this embodiment, the inventors attempted promoter capture of these genes to capture promoter sequences that mark microglia. Identifying the core promoter of a gene is a complex task. Cell-specific core promoter sequences are often unknown, and their location and length are difficult to predict before discovery. Some gene core promoters are only a few hundred base pairs long, while others are tens of thousands. Some gene promoters are close to their coding sequences, while others are far from them; some are located upstream of the coding region, while others are downstream. These factors all hinder the discovery of gene promoters. See Table 1 for the capture results of gene core promoters.

[0090] Table 1: Microglial core promoter capture status

[0091]

[0092] See fluorescence images of microglia from transgenic zebrafish containing the vmp1 gene promoter. Figure 12 (3 days post-fertilization), DsRed expression was regulated by the known promoter of the coro1a gene. The high expression of DsRed indicated that the cells in the field of view were microglia (an immune cell). However, no eGFP signal was observed in these cells, suggesting that the upstream sequence of the start codon of the cloned vmp1 gene does not have the function of initiating eGFP gene expression. The situation was similar to that of the vmp1 gene when the upstream sequence of the start codon was used in transgenic zebrafish studies (see [link to vmp1 gene study]). Figure 13 The inventors analyzed the reasons for the above phenomenon: it may be that the vmp1 gene promoter is not located 4000 bp upstream of its start codon; or it may be that although the promoter of the gene was successfully cloned, the promoter could not effectively activate the expression of the effector gene in animal models. The above experimental results illustrate that not any random sequence with promoter effects can activate the expression of effector genes, that is, not all random promoters are operable, and illustrate the difficulty of cloning promoters and applying them to control the expression of effector genes in specific cells.

[0093] against Figure 13 and Figure 14Based on the experimental results, the function of the Coro1a gene promoter in the prior art is further explained as follows: The Coro1a gene promoter can label all immune cells (including immune cells such as macrophages, monocytes, granulocytes, and lymphocytes). However, based on the current understanding that microglia are the only immune cells in the central nervous system, we believe that the Coro1a gene promoter in the brain labels panmicroglia (which may include mature, immature, and undifferentiated states).

[0094] (3) Cloning of the promoter of the lgals3bpb gene

[0095] The inventors first cloned a sequence approximately 2000 bp upstream of the start codon of the lgals3bpb gene (SEQ ID NO. 10), but this failed to initiate eGFP expression. For the lgals3bpb gene in this protocol, the high GC content and repeated polyTs in its promoter region reduced the efficiency of DNA polymerase chain reaction (PCR). This forced the inventors to amplify the sequence in segments, ultimately obtaining a 4141 bp promoter sequence, as shown in SEQ ID NO. 1. Amplifying the 4141 bp sequence before the start codon of the lgals3bpb gene via PCR successfully initiated the expression of its carried eGFP, and the cells expressing eGFP were localized to the brain (based on experimental data from Example 1), demonstrating a specific labeling of microglia – a novel discovery.

[0096] In summary, this approach provides an operable promoter sequence for labeling microglia, enabling specific manipulation of microglia. For example, carrying a nitroreductase gene after the promoter can specifically kill microglia without harming other macrophages; or the promoter can be used to specifically add or knock out a gene in microglia or Langerhans cells; furthermore, fluorescent proteins can be integrated after the promoter to locate microglia and observe the expression and function of the gene (lgals3bpb) during development.

[0097] Example 3

[0098] The inventors also cloned the promoter of the apoc1 gene (SEQ ID NO. 10) from Example 2 using the method and procedure of Example 1, and established the transgenic zebrafish strain Tg(apoc1:eGFP) using the method and procedure of Example 1. The whole-body eGFP signal of F1 generation zebrafish was detected; the experimental results are shown below. Figure 14The study found that the eGFP signal specifically colonizes in the brain and skin, and that the apoc1 gene promoter, in addition to the markers mentioned above, also marks the yolk sac and caudal fin. This indicates that the apoc1 gene promoter marks a wider range of areas compared to the lgals3bpb gene promoter, while the lgals3bpb gene promoter exhibits relatively higher specificity.

