ApoC1 gene promoter and its application and animal model and method constructed therefrom

Abstract

The present invention relates to the technical field of nerve cell labeling methods and related animal models, and specifically to an apoc1 gene promoter and its application, and an animal model and method constructed by the promoter. The nucleotide sequence of the apoc1 gene promoter is shown in SEQ ID NO.1, which can be used to label or manipulate microglia, and the promoter can be used to construct a zebrafish transgenic strain. This solution can solve the technical problem of the lack of an operable promoter sequence for labeling microglia in the prior art. The zebrafish transgenic strain constructed using the apoc1 gene promoter is a very good zebrafish animal model for studying microglia, which can be used for the study of the mechanism of the role of microglia in neurodegenerative diseases, and can also be used for drug screening and efficacy testing, and has good application prospects.

Description

Technical Field

[0001] The invention relates to the field of nerve cell labeling methods and related animal model technologies, and in particular to an apoC1 gene promoter and its application, and an animal model and method constructed thereby. Background Art

[0002] Microglia (or microglial cells) are tissue-resident macrophages of the central nervous system (CNS) that colonize the brain during early embryonic development. These phagocytes play many roles in the CNS, including clearing cell debris, transmitting signals to neurons and astrocytes to mediate synaptic connections, and maintaining the homeostasis of the total nervous system. Therefore, it is very necessary to fully understand the occurrence and development mechanism of microglia, the changes in their physiological processes under external stimuli, and their functional relationship with other cells of the CNS.

[0003] In the prior art, some marker genes specifically expressed by microglia have been found, such as apoeb gene (apolipoprotein E) and ccl34b.1 gene (chemokine (CC motif) ligand 34b, duplicate 1). The expression patterns and mechanisms of action of the above two genes have been studied more thoroughly, and can be used to study specific markers of microglia. In order to further study microglia and specifically operate microglia (for example, to regulate the biological processes of microglia at a fixed point in an in vivo model without affecting other cells or tissues), researchers usually use the promoter of a specific gene to control the expression of effector genes. Therefore, finding the promoter sequence of a gene specifically expressed by microglia is the key to achieving the above research. At present, there are only two genes for marking microglia, apoeb and ccl34b.1. The transgenic lines based on the above two genes are obtained through BAC (Bacterial artificial chromosome)-mediated transgenic technology, but the promoter sequence for marking microglia has not yet been determined reliably, and it cannot be arbitrarily manipulated according to the research purpose. At the same time, because the BAC fragment is too large, it is not easy to fuse it with the effector gene; and the efficiency of the stable strain obtained by BAC is extremely low, about 13%. This limits the in-depth study of microglia, and it is urgent to study and discover more microglia-specific genes, as well as clone and obtain promoter sequences that can mark and manipulate microglia. Summary of the invention

[0004] The present invention aims to provide an apoC1 gene promoter to solve the technical problem of the lack of an operable promoter sequence for marking microglial cells in the prior art.

[0005] In order to achieve the above object, the present invention adopts the following technical scheme:

[0006] The apoc1 gene promoter, whose nucleotide sequence is shown in SEQ ID NO.1.

[0007] This scheme also provides an application of an apoc1 gene promoter in marking or manipulating microglial cells.

[0008] This scheme also provides an application of an apoc1 gene promoter in constructing a zebrafish transgenic strain.

[0009] The principles and advantages of this solution are:

[0010] The inventors found that the apoc1 gene and nine other genes were enriched in microglial cells through sequencing and expression pattern studies of genes in microglial cells. Further studies found that the expression pattern of the apoc1 gene overlapped with the known marker genes of microglial cells. Therefore, the apoc1 gene has the potential to be a marker gene for microglial cells. Since one of the key factors controlling gene expression is the promoter of the gene, during the development of microglial cells, the expression of the apoc1 gene is regulated by regulating the promoter of the apoc1 gene, and the promoter of the apoc1 gene can also be used to mark microglial cells. In addition, the promoter of the apoc1 gene can also be used to perform a series of operations on microglial cells, which is helpful for the research on microglial cells. Among them, marking microglial cells refers to regulating the specific expression of marker genes in microglial cells through the promoter of apoc1 gene, so that microglial cells produce specific recognition signals relative to other tissue cells; operating microglial cells refers to regulating specific operating genes through the promoter of apoc1 gene, affecting the biological processes of microglial cells (including causing the proliferation, differentiation, apoptosis, killing and other processes of microglial cells) or knocking out specific genes in microglial cells and specifically expressing exogenous genes / overexpressing specific genes in microglial cells. More specifically, by using the promoter of apoc1 gene and integrating it with effector genes (including marker genes and effector genes) with special functions, the expression of effector genes can be regulated according to the development process of microglial cells; the specific expression of effector genes in microglial cells can also be controlled; that is, the expression of effector genes can be controlled in time and space.

[0011] In addition, the vector containing the apoc1 gene promoter and effector gene was genetically modified into the zebrafish. The zebrafish transgenic strain constructed using the apoc1 gene promoter is a very good zebrafish animal model for studying microglia. It can be used for mechanistic research on the role of microglia in neurodegenerative diseases, and can also be used for drug screening and efficacy testing.

[0012] In the prior art, there is no report on the use of the apoc1 gene or its promoter to mark microglia, and there is no report on the promoter sequence that can be used to specifically mark zebrafish microglia and can be operated. Through a lot of research, the inventor first discovered the relationship between the apoc1 gene and microglia. Although there are some genes that are enriched in microglia, the confirmation of the core promoter of the gene is a relatively complicated task. The core promoter sequence of cell expression specificity itself is unknown, and the position and length of the core promoter are difficult to predict. This has hindered the discovery of the promoter sequence of microglia-related genes. The inventor studied the promoters of genes that are enriched in microglia, and it is difficult to obtain a large number of promoters of enriched expression genes. After a lot of attempts, the promoter sequence of the apoc1 gene was finally cloned, which can effectively control the expression of the downstream coding region in microglia. The promoter sequence is then integrated with the effector gene, and a stable genetic zebrafish strain is prepared, which creates conditions for in-depth research on microglia and related drug screening and efficacy testing.

