Method for constructing transgenic fish system based on Tol2 transposition subsystem

Abstract

The invention belongs to the technical field of transgenic and biomedical research tools, and particularly relates to a method for constructing a transgenic fish system based on a Tol2 transposon system, which comprises the following steps: constructing a myo6b-lhfpl5b-DsRed plasmid by using myo6b as a promoter, lhfpl5b as a target gene and DsRed as a red fluorescent protein tag through a microinjection method, and constructing a Tol2 transposon system-based transgenic fish system. Co-injecting the plasmid containing the Tol2 transposase mRNA and the Tol2 transposase mRNA into a fertilized egg of an AB zebra fish strain, and screening generation by generation to obtain a transgenic fish strain with stable inheritance MET channel gene lhfpl5b marked hair cells. The spatio-temporal dynamic visualization of the living whole fish to the lhfpl5b is realized, the deafness-causing molecular pathway is clarified, and an in-vivo platform is provided for drug screening and intervention strategies.

Description

Technical Field

[0001] This invention belongs to the field of transgenic and biomedical research tools technology, specifically relating to a method for constructing a transgenic animal that utilizes the Tol2 transposon system to achieve hair cell-specific expression of the fusion protein lhfpl5b-DsRed in zebrafish. This method is applicable to the in vivo visualization of mechanoreceptor transduction (MET) channel-related components and the study of hair cell development and damage regeneration mechanisms. Background Technology

[0002] Sensorineural hearing loss is the most common and irreversible type of hearing impairment, and its pathological basis mainly involves damage to the structure and function of hair cells in the sensory epithelium of the inner ear. In mammals, hair cells are located in the organ of Corti and the vestibular sensory epithelium (utricle, saccule, ampullary crest) of the inner ear. Their apexes carry stair-step-arranged tufts of stomatal hairs, responsible for rapidly converting mechanical vibrations into electrical signals and transmitting them to the central auditory pathway via synapses. It is generally believed in the field that genetic variations, noise exposure, ototoxic drugs, and aging can all lead to hair cell damage, apoptosis, or functional abnormalities, thereby causing progressive or congenital hearing loss and vestibular dysfunction. Existing clinical interventions (such as hearing aids and cochlear implants) mainly provide acoustic and electrical replacements or compensations, but are difficult to address at the source of key molecular processes, leaving a significant treatment gap in clinical practice.

[0003] Hair cells rely on mechanoreceptive transduction channels (MET channels) located at the distal end of the stomatal tract for mechanoreceptive transduction. Under mechanical stimulation, the ciliary tip junctions are stretched, triggering the opening of the MET channel and generating a rapid, transient inward current of cations. This induces hair cell depolarization and initiates subsequent synaptic transmission. Research in this field generally considers TMC1 / TMC2 to be candidate or core components of the MET channel pores; LHFPL5 (also known as TMHS), TMIE, and CIB2, as key auxiliary subunits, participate in the assembly of the channel complex, membrane domain localization, and gating homeostasis; while the traction proteins PCDH15 / CDH23 and myosin MYO1C regulate mechanical coupling, adaptation, and repositioning processes. Disruption of any of these processes can lead to abnormal transduction currents, channel misalignment, or traction instability, resulting in congenital or progressive deafness and balance disorders.

[0004] From the perspective of basic research and translational applications, systematically analyzing the in vivo assembly, subcellular localization, and dynamic homeostasis of MET channels is of great significance for elucidating the molecular mechanisms of auditory and vestibular sensory formation, and for identifying the pathogenic sites and pathways of hereditary deafness. However, due to the limited number of hair cells, the small spatial scale, and the static characteristics of traditional sample fixation methods, current technologies are still insufficient in their ability to "continuously, specifically, and quantitatively trace key subunits of the MET complex in vivo," making it difficult to meet the research needs of assembly processes, developmental sequences, and damage regeneration dynamics.