[0099] Apart from the difference in promoter sequence and corresponding cloning primer, the operation procedures in this embodiment are the same as those in Embodiment 1. However, the green fluorescent protein signal controlled by the promoter of the lgals3bpb gene is stronger, which is more conducive to the observation of microglia.

[0100] The above descriptions are merely embodiments of the present invention, and common knowledge such as specific technical solutions and / or characteristics are not described in detail here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solutions of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims. SEQUENCE LISTING <110> Chongqing Institute of Green and Intelligent Technology, Chinese Academy of Sciences <120> A promoter for the lgals3bpb gene and its application in constructing a zebrafish model. <130> 2022.04.19 <160> 11 <170> PatentIn version 3.5 <210> 1 <211> 4141 <212> DNA <213> Danio rerio <400> 1 tgttgctctg aagtcagagg aggacgctgg gaaaatcgcc acctacattg tggcgtttac 60 ccaagatggc cgaattctgc tacctgaaac taagatcggg gatgtgaagt atgagggact 120 tgagttcatt tagtgtctgg acgtattttc atgccaagga ttggtaaatg ccttcttcta 180 ggtgatgtac atctagattt gggcattgta aggttaagaa taatgccaag tgtctgatgg 240 cagccaaaat gtggttgtaa atatcgactc ttaaaattaa agttgactct ttaattattt 300 taaatcacct gtcggagcct tcatcaaaag ttttttttat tgtttgcttc tctgtctttt 360 tcgaggttta gcgtttcacg ccagcactgt acttacagtg taatgaatgt aacagattgg 420 atgaagtgac agactcgaat agtttaccct acaaacatca ctgcagttgc catgtggttt 480 tgacgaatgt cttttttttt tttttactgt gattgtttt gttttttact ttattgacaa 540 gtgtcttaaa cagatcagtt actttttaaa aaaatctat aaatctgaac tgtcacaaaa 600 tgaacatcat gtttcatcga ttcaccagag ctatttattt gttgttgctg tattttgttt 660 ctgaagattg aaggttcata agaacggcac ttgtgtactc tggttatttc atccagttgt 720 ttgattcact cagttttgtt aaatcaaata tcacattaag ctaaatacac aggacagact 780 gcacccacag tgtggatgta cacagagttc atgaatttat tactcttaat cagtgtcttt 840 attgggtaca ccttactagt agcgagttga accccccttt gccttcagaa ctgccttaat 900 tctccgtggc ataaatattc ctcagagact ttggtccata ttgacattat agcatcatgc 960 atttgctgca gatttgttgg ctgcacatcc atgatgtgaa tctcccgttc caccacatcc 1020 caaaggtgct ctattggatt gagctctggt gactgtggag accatttgag taccgtgaac 1080 tcattgtcat gttcaagaaa ccagtctgag atgattcgga gatggttatc ctgctggaag 1140 tagatatcag aagatgggta cacagtggtc ataaagggat ggacattgtc ggcaacaata 1200 ctcaggtagg ctgtggcgtt gtcatgatgc tcagttggta ctaatgggcc caaagtgtgc 1260 caagacaata tccccatacc attacaccac caccacaagc ctgaaccgtt gatacaaggc 1320 tggatggatc catgctttta tgttgttgat gccaaattct gaccctacca ttcgaaaatc 1380 gcagcagaag tcgagactca tcagaccagg caacgtttgt ccaatcttct attgtccaat 1440 tttgctgagc ctgtgtgaat tgtagcctca gtttcctgtt cttagcggac aggattggca 1500 cccattgtgg tcttctgctg ctgtagccca tccgcctcaa ggttggatgt