[0013] Furthermore, the apoC1 gene promoter is used to control the expression of effector genes in microglial cells.

[0014] This scheme provides an operable promoter sequence for marking microglia, which can be used to specifically operate microglia. For example, carrying a nitroreductase gene after the promoter can specifically kill microglia without damaging other macrophages; or using the promoter to specifically supplement or knock out a gene in microglia or Langerhans cells; fluorescent protein can also be integrated after the promoter to locate microglia. For the specific knockout of a gene, the following example is given: adding a gene cDNA sequence after the promoter sequence through molecular cloning, and introducing the constructed plasmid into the model animal can achieve the supplementation of a gene in microglia or Langerhans cells. At the same time, by combining with the existing CRISPR / Cas9, the Cas9 gene is specifically expressed after the promoter, and the marker gene is fused at the same time (to facilitate the detection of whether the Cas9 gene is expressed), and then by interacting with the target RNA that stably expresses a gene target site systemically, it can achieve the knockout of a gene in microglia or Langerhans cells.

[0015] Furthermore, the effector gene is one of a nitroreductase gene, a fluorescent protein gene and a conditionally knocked-out Cas9 gene.

[0016] By adopting the above technical scheme, nitroreductase can reduce non-toxic metronidazole to cytotoxic metabolites by nitroreductase, which can specifically induce apoptosis of cells expressing metronidazole nitro controlled by the apoc1 gene promoter. Under the regulation of the apoc1 gene promoter, microglial cells can produce fluorescent signals to distinguish them from other types of cells, making it easier to observe microglial cells. In microglial cells, the Cas9 gene conventionally conditionally knocked out in the prior art is specifically expressed, which can achieve gene knockout in microglial cells without affecting other types of cells.

[0017] This protocol also provides a method for constructing an animal model using the apoc1 gene promoter, comprising the following steps in sequence:

[0018] S1: Obtain the apoc1 gene promoter;

[0019] S2: Plasmid construction: The apoc1 gene promoter, effector gene and empty plasmid were integrated by homologous recombination, and then transformed into competent cells. After screening and plasmid extraction, the plasmid used for transgenic operation was obtained;

[0020] S3: Obtain F0 generation zebrafish;

[0021] S4: Obtaining stably inherited transgenic zebrafish lines.

[0022] The above technical scheme is adopted to obtain the promoter fragment (4212 bp) of the apoc1 gene by DNA polymerase chain reaction; then the expression vector is constructed and genetically modified into zebrafish fertilized eggs, and finally a stable transgenic fish line is obtained through multi-generation hybridization.

[0023] Further, in S1, the apoc1 gene promoter is obtained by polymerase chain reaction; the primers include upstream primers with sequences shown in SEQ ID NO.3 and SEQ ID NO.4 and downstream primers with sequences shown in SEQ ID NO.5 and SEQ ID NO.6. Since the sequence of the apoc1 gene promoter is relatively long, segmented PCR is used for cloning.

[0024] Furthermore, in S3, the plasmid and transposase used for transgenic operation were mixed and injected into zebrafish single-cell fertilized eggs; 3 days after fertilization, fertilized eggs with integrated effector genes were selected; and then cultured to sexual maturity to obtain F0 generation transgenic zebrafish. The use of transposase can improve the efficiency of inserting the target gene into the zebrafish genome.

[0025] Further, in S4, the F0 generation transgenic zebrafish is mated with the wild-type zebrafish to obtain the F1 generation transgenic zebrafish; the F1 generation transgenic zebrafish is mated with the wild-type zebrafish to obtain a stable genetic transgenic zebrafish strain. Through multiple matings and screening, a stable genetic transgenic zebrafish strain can be obtained. The above means are conventional means of obtaining a stable transgenic zebrafish strain in the prior art.

[0026] Furthermore, a transgenic zebrafish strain was obtained by constructing an animal model using the apoc1 gene promoter. In the transgenic zebrafish strain obtained in this scheme, the apoc1 gene promoter controls the expression of effector genes specifically in microglial cells, creating conditions for studying the occurrence and development mechanism of microglial cells and developing related drugs.

[0027] In summary, the inventors found that the immune cells in the brain of zebrafish can be divided into multiple groups by single cell sequencing analysis, and screened out multiple genes similar to the expression patterns of apoeb gene and ccl34b.1 gene (microglia marker gene recognized by the prior art), and used these genes as candidate genes for cloning promoters. And although apoeb gene and ccl34b.1 gene are expressed in large quantities in microglia, their expression is not concentrated in the immune cells of the brain. Taking apoeb gene as an example, it also has a certain expression in neuronal cells. So it is very necessary to screen out genes that can be specifically expressed in microglia.

[0028] Due to the complexity of the core promoter sequence with cell expression specificity, the inventors have only obtained the sequences upstream of the start codons of some candidate genes through a large number of attempts. The sequences upstream of the start codons obtained were integrated into the expression plasmid and transgenic into zebrafish. It was found that not all cloned sequences upstream of the start codons have the activity of initiating the expression of effector genes. Finally, it was found that only the sequences upstream of the start codon of the apoc1 gene obtained by cloning and the sequences upstream of the start codon of the lgals3bpb gene can initiate the expression of effector genes, that is, the core promoter sequence was obtained. And in further experiments, we found that the promoter of the apoc1 gene and the promoter of the lgals3bpb gene can initiate the specific expression of effector genes in microglia, not in neurons, nor in other immune cells other than microglia. Such an effect makes it possible for us to observe and operate microglia specifically. The discovery of the expression pattern of the apoc1 gene and the lgals3bpb gene and the promoter of the apoc1 gene and the lgals3bpb gene has achieved unexpected technical effects, overcoming the technical problems in the prior art caused by the lack of an animal model capable of locating and operating microglial cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 These are the results of a study on the expression patterns of the apoc1 gene in Example 1 and known microglial marker genes.