[0005] In this technological context, the LHFPL5 family, as conserved helper subunits of the MET channel, has been proven crucial for channel assembly and functional maintenance. Zebrafish possess lhfpl5a / b homologs, with lhfpl5b expressed in the inner ear and lateral line hair cells, participating in the localization and stability of the MET complex and believed to interact functionally with proteins such as Tmc and Tmie. Therefore, establishing an in vivo model capable of specifically and visually tracing lhfpl5b within hair cells is necessary and valuable for elucidating the spatial localization and membrane domain distribution of the MET complex, and characterizing its expression and translocation dynamics at different developmental and maturation stages. Summary of the Invention

[0006] The technical problem to be solved by this invention is to provide a method for constructing transgenic fish lines based on the Tol2 transposon system, so as to realize the in vivo visualization of the lhfpl5b protein fusion in hair cells, and can be used in conjunction with the existing hair cell marker line Tg(pou4f3:EGFP) for intuitive evaluation of co-expression and localization relationship, providing a tool for MET channel research.

[0007] To address the aforementioned technical problems, embodiments of the present invention provide a method for constructing transgenic fish lines based on the Tol2 transposon system. The method involves constructing a myo6b-lhfpl5b-DsRed plasmid using myo6b as the promoter, lhfpl5b as the target gene, and DsRed as the red fluorescent protein tag via microinjection. The plasmid containing the Tol2 transposition site and Tol2 transposase mRNA are co-injected into fertilized eggs of the AB zebrafish strain. Through successive generations of screening, a transgenic fish line stably inheriting the MET channel gene lhfpl5b-labeled hair cells is obtained. The method includes the following steps:

[0008] S1. Construction of myo6b-lhfpl5b-DsRed plasmid: Using myo6b promoter, lhfpl5b as the target gene, and DsRed as the red fluorescent protein tag, the myo6b-tbx1-DsRed original plasmid was linearized and purified, and then myo6b-lhfpl5b-DsRed plasmid was obtained by homologous recombination.

[0009] S2. Preparation of Tol2 mRNA: The Tol2 transposase plasmid was linearized, transcribed, and purified to obtain Tol2 transposase mRNA;

[0010] S3. Microinjection: The myo6b-lhfpl5b-DsRed plasmid obtained in step S1 and the Tol2 transposase mRNA obtained in step S2 were mixed and injected into the fertilized eggs of the AB zebrafish strain by microinjection, and cultured in E3 culture medium and incubated in a constant temperature incubator at 28.5℃.

[0011] S4. Screening for stable genetically inherited transgenic fish lines: Select juvenile fish with hair cells marked with DsRed fluorescent protein, and after diplomatic screening, obtain transgenic fish lines with stable genetically inherited lhfpl5b-marked hair cells.

[0012] The construction of the myo6b-lhfpl5b-DsRed plasmid in step S1 includes the following steps:

[0013] S1.1 Linearize the original plasmid myo6b-tbx1-DsRed by double enzyme digestion;

[0014] S1.2, The linearized product of myo6b-tbx1-DsRed double digestion was subjected to electrophoresis, and the bands of the size of the double digestion were purified by gel extraction.

[0015] S1.3, Overlap extension PCR technology was used to construct the target gene lhfpl5b cds region and the sequence containing the fluorescent protein DsRed;

[0016] S1.4 Using the myo6b-tbx1-DsRed original plasmid as a template, extract the sequence containing DsRed fluorescent protein and purify the band containing DsRed fluorescent protein;

[0017] S1.5. By using overlapping extension PCR to achieve seamless ligation of the target gene lhfpl5b and DsRed fluorescent protein using complementary sequences between the two fragments, a fusion expression fragment of the target gene lhfpl5b and DsRed fluorescent protein is obtained. The linearized myo6b-tbx1-DsRed plasmid is then overlapped and extended with the lhfpl5b-DsRed fragment to construct the myo6b-lhfpl5b-DsRed plasmid.

[0018] The preparation of Tol2 mRNA in step S2 includes the following steps:

[0019] S2.1 Linearize pCS2-transposase by single enzyme digestion, and purify the bands cut from the single enzyme digested plasmid;

[0020] S2.2, Transcribe Tol2 mRNA;

[0021] S2.3. The transcript obtained in step S2.2 is purified using the LiCl purification method.