gttgtgtgtt 1560 cagagatgct tttctgcaga cctcggttgt agcgagtgca tatttgagtt actgttgcct 1620 ttctgtcagc tcaacccagt ctggccattc tcctctgacc tcttgtatta acaaggcatt 1680 tgcatccaca gaactgccgc accaacaacc ctgccacaat caaagttgct gccatgtgat 1740 tggctgatta gaaatttgca ttgagcattt tgacaggtgt gcctaataaa gtggctaggg 1800 agtgtatata taaacacaaa caaagacaaa attattagcc ctccttggaa atcataaatc 1860 cttttttaaa tatgtcctaa gagatgttta acagagccgt tacattttta tgatattttc 1920 tattatatga ttattcttta gaaagtcata tttctttgg tttgtcttaa ataatagaat 1980 aaaaaaaa agagtaaaaa ataaaggctg tgtttgaaca cgcctactac tcagtgggta 2040 ctacatttga atttgaattt actacctgac cattagaaaa gtaagatcta tacagtataa 2100 atgtgaatag tatgaatggt acttggacgt acaacagccg tcattttctg acctacccac 2160 atcagttggt cgcttcactc ccgttcatga attctctcac tgtgcattat gggatagcgt 2220 agcgtccatc tgatgtgcac ttcaaaatct agtcagaagt agtaggtcat ccgggtactt 2280 ctcgcataca gtttttg aattctgtga atgaattcgg acatactact tgactcgcat 2340 actgttttta acttactata aagtatggaa gtatgcgatt tcggacgcag ctaaagtca 2400 taaaaattag ccccttttt ttcaattgg aaaaaaaa tgattgcct acagaacaat 2460 ccactgttgt ccaatgactt acctagttga cctaattaac ctagttaagt ctcaattg 2520 cacttttagc tgaatattat tgtcacaca tattgtgtac atcatggcaa agacgaaaga 2580 aattggttat tagaatcag ttattaaaat ctgtagtttt aaaatatgg ccaccacctg 2640 gttttaccc atgctgtcct ccaccccac aacattatt atggtttaac agaagggtcc 2700 cagaaacata ctgtccctgg tagagatggg tctcaagt gccttgaagc tgaccagaga 2760 thunderstorm aggaatg thunderstorm thunderstorm aggaatt 2820 ccctgaacta ttgggtgaag acagctgta actttgaa ctcagttacc aaaatgtaga 2880 tcggttttac tttaatctga aaagtaagta aaagttgtaa tatgttgcca agtatggtga 2940 cctatactaa taatttctgc tctgcttta accatccaa gtacaacac acaggagtaa 3000 acacaaacac ctgggaaaca attgcctaaa cagccactat gttaagaact aattacacca 3060 gatcaatcaa tgccttactc aagggaatca cctcagccat gtcattgagg gtggtaagtt 3120 accacctaca accctgacag gactcaaacc tgcaaccttc ttatcataag tccaacacta 3180 acccttagat ggtgtaagtg ttgtaggtgg tgggagagat caatcggtcc cttgctcaag 3240 gaactcacct cagccatggc attgagggtg gtgagttacc acctacaacc ctgacagaac 3300 ttgagcctct gcctttcgat cacaagtcca attctttaac cattaggcca cagctgcccc 3360 ttgtatggtg taagggttgt agctggtggg agagatcaat gggtcccttg ctcaaggaac 3420 tcatctcagc catggtattg agggtggtga gttaccacct acaaccctga cagaacttga 3480 gcctctgcct ttcgatcaca agtacaattc tttaaccatt aggccacagc tgccccttgg 3540 atggtgtaag ggttaaaggt ggtgggagag atcaatcagt gcctagctca agaaactcac 3600 ctcagccagg gtattgagag tggtgagtcc cttaagcaag gcaccgattg atctagtgta 3660 actagtactt cacatagtgg ctgttaatga aaccagacac agatgagact gactggaaac 3720 aaatgatcag gagcagcctc tgggaaaaca ggctctacag accccaggaa agcagaccta 3780 acccctattc tggcaattca cttgccagca aagagattca taagttatta tgggttatca 3840 atagggcaag tgactggtga ttgttttggg gaacactgta ttgtgggatt ttagtttgac 3900 taacacctca tgtggctaaa cttaacccag