[0030] Figure 2 This is the expression of green fluorescent protein regulated by the Apoeb gene promoter in prior art research.

[0031] Figure 3 This is a fluorescence microscopic image of the zebrafish Tg (coro1a:DsRed; vmp1-eGFP) strain of Example 2.

[0032] Figure 4 This is a fluorescence microscopic image of the zebrafish Tg (coro1a:DsRed; havcr1-eGFP) strain of Example 2.

[0033] Figure 5 This is the electrophoresis diagram of the two DNA fragments of the apoc1 gene promoter in Example 3.

[0034] Figure 6 Schematic diagram of the construction process of the pT2AL-cryaa-apoc1-eGFP plasmid in Example 3.

[0035] Figure 7This is a schematic diagram of the pT2AL-cryaa-apoc1-eGFP plasmid in Example 3.

[0036] Figure 8 This is the colony PCR experimental result of Example 3.

[0037] Fig. 9 This is a microscopic image of the preliminary screening cryaa-cerulean results of Example 3.

[0038] Fig.10 This is a schematic diagram of the construction and screening of transgenic zebrafish in Example 3.

[0039] Fig.11 This is a fluorescence microscopic image of the whole-body eGFP positive signal detection of zebrafish in Example 3.

[0040] Fig.12 This is the co-localization result of transgenic zebrafish eGFP and midbrain microglia in Example 3.

[0041] Fig.13 The figure shows the co-localization result of eGFP and pan-microglia in the midbrain of the transgenic line of Example 3 when it develops to 12 months. DETAILED DESCRIPTION

[0042] The present invention is further described in detail below in conjunction with examples, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the technical means used in the following examples and experimental examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used can be obtained from commercial sources.

[0043] Example 1: Study on the expression pattern of apoc1 gene

[0044] The full name of the apoc1 gene is Apolipoprotein C1, and its receiving number is NM_001291886.1. The inventors found through sequencing that the expression pattern of the apoc1 gene overlaps with the known marker genes of microglia (see Figure 1 ).

[0045] Through single-cell sequencing analysis of immune cells in the zebrafish brain, we found that immune cells in the brain can be divided into multiple groups. Then, when we detected the apoeb gene (apolipoprotein E, receiving number NM_131098.2) and the ccl34b.1 gene (chemokine (CC motif) ligand 34b, duplicate 1, receiving number NM_001115054.2), we found that these two genes were only expressed in some groups, such as Figure 1 As shown. Figure 1In the figure, the coordinate axis represents the gradient between data points, which is used to simulate their distance function in high-dimensional and low-dimensional space. Among them, the positive value of the gradient represents the attraction between data points in low-dimensional space, while the negative value represents the repulsion. The 0-6 in the gradient diagram on the right represents the expression level of the gene in different groups, and the depth of the color is used to represent the expression level. Figure 1 In the figure, the genes in the right box have the same expression pattern as the apoeb gene and the ccl34b.1 gene in the immune cells of the zebrafish brain, that is, the cell groups enriched in expression are the same, including the apoc1 gene, the p2ry12 gene, the vmp1 gene, the fcer1gl gene, the havcr1 gene, etc. For reports on apoeb and ccl34b.1 genes as marker genes for microglia, please refer to the prior art literature for details (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; Shuting Wu, Two phenotypically and functionally distinct microglial populations in adult zebrafish, Sci Adv. 2020 Nov 18; 6(47): eabd1160. doi: 10.1126 / sciadv.abd1160.).

[0046] 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.) show that in the existing BAC-constructed apoeb transgenic expression pattern, the effector gene (GFP) carried by apoeb is not only expressed in microglia, but also detected in brain neurons (not immune cells in the zebrafish brain) (see Figure 2 , which comes from the above-mentioned paper by Francesca Peri). Figure 2The thin arrows in the middle refer to microglia, and the thick arrows refer to neurons. The morphological characteristics of neurons are cells with long linear branches. This shows that the specificity of using the apoeb gene to mark microglia is not strong, and it will also mark neurons, which is not conducive to the observation and operation of microglia. Through single-cell sequencing analysis, we found that the expression pattern of the apoc1 gene in brain immune cells is consistent. Those skilled in the art may expect that the expression patterns of the apoc1 gene and the apoeb gene in non-brain immune cells will also be consistent. In this case, the apoc1 gene is not a specific gene for microglia in the brain microenvironment, and may be expressed in other brain neuronal cells. However, the inventors have found that the apoc1 promoter cloned in the present invention is not expressed in neuronal cells, and can effectively distinguish microglia from neuronal cells. Therefore, the apoc1 gene (and its promoter) has the potential to be a microglia marker gene (promoter). Before the present patent application, there was no report on the study of apoc1 marking microglia, and there was no report on the promoter sequence that was used to specifically mark zebrafish microglia and could be operated.

[0047] Example 2: Acquisition of the promoter of the apoc1 gene

[0048] Based on the highly expressed genes in microglia screened out in Example 1, 10 of them were selected as candidate genes to capture the promoter sequence of labeled microglia. The confirmation of the core promoter of a gene is a relatively complicated task. The core promoter sequence with cell expression specificity itself is unknown, and the position and length of the core promoter are difficult to predict before the research discovery. The core promoters of some genes are only a few hundred base pairs, while some are tens of thousands of base pairs. The promoters of some genes are closer to the coding sequence, the promoters of some genes are farther from the coding sequence, the promoters of some genes are located upstream of the coding region, and some are located downstream. These factors have hindered the discovery of the promoters of genes.