[0022] The microinjection in step S3 includes the following steps:

[0023] S3.1. The night before the injection, place the AB wild-type zebrafish in the hybridization tank and separate the males and females with a baffle. The next morning, remove the baffle to allow them to lay eggs and fertilize externally. After obtaining a certain number of embryos, use a filter to remove the embryos and rinse them with system water onto the injection plate.

[0024] S3.2. The myo6b-lhfpl5b-DsRed plasmid obtained in step S1 and the Tol2 transposase mRNA obtained in step S2 are mixed and injected into zebrafish embryos.

[0025] S3.3. The injection system is injected into a capillary glass needle using a micro-sample needle until it reaches the appropriate diameter. The injection is then performed at the 1-cell stage of the embryo (to maximize transposition efficiency). After injection, the needle is immersed in E3 culture medium and placed in a constant temperature incubator at 28.5℃.

[0026] In step S3.2, the mRNA concentrations of myo6b-lhfpl5b-DsRed plasmid and Tol2 transposase were both 16 ng / µL, with a total volume of 5 µL.

[0027] The step S4, screening for stable genetically inherited transgenic fish lines, includes the following steps:

[0028] S4.1 Take 4 dpf injections of myo6b-lhfpl5b-DsRed plasmid and place them under a confocal microscope to observe whether there are hair cells marked with DsRed fluorescent protein. Select the fish with marked hair cells and raise them.

[0029] S4.2. Diplomatic screening of F1 generation transgenic fish: After the G0 generation is raised to adulthood, the G0 generation fish are mated with AB wild-type zebrafish to produce eggs and obtain F1 generation embryos. At 4 dpf, they are observed under a confocal microscope to screen for juvenile fish that can be labeled with zebrafish inner ear and neurothalamic hair cells and continue to be raised.

[0030] S4.3. Diplomatic selection to obtain F2 generation of transgenic fish with stable inheritance: F1 generation is raised to adulthood, and then diplomacy is continued to obtain F2 generation of transgenic fish with stable inheritance of lhfpl5b marker hair cells.

[0031] The beneficial effects of the above technical solution of the present invention are as follows:

[0032] (1) This invention utilizes the myo6b promoter to drive expression in hair cells with high specificity. The lhfpl5b-DsRed fusion can be continuously visualized at various developmental stages in vivo, avoiding the problems of high background and poor reproducibility caused by in vitro staining and antibody dependence, and achieving long-term and stable in vivo tracing.

[0033] (2) The present invention uses Lhfpl5b protein as a backbone to construct fluorescent fusion, which can intuitively present the real location and spatiotemporal changes of MET channel-related components and support the dynamic tracking of fine hair cell structure;

[0034] (3) The transgenic fish line constructed in this invention can be crossbred with green fluorescent transgenic lines such as Tg(pou4f3:EGFP) to form a two-color co-expression system, which facilitates co-localization analysis and improves spatial resolution and interpretation accuracy. Attached Figure Description

[0035] Figure 1 This is a schematic diagram of the step S1 in this invention for constructing the myo6-lhfpl5b-DsRed plasmid;

[0036] Figure 2 This is the plasmid map of myo6-lhfpl5b-DsRed used in this invention;

[0037] Figure 3 This is a schematic diagram of step S3, microinjection, in this invention;

[0038] Figure 4 This is a flowchart of the stable genetic fish line screening process in step S4 of the present invention;

[0039] Figure 5 This invention presents the expression results of the lhfpl5b gene in zebrafish hair cells and their precursor cells, wherein... Figure 5 A represents the grouping results of zebrafish hair cells and their precursor cells. Figure 5 B. Figure 5 C represents the expression of the zebrafish lhfpl5b gene in hair cells and its precursor cells;

[0040] Figure 6 This is a diagram showing the in vivo fluorescence tracing of Tg(myo6b:lhfpl5b-DsRed) transgenic zebrafish in Example 2. Figure 6 A- Figure 6 B is a schematic diagram of in vivo labeling of hair cells in the whole zebrafish; Figure 6 C is a magnified view of the zebrafish inner ear region to show the markings of hair cells at the utricle / saccule and the ampulla of the semicircular canals; Figure 6 D- Figure 6 D' is a magnified view of the lateral line thalamus region to show the markings of single clusters of thalamic hair cells;