actttgctga caaacagttc ttatcgaaac 3960 ttcccaaatt gtacattttc atgaaacgaa acttttggtg tcctagaaat gtgattgcgc 4020 agcgtttcgt taaagtttca caattgcaaa cttgatttgg ccgttattta aaggaaagcc 4080 aagatgcttt gtaagtcatt cagttgtgaa ttgcatcctt gtcgttgctg tttctgacat 4140 c 4141 <210> SEQ ID NO: 2 <211> 40 <212> DNA <213> Artificial Sequence <400> SEQ ID NO: 2 tcgaattcct gcagcccggg tgttgctctg aagtcagagg 40 <210> SEQ ID NO: 3 <211> 40 <212> DNA <213> Artificial Sequence <400> SEQ ID NO: 3 gctgttgtac gtccaagtac cattcatact attcacattt 40 <210> SEQ ID NO: 4 <211> 20 <212> DNA <213> Artificial sequence <400> 4 gtacttggac gtacaacagc 20 <210> 5 <211> 40 <212> DNA <213> Artificial sequence <400> 5 tcctcgccct tgctcaccat gatgtcagaa acagcaacga 40 <210> 6 <211> 720 <212> DNA <213> Artificial sequence <400> 6 atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180 ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420 aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 480 ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 540 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600 tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660 ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa 720 <210> 7 <211> 20 <212> DNA <213> Artificial sequence <400> 7 atggtgagca agggcgagga 20 <210> 8 <211> 41 <212> DNA <213> Artificial sequence <400> 8 tggatcatca tcgataggcc tttacttgta cagctcgtcc a 41 <210> 9 <211> 1950 <212> DNA <213> Artificial sequence <400> 9 atggaagagg tgtgcgactc ttccgccgca gcttcatcta ccgtccagaa ccagcctcag 60 gaccaagagc acccctggcc atatctgaga gagttcttct ccctgagcgg agtgaataag 120 gactcattta aaatgaagtg tgtgctgtgt cttccactca acaaagagat tagcgcgttt 1​aaatcctcac cctcgaacct gaggaagcac attgagcgta tgcacccta ctacctgaaa 240 aattactcca agctgaccgc ccagagaga aagataggca ctagtactca cgcatccagc 300 agtaagcagt tgaaagtaga ctcagtgttc cctgttaagc atgtgtcccc tgtaacagtt 360 aacaaggcta ttctccggta cattatccaa ggtctgcacc cgttcagcac cgtcgaccta 420 ccctccttca aggagctgat tagtacgctt cagcctgga tagtgtgat tactcgccct 480 actctgagaa gcaaatcgc agaagcggcg ctgatcatga aacaagaagt gactgctgca 540 atgagcgagg tggaatgat cgcgacgaca accgactgtt ggacagcacg tggagtcc 600 tttataggcg tcactgccca ctggattaac cccggttcac tggagcgaca ctcagcagcc 660 ctggcctgca aacgccttat gggatctcac acttttgagg tgttagcgtc tgctatgaac 720 gataccact cggaatacga aattagggac aaggtggtct gtaccacac agactctgga 780 tctaactta tgaagcgtt tcgcgttttt ggcgtggaga acatgatat cgaaacggag 840 gccagacgct gtgagtcaga cgacactgac agcgaaggct gcggcgaagg ttcagacggc 900 gtcgaatttc aggacgcgag tcgcgttctc gaccaggatg atggatttga atttcagtta 960 ccaaagcacc agaagtgtgc ctgtcatctt ctaaacctcg tctcttcggt cgacgcacaa 1020 aaagctctgt ccaacgagca ttacaagaag ctctaccggt ctgtcttcgg caagtgtcag 1080 gccctttgga acaagtcctc cagatccgct ctcgccgcag aggcggtaga gagtgagtcc 1140 agacttcagc