[0049] In the attempt to study the promoter of alternative genes, the inventors designed primers for the fcer1gl gene, p2ry12 gene, etc., in order to clone the base sequence information before the start codon, but the amplification ultimately failed and the above sequence information could not be obtained. In addition, the inventors cloned the base sequence before the start codon (about 4000bp) for the vmp1 gene, etc. However, after connecting eGFP and pT2AL vectors and then injecting zebrafish, the inventors found that the transgenic zebrafish constructed in parallel with other genes including the vmp1 gene and havcr1 gene did not have eGFP signals, indicating that the promoter capture failed. Fluorescent images of microglial cells in transgenic zebrafish containing the vmp1 gene promoter are shown in Figure 3(3 days after fertilization), the expression of DsRed is regulated by the promoter of the known coro1a gene. DsRed is expressed in large quantities, indicating that the cells in the field of view are microglial cells, while no eGFP signal appears in these cells, indicating that the upstream sequence of the start codon of the cloned vmp1 gene does not have the function of initiating the expression of the eGFP gene. Transgenic zebrafish studies were conducted on the upstream sequence of the start codon of the havcr1 gene, and the situation is the same as that of the vmp1 gene (see Figure 4 ). The inventors analyzed the reasons for the above phenomenon: it may be that the promoter of the vmp1 gene is not located 4000bp upstream of its start codon; or it may be that although the promoter of the gene was successfully cloned, the promoter could not effectively initiate the expression of the effector gene in the animal model. The above experimental results show that not all sequences with promoter effects can initiate the expression of effector genes, that is, not all promoters are operable, and the difficulty of cloning promoters and applying them to control the expression of effector genes in specific cells. In addition, the function of the promoter of the Coro1a gene in the prior art is further explained as follows: The promoter of the Coro1a gene can mark all immune cells (including immune cells such as macrophages, monocytes, granulocytes, lymphocytes, etc.), but based on the current belief that microglia are the only immune cells in the central nervous system, we believe that the promoter of the Coro1a gene marks pan-microglia in the brain (which may include mature, immature and undifferentiated states).

[0050] For the apoc1 gene in this scheme, the base sequence before its start codon is rich in multiple consecutive Ts, which reduces the amplification effect of the DNA polymerase chain reaction. Through Dalian attempts, a 4212bp promoter sequence was finally obtained, and its sequence is shown in SEQ ID NO.1. The 4212bp sequence before the start codon of the apoc1 gene amplified by DNA polymerase chain reaction can initiate the expression of the carried eGFP, and the eGFP-expressing cells are located in the brain, indicating specific labeling of microglia, which is a brand-new discovery. In contrast, the inventors also tried to clone the 2000bp sequence before the start codon of the apoc1 gene (SEQ ID NO.2), but this partial sequence was not able to initiate the expression of the reporter gene eGFP (see Example 3 for the operation method).

[0051] This scheme provides an operable promoter sequence for marking microglia, which can be used to specifically operate microglia. For example, carrying a nitroreductase gene after the promoter can specifically kill microglia without damaging other macrophages; or using the promoter to specifically supplement or knock out a gene in microglia or Langerhans cells; fluorescent protein can also be integrated after the promoter to locate microglia and observe the expression and function of the gene (apoc1 gene) during development. The following examples use the integration of eGFP as an example to illustrate the function of the promoter of this scheme.

[0052] Example 3: Zebrafish microglial cell labeling model based on apoc1 gene promoter sequence

[0053] The zebrafish microglia labeling model based on the apoc1 promoter sequence was achieved by the following steps:

[0054] (1) The 4212 bp sequence before the start codon of the apoc1 gene was amplified by DNA polymerase chain reaction as a promoter (SEQ ID NO.1). The primer sequences used were:

[0055] F1-apoc1: 5'-AGACAGCAGGAATAAAGGTT-3', SEQ ID NO.3;

[0056] R1-apoc1: 5'-tcctcgcccttgctcaccatGGAATCACTGTTAGAGAGCT-3', SEQ ID NO. 4.

[0057] F2-apoc1: 5'-atcgaattcctgcagcccgggCAGTTTGTTTGGACTCCTTGC-3', SEQ ID NO.5;

[0058] R2-apoc1: 5'-AACCTTTATTCCTGCTGTCTACAAACATAGATACATGCAG-3', SEQ ID NO. 6.

[0059] The PCR system (using the PCR kit of Yisheng Biotechnology (Shanghai) Co., Ltd.) is:

[0060] 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.

[0061] The PCR program was: 98°C, 3 min, ×1; (98°C, 10 s)→(60°C, 20 s)→(72°C, 2 min), ×40; 72°C, 5 min, ×1.

[0062] The purified and enriched apop1 gene promoter amplification product was obtained by conventional agarose electrophoresis and gel recovery methods, and sent to a third-party company for sequencing to confirm that the recovered product was the target sequence. The electrophoresis diagram of the two DNA fragments is shown in Figure 5 .

[0063] (2) The eGFP fluorescent protein sequence was obtained as a marker gene by DNA polymerase chain reaction. The sequence of eGFP is shown in SEQ ID NO.7. The primers used are:

[0064] F1-eGFP: 5'-atggtgagcaagggcgagga-3', SEQ ID NO.8;

[0065] R1-eGFP: 5'-tggatcatcatcgataggcctttacttgtacagctcgtcca-3', SEQ ID NO. 9.

[0066] The PCR system was as follows: 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. The PCR procedure was the same as (1).

[0067] The purified and enriched eGFP fluorescent protein gene amplification product was obtained by conventional agarose electrophoresis and gel excision recovery methods, and was sent to a third-party company for sequencing to confirm that the recovered product was the target sequence.

[0068] (3) The promoter of the apoc1 gene, the eGFP fluorescent protein sequence and the pTAL2-cryaa vector (Cat. No.: #31823; see 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 by homologous recombination to obtain the pT2AL-cryaa-apoc1-eGFP plasmid. The general process is shown in Figure 6 The specific process is as follows:

[0069] The pTAL2-cryaa vector was treated with restriction endonucleases Xma1 and Cla1, and the apoc1 gene promoter amplification product 1 was obtained by DNA polymerase chain reaction, and PCR product A was obtained after purification and recovery; the apoc1 gene promoter amplification product 2 was obtained by DNA polymerase chain reaction, and PCR product B was obtained after purification and recovery; the eGFP sequence amplification product was obtained by DNA polymerase chain reaction amplification of the plasmid containing the eGFP fluorescent protein sequence, and PCR product C was obtained after purification and recovery. Then, the PCR product A, PCR product B and PCR product C were connected using homologous recombination enzymes to form the pT2AL-cryaa-apoc1-eGFP plasmid. The schematic diagram of the plasmid can be found in Figure 7 .