tgctgagacc taatcagaca cgctggaaca gcactttcat ggcagtggac 1200 cgaatcttac aaatctgtaa agaagccggt gagggcgctc tccgcaacat ctgtacatca 1260 ctggaagtcc ctatgtttaa cccagctgag atgctcttct tgacagagtg ggctaacact 1320 atgcggccag tcgctaaagt cctcgacatc cttcaggccg aaaccaatac acagttaggc 1380 tggctccttc caagcgtgca ccaactcagt ctgaagctcc aaagactgca tcatagtctg 1440 cggtactgcg atcccctcgt ggatgctctg cagcagggaa tccagactcg gttcaaacac 1500 atgttcgaag accctgagat tatcgctgca gccatccttc tcccaaaatt tcgaacctcg 1560 tggacaaacg atgagaccat tattaagcgc ggcatggatt atatccgcgt ccaccttgaa 1620 cccctggacc acaaaaaaga gctggctaat tcaagtagtg acgatgagga tttctttgcc 1680 agcttgaagc cgacgacaca cgaggcctca aaggagctgg acggttacct ggcctgcgtc 1740 agcgatacaa gggagtcact gttgaccttc cctgcaatct gttcgctgtc gatcaagaca 1800 aacactccct tgcctgctag tgcagcgtgt gagagactct ttagcacagc aggactcctg 1860 ttcagcccta agagagcaag gcttgatacc aacaactttg agaaccagtt gcttctgaaa 1920 ctgaatttac gattttacaa cttcgagtga 1950 <210> 10 <211> 2023 <212> DNA <213> Danio rerio <400> 10 gtacttggac gtacaacagc cgtcattttc tgacctaccc acatcagttg gtcgcttcac 60 tcccgttcat gaattctctc actgtgcatt atgggatagc gtagcgtcca tctgatgtgc 120 acttcaaaat ctagtcagaa gtagtagtc atccgggtac ttctcgcata cagtttttta 180 tgaattctgt gaatgaattc ggacatacta cttgactcgc atactgtttt taacttacta 240 taaagtatgg aagtatgcga ttcggacgc agctaaagtc aataaaaatt agccccttt 300 ttttcaaatt ggaataaaaa aattgattgc ctacagaaca atccactgtt gtccaatgac 360 ttacctagtt gacctaatta acctagttaa gtcttcaaat tgcactttta gctgaatatt 420 attgtcacac aatattgtgt acatcatggc aaagacgaaa gaaattggtt attagaaatc 480 agtttaaa atctgtatgt ttaaaaatat ggccaccacc tggtttttac ccatgctgtc 540 ctccaacccc acaaacatta ttatggttta acagaagggt cccagaaaca tactgtccct 600 ggtagagatg ggtcttcaaa gtgccttgaa gctgaccaga gaaaagga aacaacccca 660 tgagtaaatg atgacagaat tttcaagttt tagtaaact atccctgaac tattgggtga 720 agacagcttg taactttttg aactcagtta ccaaaatgta gatcgggttt actttaatct 780 gaaagtag taaaagttgt aatatgttgc caagtatggt gacctatact aataatttct 840 gctctgcttt taacccatcc aagtacaaac acacaggagt aaacacaaac acctgggaaa 900 caattgccta aacagccact atgttaagaa ctaattacac cagatcaatc aatgccttac 960 tcaaggaat cacctcagcc atgtcattga gggtggtaag ttaccaccta caaccctgac 1020 aggactcaaa cctgcaacct tcttatcata agtccaacac taacccttag atggtgtaag 1080 tgttgtaggt ggtgggagag atcaatcggt cccttgctca aggaactcac ctcagccatg 1140 gcattgaggg tggtgagtta ccacctacaa ccctgacaga acttgagcct ctgcctttcg 1200 atcacaagtc caattcttta accattaggc cacagctgcc ccttgtatgg tgtaagggtt 1260 gtagctggtg ggagagatca atgggtccct tgctcaagga actcatctca gccatggtat 1320 tgagggtggt gagttaccac ctacaaccct gacagaactt gagcctctgc ctttcgatca 1380 caagtacaat tctttaacca ttaggccaca gctgcccctt ggatggtgta agggttaaag 1440 gtggtgggag agatcaatca gtgcctagct caagaaactc acctcagcca gggtattgag 1500 agtggtgagt cccttaagca aggcaccgat tgatctagtg taactagtac ttcacatagt 1560 ggctgttaat gaaaccagac acagatgaga ctgactggaa acaaatgatc aggagcagcc 1620 tctgggaaaa caggctctac agaccccagg aaagcagacc taacccctat tctggcaatt 1680 cacttgccag caaagagatt cataagttat tatgggttat caatagggca agtgactggt 1740 gattgttttg gggaacactg tattgtggga ttttagtttg actaacacct catgtggcta 1800 aacttaaccc agactttgct gacaaacagt tcttatcgaa acttcccaaa ttgtacattt 1860 tcatgaaacg aaacttttgg tgtcctagaa atgtgattgc gcagcgtttc gttaaagtttt 1920 cacaattgca aacttgattt ggccgttatt taaaggaaag ccaagatgct ttgtaagtca 1980 ttcagttgtg aattgcatcc ttgtcgttgc tgtttctgac atc 2023 <210> 11 <211> 4212 <212> DNA <213> Danio rerio <400> 11 cagttgtttg gactccttgc cctcaactgt ccacactata ttttataggg ccaaagagat 60 tgcaatttag taaataaaga ctgaggacga aaataataat aacatttatt acttgataaaa 120 tgcttctgtt ggggcagcac ggtggcgcag tgggtagcac agtcggctcg cagcaagaag 180 gttgctagtt tgagccccgg ctgggtcagt tggcatttct gtgtggagtt tgcatgttcg 240 ccctatgttg gcgtagttt cctcttggtg ctccggtttc gccctcagtc caaaatcatg 300 tggtacaggt gaattgggca agctaaattg tccgtcgtgt atgtgtagga atgagtgtgt 360 atggatgttt cccagtaatg ctgagtaaaa tatatgctgg ctaagttggc agttaatttcc 420 gctgtggcaa aaatgaata attackactg ttatacaca caacacaca cctacacaca 480 ggtttgttt tgtgaatttg aggtttcat tgtttctata ctgtacaaac tgttattttca 540 ttgccctaca ccttacatga agctgtgtgc tgttttact tcagaaataa atcattata 600 cacgtcatta tacaagtcat tatacacatt tgcatcctaa tgtgacacaa aaacacgtac 660 atacacatttt ttgctgatgat atgtcacaa aaggcatact gtacattcag acacacatac 720 tgtatttata gctatggaa aaagtttaag gtaaaataa aaagttatgt gtaattttg 780 gttaatttg gttttattct gtaaactact atgcctttt cttccaaacc cttttaggga 840 agagctttgc tcacaaggt taaacaaaa gcttgagatg gcacagcagg tcactgtcta 900 gactcataa atgaaaacc aagggccgtt gactcataa agtacataa actaaagaa 960 caacaggtga ctattatgt gtgtgtgaga cgagctgttt gatggcttt ggtcttaaag 1020 tgttctgcaa agcctgcaga taagtgacaa agggacact ggaggcccg tcctccacag 1080 actgcaagtg gtcagatgtg agcagaggta actgcagatc aatgcctctt ggtgtgatcc 1140 tctataaaca tcatgtcctc acggagcctc gcttcccctt attccagtct agacgctttc 1200 aacacttttc aagctgtcag cttttccgat cacgatcaag tcttttaata gatgcactat 1260 agtacaagaa agtattttgc aaaataaaga aaaatctatt aaacaggaaa aaaaatctcc 1320 ctgacctgag tgataaaaaa aataaacagg tcattgacca acaagaactg ctgattttgt 1380 gtgtgtgttt tgtgcccaca ctggattggg aaatacataa gatcaatgct gacaagatgt 1440 taactcttga ccttatttta aagtgtaaaa atgcaaatta cataaagtgc gatcaattat 1500 tatatgctac tataagatgc ttttttatta aataaattac tttgtcattt attgtttagt 1560 cttcatatca aacgttgatt tgaccctcaa aaacgtttat tttcaaaaag tccatacgtt 1620 gctctgatta tcacattatt ctgtcaatat ttttacattt tcatgattct ctgatgaaca 1680 aaatgttaaa ataacagcat atagaatgtt ttaatttcat ctaaaataat tttgtatttt 1740 aactgtttaa tctaccgcag gcaatattgc taggtagaaa caaaaacaat atgaccactt 1800 tcaattcac aaccttcat agcttcatgg