[0070] The connection system is: 2× homologous recombination enzyme: 10 μL; pTAL2-cryaa vector after enzyme digestion: 100 μg; PCR product A: 20 μg; PCR product B: 25 μg; PCR product C: 30 μg. The connection time is: 15 min; the temperature is: 50°C.

[0071] (4) The constructed plasmid was transferred into E. coli DH5 competent cells by conventional means in the prior art for transformation. Single clones were picked and added to 40 ml of culture medium containing ampicillin antibiotics, cultured at 37°C and 180 rpm for 12-16 hours, and the plasmid was purified and extracted by using an endotoxin-free plasmid extraction kit to obtain a plasmid vector pT2AL-cryaa-apoc1-eGFP with high purity that was free of E. coli cell bodies and other substances. The colony PCR screening positive colony image can be found in Figure 8 .

[0072] (5) The pT2AL-cryaa-apoc1-eGFP plasmid (40 ng / μL) and transposase (100 ng / μL, Tol2transposase mRNA, whose template DNA sequence is shown in SEQ ID NO.10) were mixed in a volume ratio of 1:1 and injected into a single-cell fertilized zebrafish egg (AB strain zebrafish, injection volume 1 nL), thereby randomly integrating the plasmid vector into the zebrafish genome through the action of the transposase. A transposon is a sequence that can change its position, including an intermediate sequence and a terminal repeat sequence, and the intermediate sequence can express a transposase. The transposase can recognize the terminal repeat sequence, and then remove the transposition sequence and transfer it to other locations in the genome. The transposase in this scheme is a tool to improve the efficiency of plasmid insertion. If it is not used, the insertion of exogenous genes into the zebrafish genome can also be carried out, but the efficiency will be reduced.

[0073] (6) Microinjection of single-cell fertilized egg embryos. Detect the expression of cryaa fluorescent protein signal in the eye under a fluorescence microscope 2 days after fertilization to determine whether the microinjection is successful. Then, 3 days after fertilization, detect the expression and location of eGFP under a fluorescence microscope to determine whether the recombinant plasmid can drive eGFP expression and whether it can specifically label microglia. The cryaa-cerulean labeling results 3 days after fertilization are shown in Fig. 9 (cryaa-cerulean can specifically express blue fluorescence in the zebrafish lens and is usually used to detect whether the plasmid is integrated into the chromosome after being injected into the zebrafish).

[0074] (7) Fertilized eggs expressing fluorescence were selected and cultured at a standard temperature of 28.5°C until sexual maturity (F0 generation; founder). The F0 generation was mated with wild-type zebrafish to obtain fluorescent offspring individuals, the F1 generation. The F1 generation was further cultured to sexual maturity and mated with wild-type zebrafish to obtain the fluorescent F2 generation. Thus, the construction of the fluorescent zebrafish strain Tg (apoc1: eGFP) was completed.

[0075] For transgenic construction and screening schemes of (4)-(7), see Fig.10 . Detect the whole body eGFP signal of F1 generation zebrafish. The experimental results can be found in Fig.11 , it was found that the eGFP signal was specifically colonized in the brain and skin. In addition to the above-mentioned markers, the promoter of the apoc1 gene also marks the yolk sac and the caudal fin. Among them, the cells expressing the eGFP signal in the skin are mainly Langerhans cells, which are tissue-resident macrophages. The apoc1 gene is involved in lipoprotein transport. In addition to microglia, some other non-brain cells also express the apoc1 gene. If the promoter of the apoc1 gene is used to operate cells expressing the gene, in microglia, these gene operations will more likely affect the brain microenvironment, such as neuronal development and function; in other non-brain cells, these gene operations will not affect the brain microenvironment. Therefore, using the promoter of the apoc1 gene to start the expression of effector genes in the corresponding cells will not affect the operation of microglia and the experimental results. In addition, if we use MTZ-NTR to specifically eliminate cells containing the promoter of the apoc1 gene, we can control the concentration of MTZ to ensure that only microglia in the brain are eliminated without affecting cells in other parts.

[0076] The zebrafish Tg(coro1a:DsRed) strain was crossed with the aforementioned F0 generation (Tg(apoc1:eGPF)) expressing eGFP to obtain the hybrid strain Tg(coro1a:DsRed;apoc1:eGPF). Fig.12At 3dpf, coro1a-DsRed+ cells (red, left picture) and apoc1-eGFP+ cells (green, middle picture) were observed, and it was found that the cells marked by the promoter of the apoc1 gene and the cells marked by the promoter of the Coro1a gene had a high degree of overlap (the fused image is the right picture), indicating that the promoter of the apoc1 gene can activate the specific expression of the effector gene in microglial cells. Among them, the method for obtaining the zebrafish Tg (coro1a: DsRed) strain is described above in this embodiment, except that the promoter and effector gene are replaced with the promoter of the coro1a gene in the prior art (the information on the promoter can be found in the literature Li, L., Live imaging reveals differing roles of macrophages and neutrophils during zebrafish tail fin regeneration. 2012, Journal of Biological Chemistry 287, 25353-25360.) and DsRed fluorescent protein. In Fig.12 In the figure, the arrowhead indicates cells co-stained with DsRed and eGFP, and the arrow indicates some cells that are not co-stained (only DsRed signal). The promoter of the Coro1a gene is mainly used to specifically mark immune cells (leukocytes), including microglia, while the promoter of the apoc1 gene mainly marks microglia. The immune cells in the brain are mainly microglia, so there is a situation where the marked cells overlap. In the fluorescent image, there are cells that are only marked by the promoter of the Coro1a gene but not by the promoter of the apoc1 gene. The inventor analyzed that the reason is that these cells marked by the promoter of the non-apoc1 gene are not tissue-resident cells (i.e., non-microglia), but macrophages patrolling there and marked by the promoter of the Coro1a gene. The experimental results further illustrate the specificity of the promoter marking of the apoc1 gene, which only marks microglia, but does not mark other immune cells and neurons in the brain. We can use the characteristics of the promoter of the apoc1 gene to conduct specific research on microglia and related operations.

[0077] The co-localization results of the newly constructed transgenic strain (fluorescent strain zebrafish Tg(apoc1:eGFP)) with pan-microglia in the midbrain at 12 months of development are shown in Fig.13(The left picture is the red Lcp1 marker, the middle picture is the green eGFP marker, and the right picture is a fusion image of the two pictures). Anti-Lcp1 refers to the antibody against LCP1 (lymphocyte cytosolic protein 1). The promoters of the lcp1 and coro1a genes mark a type of cell, and this antibody is generally used to mark the pan-microglia in the zebrafish brain. The above experimental results show that the expression of effector genes controlled by the promoter of the coro1a gene is concentrated in the pan-microglia in the midbrain. Fig.13 In the figure, arrowheads indicate cells co-stained with Lcp1 and eGFP, and arrows indicate some cells that are not co-stained (only Lcp1 signal). Adult zebrafish have macrophages from other sources to replace some microglia in the brain, so there will be cells that do not express eGFP, which further illustrates the specificity of the promoter marking of the apoc1 gene, which only marks microglia, but not other immune cells and neurons.

[0078] The above is only an embodiment of the present invention, and the common knowledge such as the known specific technical solutions and / or characteristics in the solution is not described in detail here. It should be pointed out that for those skilled in the art, without departing from the technical solution of the present invention, several modifications and improvements can be made, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records 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> ApoC1 gene promoter and its application and animal model and method constructed therefrom <130> 2022.4.19 <160> 10 <170> PatentIn version 3.5 <210> 1 <211> 4212 <212> DNA <213> Danio Rerio <400> 1 cagttgtttg gactccttgc cctcaactgt ccacactata ttttataggg ccaaagagat 60 tgcaatttag taaaataaga ctgaggacga aaaataat aacatttatt acttgataa 120 tgcttctgtt ggggcagcac ggtggcgcag tggtagcac agtcggctcg cagcagaag 180 gttgctagtt tgagccccgg ctggtcagt tggcatttct gtgtggatt tgcatgttcg 240 ccctatgttg gcgtaggttt cctcttggtg ctccggttc gccctcagtc caaatcatg 300 tggtacaggt gattgggca 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 ggtaattttg gttttattct gtaaactact aatgcctttt cttccaaacc cttttaggga 840 agagctttgc tcacaaaggt taaacaaaaa gcttgagatg gcacagcagg tcactgtcta 900 gactcataaa atgaaaaacc aagggccgtt gacttaacaa agtacataaa actaaaagaa 960 caacaggtga ctaattatgt gtgtgtgaga cgagctgttt gaatggcttt ggtcttaaag 1020 tgttctgcaa agcctgcaga taagtgacaa agggaacact ggaaggcccg 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 aaacgttat tttcaaaaag tccatacgtt 1620 gctctgatta tcacattatt ctgtcaatat tttacattt tcatgattct ctgatgaaca 1680 aaatgttaaa ataacagcat atagaatgtt ttaatttcat ctaaaataat tttgtatttt 1740 aactgtttaa tctaccgcag gcaatattgc taggtagaaa caaaaacaat atgaccactt 1800 tcaaattcac aaaccttcat agcttcatgg ctgggacttt acataaatgt ctaatgtgtt 1860 tgaacattta agcacagaat atggcaaaaa atagtgtaca gcattgacga tgcagtgctt 1920 taattaaaat aaatcaaatg acaccatagg ccaatataag ataacatcta atgattctcc 1980 tgacctagaa tccaaaggtg gtttcaatgc agttagcaga atcctgcatg tatctatgtt 2040 tgtagacagc aggaataaag gttatgaaac tactaagaga taaacaactt caatttactt 2100 tagtccaaat ccagactctt tatctcatgt tgtgatgtgg ctggaaggtt aaagaactgt 2160 ggagtaaatc agtgtctcat gacattaaac aactacaaag ttacaccc 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 agggttgtt gatagtact tgcaaggtt cttatactca 2580 gttaaacgtt cttatataca gttaaatgtc tgttgaagga gcagtatca cagatattaa 2640 gcagtctatt atactcaa tggaccatca aataaagtg ttacctaaa gtgaatggtc 2700 agggggatac catgggatgg tgtatatatgc attttatttg tataaaatgc tgccatgtgc 2760 aatgtctgct acgggtcagt ttcttgtc actggtcaa caagcagtat gatgatcat 2820 tattgaactc tttatagata gtaatgaat gtaaatgaaaaaaata ttctctctca 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 gataataaat ggactttgta ccatatatgc aaggcaaggg 3300 taccagataa agaaagtacc cactttttta taacattgtt ttatttttga cagatgttgt 3360 ttcaaaggtc actggcactg aagtaaaata aagtagctca taacgaataa tcaaagaaaa 3420 tctccgtgaa aacagctgtt gtggcaaacg gagtttactt acctctaaag gatgtttact 3480 atagtcattt accacatcgc aaagctgttaa ggagaacgag agatgaacgc gaggaaagcg 3540 agtgattgca ggaggtcttg ataggtcaga ggtggtgtta ttacacaacg tcatctctgt 3600 gtccctataa agggacaagc catctgtggg acatcacaaa ctcagcccaa ttctttcgag 3660 gactgcgaag aggtacgcaa acttatttt gttgtggtca ttttcaagc agagcgaatc 3720 tagagctt taacctgaaa cgacagaata acctttcttt attaggttca gtgcgcaaac 3780 acgcagtata ttggcgacat ctagccgcca tccctgcta tgattggaaa atttagcata 3840 tttttgtaaa tgttagagga agataataa aacactatta aacgtttaca aacaatgcgc 3900 aaacatattt ttaaaagcgc gaaatatata ttatacactt cattagaacc tttgtaataa 3960 attttaatct tatgtttatt gtttagtttt caaagcaaa taacgtctt gttttgatta 4020 atgacaaaaa tgattaatga caaaaaaatc ttctgttca accagaagtt tttttttt 4080 attttttatt ttttaga ttacattag cgtaaaccat aaattgttta ataatatct 4140 taggaataa ttacaccg aatcggtcaa attatgatg cctgagattt ctagctctct 4200 