ctgggactt actaaatgt ctaatgtgtt 1860 tgaacattta agcacagaat atggcaaaaa atagtgtaca gcattgacga tgcagtgctt 1920 taattaaat aaatcaatg acaccatagg ccaataag atacatcta atgattctcc 1980 tgacctagaa tccaaagtg gttcaatgc agttagcaga atcctgcatg tatctatgtt 2040 tgtagacagc aggaataag gttatgaac tactaagaga taacaactt caatttactt 2100 tagtccaaat ccagactctt tatctcatgt tgtgatgtgg ctggaggtt aagaactgt 2160 ggagtaaatc agtgtctcat gatacaac aactacaag ttatacacccc ttacaattgc 2220 gcagtcacaa ataataca cagtaataat attttaaag ttgaattatg tttttgaaa 2280 actaaggctg agattcaaga agtaggctat caatctctt gctcgtgtgt tcagacatcc 2340 atattcacc atacaaaa agaaacctgc atacagacag tgatcatttt gtataat 2400 aaaggagcag attcctaaaa agtgaccact aaagcgggtc acacttatt ttgataggtc 2460 gtttgttgaa tttgttac attgcatcta catgccaact aattctcatt agattataag 2520 tagactgtta ggttggggtt agggtttgtt gacatgtact tgcaaaggtt cttatactca 2580 gttaaacgtt cttatataca gttaaatgtc tgttgaagga gcagtatcaa cagatattaa 2640 gcagtctatt aatactcaaa tggaccatca aaataaagtg ttaccctaaa gtgaatggtc 2700 agggggatac catgggatgg tgtaatatgc attttatttg tataaaatgc tgccatgtgc 2760 aatgtctgct acgggtcagt ttctttagtc actggtcaaa caagcagtat gaatgatcat 2820 tattgaactc tttatagata gtaatgaaat gtaaatgaaa acaaaaataa tttcttctca 2880 aaaattctgt tttacactgt aaacatgctg aggtccacac aattccttct tgttatccca 2940 acacaaactg attaagtaaa cttaattaaa tttacaaatg aaagtagctt gaacataaaa 3000 caattaagtt gtcccaaaaa aactcaggaa ttgtgttgat tcagctcatt ttagagtcat 3060 gattagtttg aacaagcagg aaaaatagtt ttgagcatac ataccagaat taactgtatc 3120 agtgaagtat gatgtatttt tatgccataa cactgatgct gtagtatatt tactagagga 3180 gatattacac aattgttttg tccttcagag tcatgtactg tagatgtaga aaaaagcacg 3240 taaccaactg taaaatccat gataataat ggactttgta ccatatatgc aaggcaaggg 3300 taccagataa agaagtacc cacttttta taacattgtt ttattttga cagatgttgt 3360 ttcaaggtc actggcactg aagtaaaata aagtagctca taacgaataa tcaaagaaaa 3420 tctccgtgaa aacagctgtt gtggcaaacg gagtttactt acctctaaag gatgtttact 3480 atagtcattt accacatcgc aaagctgtaa ggagaacgag agatgaacgc gaggaagcg 3540 agtgattgca ggaggtcttg ataggtcaga ggtggtgtta tacacaacg tcatctctgt 3600 gtcccttaa agggacaagc catctgtggg acatcacaaa ctcagcccaa ttctttcgag 3660 gactgcgaag aggtacgcaa acttatttt gttgtggtca ttttcaagc agagcgaatc 3720 tagagctt taacctgaa cgacagaata acctttcttt attaggttca gtgcgcaaac 3780 acgcagtata ttggcgacat ctagccgcca tccctgcta tgattggaaa atttagcata 3840 ttttgtaaa tgttagagga agataataa aacactatta aacgtttaca aacaatgcgc 3900 aaacatattt ttaaaagcgc gaaatatata ttatacactt cattagaacc tttgtaataa 3960 attttaatct tatgtttatt gtttagttt caaaagcaaa taacgtcttt gttttgatta 4020 atgacaaaaa tgattaatga caaaaaaaatc ttcttgttca accagaagtt ttttttattt 4080 attttttatt ttttttaaga ttaacattag cgtaaaccat aaattgttta aataatatct 4140 taggaaataa tttaacaccg aatcggtcaa aattatgatg cctgagattt ctagctctct 4200 aacagtgatt cc 4212