cc 4212 <210> 2 <211> 2169 <212> DNA <213> Actual damage <400> 2 agacagcagg aaaaggtt atgaactac window acaacttcaa tttactttag 60 tccaaatcca gactcttat ctcatgttgt gatgtggctg gaaggttaaa gaactgtgga 120 gtaaatcagt gtctcatgac attackacac tacaaagtta tacacctta caatgcgca 180 gtcacaaata atacacag taataat ttaagttg atttatgtt ttgaaaaact 240 aaggctgaga ttcagaagt aggctatcaa tctctttgct cgtgtgttca gataccata 300 attcaccata tcaaaaaga aacctgcata cagacagtga tcattttgta tattaataa 360 ggagcagatt cctaaaaagt gaccactaa gcgggtcaca ctttattg ataggtcgtt 420 tgttgaattt atgttacatt gcatctacat gccaactaat tctcattaga ttataagtag 480 actgttaggt tggggttagg gttgttgac atgtacttgc aaaggttctt attackcagtt 540 aaacgttctt atacagtt aaatgtctgt tgaaggagca gtatcacag attackagca 600 gtctattaat actcaaatgg accatcaaaa taaagtgtta ccctaaagtg aatggtcagg 660 gggataccat gggatggtgt atatgcatt ttatttgtat aaatgctgc catgtgcaat 720 gtctgctacg ggtcagtttc tttagtcact ggtcaacaa gcagtatgaa tgatcattat 780 tgaactcttt atagatagta atgaaatgta aatgaaaaca aaaataattt cttctcaaaa 840 attctgtttt acactgtaaa catgctgagg tccacacaat tccttcttgt tatcccaaca 900 caaactgatt aagtaaactt aattaaattt acaaatgaaa gtagcttgaa cataaaacaa 960 ttaagttgtc ccaaaaaaaac tcaggaattg tgttgattca gctcatttta gagtcatgat 1020 tagtttgaac aagcaggaaa aatagttttg agcatacata ccagaattaa ctgtatcagt 1080 gaagtatgat gtatttttat gccataacac tgatgctgta gtatatttac tagaggagat 1140 attacacaat tgttttgtcc ttcagagtca tgtactgtag atgtagaaaa aagcacgtaa 1200 ccaactgtaa aatccatgat aataaatgga ctttgtacca tatatgcaag gcaagggtac 1260 cagataaaga aagtacccac ttttttataa cattgtttta tttttgacag atgttgtttc 1320 aaaggtcact ggcactgaag taaaataaag tagctcataa cgaataatca aagaaaatct 1380 ccgtgaaaac agctgttgtg gcaaacggag tttacttacc tctaaaggat gtttactata 1440 gtcatttacc acatcgcaaa gctgtaagga gaacgagaga tgaacgcgag gaaagcgagt 1500 gattgcagga ggtcttgata ggtcagaggt ggtgttatta cacaacgtca tctctgtgtc 1560 cctataaagg gacaagccat ctgtgggaca tcacaaactc agcccaattc tttcgaggac 1620 tgcgaagagg tacgcaaact tatttttgtt gtggtcattt ttcaagcaga gcgaatctag 1680 agagctttaa cctgaaacga cagaataacc tttctttatt aggttcagtg cgcaaacacg 1740 cagtatattg gcgacatcta gccgccatcc cttgctatga ttggaaaatt tagcatattt 1800 ttgtaaatgt tagaggaaga taaataaaac actattaaac gtttacaaac aatgcgcaaa 1860 catattttta aaagcgcgaa atatatattt aacacttcat tagaaccttt gtaataaatt 1920 ttaatcttat gtttattgtt tagttttcaa aagcaaataa cgtctttgtt ttgattaatg 1980 acaaaaatga ttaatgacaa aaaaatcttc ttgttcaacc agaagttttt tttatttatt 2040 ttttattttt tttaagatta acattagcgt aaaccataaa ttgtttaaat aatatcttag 2100 gaaataattt aacaccgaat cggtcaaaat tatgatgcct gagatttcta gctctctaac 2160 agtgattcc 2169 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <400> 3 agacagcagg aataaaggtt 20 <210> 4 <211> 40 <212> DNA <213> Artificial Sequence <400> 4 tcctcgccct tgctcaccat ggaatcactg ttagagagct 40 <210> 5 <211> 41 <212> DNA <213> Artificial Sequence <400> 5 atcgaattcc tgcagcccgg gcagttgttt ggactccttg c 41 <210> 6 <211> 40 <212> DNA <213> Artificial Sequence <400> 6 aacctttatt cctgctgtct acaaacatag atacatgcag 40 <210> 7 <211> 720 <212> DNA <213> Artificial Sequence <400> 7 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> 8 <211> 20 <212> DNA <213> Artificial Sequence <400> 8 atggtgagca agggcgagga 20 <210> 9 <211> 41 <212> DNA <213> Artificial Sequence <400> 9 tggatcatca tcgataggcc tttacttgta cagctcgtcc a 41 <210> 10 <211> 1950 <212> DNA <213> Artificial sequence <400> 10 atggaagagg tgtgcgactc ttccgccgca gcttcatcta ccgtccagaa ccagcctcag 60 gaccaagagc acccctggcc atatctgaga gagttcttct ccctgagcgg agtgaataag 120 gactcattta aaatgaagtg tgtgctgtgt cttccactca acaaagagat tagcgcgttt 180 aaatcctcac cctcgaacct gaggaagcac attgagcgta tgcaccctaa ctacctgaaa 240 aattactcca agctgaccgc ccagaagaga aagataggca ctagtactca cgcatccagc 300 agtaagcagt tgaaagtaga ctcagtgttc cctgttaagc atgtgtcccc tgtaacagtt 360 aacaaggcta ttctccggta cattatccaa ggtctgcacc cgttcagcac cgtcgaccta 420 ccctccttca aggagctgat tagtacgctt cagcctggaa ttagtgtgat tactcgccct 480 actctgagaa gcaaaatcgc agaagcggcg ctgatcatga aacagaaagt gactgctgca 540 atgagcgagg tggaatggat cgcgacgaca accgactgtt ggacagcacg taggaagtcc 600 tttataggcg tcactgccca ctggattaac cccggttcac tggagcgaca ctcagcagcc 660 ctggcctgca aacgccttat gggatctcac acttttgagg tgttagcgtc tgctatgaac 720 gacatccact cggaatacga aattagggac aaggtggtct gtaccacaac agactctgga 780 tctaacttta tgaaagcgtt tcgcgttttt ggcgtggaga acaatgatat 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 ccaaccagt ctgaagctcc aaagactgca tcatagtctg 1440 cggtactgcg atcccctcgt ggatgctctg cagcagggaa tccagactcg gttcaacac 1500 atgttcgaag accctgagat tatcgctgca gccatccttc tcccaaatt tcgaacctcg 1560 tggacaacg atgagaccat tattaagcgc ggcatggatt atatccgcgt ccaccttgaa 1620 cccctggacc acaaaaaaga gctggctaat tcaagtagtg acgatgagga ttctttgcc 1680 agctgaagc cgacgacaca cgaggcctca aaggagctgg acggttacct gggctgcgtc 1740 agcgatacaa gggagtcact gttgaccttc cctgcaatct gttcgctgtc gatcagaca 1800 aacactccct tgcctgctag tgcagcgtgt gagagactct tgcacagc aggactcctg 1860 ttcagcccta aggagcaag gcttgatacc aaaactttg agaaccagtt gcttctgaaa 1920 ctgaatttac gattttaca ctcgagtga 1950

Claims

1. apoc1 A gene promoter, characterized in that: Its nucleotide sequence is shown in SEQ ID NO.

1.

2. according to claim 1 apoc1 The use of a gene promoter for controlling the expression of effector genes in microglial cells is characterized by: The effector gene is one of a nitroreductase gene, a fluorescent protein gene and a conditionally knocked-out Cas9 gene.

3. According to claim 1 apoc1 The application of a gene promoter in constructing a zebrafish transgenic strain is characterized by: Said apoc1 The gene promoter is used to control the expression of the effector gene in microglial cells; the effector gene is one of a nitroreductase gene, a fluorescent protein gene and a conditionally knocked-out Cas9 gene; Zebrafish transgenic strains were obtained by the following method: S1: Acquired by polymerase chain reaction apoc1 Gene promoter; The primer sequences used are: F1-apoc1: 5'-AGACAGCAGGAATAAAGGTT-3'; R1-apoc1:5'-tcctcgcccttgctcaccatGGAATCACTGTTAGAGAGCT-3'; F2-apoc1: 5'-atcgaattcctgcagcccgggCAGTTGTTGGACTCCTTGC-3'; R2-apoc1: 5'-AACCTTTATTCCTGCTGTCTACAAACATAGATACATGCAG-3'; After polymerase chain reaction and purification recovery, PCR product A and PCR product B were obtained; S2: Plasmid construction: apoc1 The PCR products A and B of the gene promoter, the effector gene and the empty plasmid are integrated by homologous recombination, and then transformed into competent cells. After screening and plasmid extraction, a plasmid for transgenic operation is obtained; S3: The plasmid and transposase used for transgenic operation are mixed and injected into the single-cell fertilized eggs of zebrafish; 3 days after fertilization, the fertilized eggs with integrated effector genes with fluorescent expression are selected; then they are cultured at the standard temperature of 28.5℃ until sexual maturity, and the F0 generation of transgenic zebrafish are obtained; S4: F0 transgenic zebrafish are mated with wild-type zebrafish to obtain fluorescent offspring F1 transgenic zebrafish; The F1 generation of transgenic zebrafish were mated with wild-type zebrafish to obtain the fluorescent F2 generation, which is a stably inherited transgenic zebrafish strain.

4. Use of the method according to claim 1 apoc1 A method for constructing an animal model using a gene promoter, characterized in that: The method includes the following steps in sequence: S1: Acquired by polymerase chain reaction apoc1 Gene promoter; The primer sequences used are: F1-apoc1: 5'-AGACAGCAGGAATAAAGGTT-3'; R1-apoc1:5'-tcctcgcccttgctcaccatGGAATCACTGTTAGAGAGCT-3'; F2-apoc1: 5'-atcgaattcctgcagcccgggCAGTTGTTGGACTCCTTGC-3'; R2-apoc1: 5'-AACCTTTATTCCTGCTGTCTACAAACATAGATACATGCAG-3'; After polymerase chain reaction and purification recovery, PCR product A and PCR product B were obtained; S2: Plasmid construction: apoc1 The PCR products A and B of the gene promoter, the effector gene and the empty plasmid are integrated by homologous recombination, and then transformed into competent cells. After screening and plasmid extraction, a plasmid for transgenic operation is obtained; S3: The plasmid and transposase used for transgenic operation are mixed and injected into the single-cell fertilized eggs of zebrafish; 3 days after fertilization, the fertilized eggs with integrated effector genes with fluorescent expression are selected; then they are cultured at the standard temperature of 28.5℃ until sexual maturity, and the F0 generation of transgenic zebrafish are obtained; S4: F0 transgenic zebrafish are mated with wild-type zebrafish to obtain fluorescent offspring F1 transgenic zebrafish; The F1 generation of transgenic zebrafish were mated with wild-type zebrafish to obtain the fluorescent F2 generation, a stably inherited transgenic zebrafish strain.

Images