Claims

1. A lgals3bpb The application of the promoter of a gene in constructing a zebrafish model is characterized in that: The method includes the following steps in sequence: S1: obtained by PCR reaction lgals3bpb Gene promoter; lgals3bpb The nucleotide sequence of the gene promoter is shown in SEQ ID NO.1; The primers for the PCR reaction include: F1- lgals3bpb :5’- tcgaattcctgcagcccgggTGTTGCTCTGAAGTCAGAGG-3’; R1- lgals3bpb :5’- GCTGTTGTACGTCCAAGTACCATTCATACTATTCACATTT-3’; F2- lgals3bpb :5’- GTACTTGGACGTACAACAGC-3’; R2- lgals3bpb :5’- tcctcgcccttgctcaccatGATGTCAGAAACAGCAACGA-3’; S2: lgals3bpb The gene promoter and the fluorescent protein gene are integrated into the vector to obtain an expression vector; lgals3bpb Gene promoters are used to control the expression of fluorescent protein genes; S3: microinjecting the expression vector and the transposase into zebrafish fertilized eggs simultaneously; selecting fertilized eggs expressing the fluorescent protein after fertilization; and culturing them until sexual maturity to obtain F0 generation zebrafish; First, the F0 generation zebrafish are mated with wild-type zebrafish, and the offspring expressing fluorescent protein are screened to obtain the F1 generation zebrafish; then the F1 generation zebrafish are mated with wild-type zebrafish to obtain a stable genetically modified zebrafish strain.

2. A method according to claim 1 lgals3bpb The application of the promoter of a gene in constructing a zebrafish model is characterized in that: In S3, the sequence of the DNA template of the transposase is shown in SEQ ID NO.

9.

3. A method according to claim 2 lgals3bpb The application of the promoter of a gene in constructing a zebrafish model is characterized in that: In S3, the culture temperature was 28.5°C.

4. A method according to claim 3 lgals3bpb The application of the promoter of a gene in constructing a zebrafish model is characterized in that: In S1, the PCR reaction system includes: 18 μL water, 25 μL buffer, 0.5 μL DNA polymerase, 1.5 μL template, 2.5 μL upstream primer and 2.5 μL downstream primer.

5. A method according to claim 4 lgals3bpb The application of the promoter of a gene in constructing a zebrafish model is characterized by , in S2, the sequence of the fluorescent protein gene is shown as SEQ ID NO.

6.

6. lgals3bpb A promoter of a gene, characterized in that Its nucleotide sequence is shown in SEQ ID NO.

1.

7. According to claim 6 lgals3bpb A promoter of a gene, characterized in that Used to control the expression of fluorescent proteins in zebrafish microglia.

Images