[0041] Figure 7 This is a schematic diagram of the chimeric marker results after hybridization of Tg(myo6b:lhfpl5b-DsRed) and Tg(pou4f3:EGFP) fish strains according to the present invention, wherein, Figure 7 A- Figure 7 B is a schematic diagram of in vivo chimeric markers in the hair cells of zebrafish. Figure 7 C- Figure 7 C'' is a magnified view of the zebrafish inner ear region, showing the chimeric markers of the hair cells at the utricle / saccule and the ampulla of the semicircular canals. Figure 7 D- Figure 7 D''、 Figure 7 E- Figure 7 E'' is a magnified view of the lateral line thalamus region, showing the chimeric markers of single clusters of thalamic hair cells. Detailed Implementation

[0042] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with the accompanying drawings and specific embodiments.

[0043] Example 1: Construction of transgenic fish lines based on the Tol2 transposon system

[0044] This invention provides a method for constructing transgenic fish lines based on the Tol2 transposon system. Specifically, it is a method for constructing transgenic animals that utilize the Tol2 transposon system to achieve hair cell-specific expression of the fusion protein lhfpl5b-DsRed in zebrafish. The method involves microinjection of a plasmid containing myo6b as the promoter, lhfpl5b as the target gene, and DsRed as the red fluorescent protein tag. The plasmid containing the Tol2 transposition point and Tol2 transposase mRNA are co-injected into fertilized eggs of the AB zebrafish strain. Through successive generations of screening, a transgenic fish line stably inheriting the MET channel gene lhfpl5b-tagged hair cells is obtained. The method includes the following steps:

[0045] S1. Construction of myo6b-lhfpl5b-DsRed plasmid: Using myo6b promoter, lhfpl5b as the target gene, and DsRed as the red fluorescent protein tag, the myo6b-tbx1-DsRed original plasmid was linearized and purified, and then myo6b-lhfpl5b-DsRed plasmid was obtained by homologous recombination.

[0046] S2. Preparation of Tol2 mRNA: The Tol2 transposase plasmid was linearized, transcribed, and purified to obtain Tol2 transposase mRNA;

[0047] S3. Microinjection: The myo6b-lhfpl5b-DsRed plasmid obtained in step S1 and the Tol2 transposase mRNA obtained in step S2 were mixed and injected into the fertilized eggs of the AB zebrafish strain by microinjection, and cultured in E3 culture medium and incubated in a constant temperature incubator at 28.5℃.

[0048] S4. Screening for stable genetically inherited transgenic fish lines: Select juvenile fish with hair cells marked with DsRed fluorescent protein, and after diplomatic screening, obtain transgenic fish lines with stable genetically inherited lhfpl5b-marked hair cells.

[0049] In this embodiment, the myo6b-lhfpl5b-DsRed plasmid is constructed in step S1 as follows: Figure 1 As shown, the plasmid map of myo6-lhfpl5b-DsRed is as follows. Figure 2 As shown, the process includes the following steps:

[0050] S1.1 Linearize the original plasmid myo6b-tbx1-DsRed by double enzyme digestion. The enzyme digestion system is shown in the table below:

[0051] Components volume 10×Cutsmart Buffer 5µL Age I 2µL Cla I 2µL myo6b-tbx1-DsRed plasmid 4µg <![CDATA[ddH2O]]> XµL Total 50µL

[0052] The reaction conditions are: 37℃ ≥ 2 h;

[0053] S1.2. The linearized product of myo6b-tbx1-DsRed double digestion was subjected to electrophoresis. The bands of the size of the double digestion were purified by gel extraction. The purification included the following steps:

[0054] Prepare 1% agarose gel: Weigh 0.4g of agarose into an Erlenmeyer flask using an electronic balance, add 45mL of 1×TAE, heat and boil repeatedly until the liquid is clear. After slightly cooling, add 1.5µL of GelRed, mix well, pour into a gel mold, and wait for solidification. Place the agarose gel into the electrophoresis buffer, add 10µL of 6×DNAloading buffer to the double enzyme digestion product obtained in step S1.1, mix well, load the sample, and perform electrophoresis at a constant voltage of 150 V for 20 min.

[0055] After electrophoresis, based on the position of the bands in the double enzyme digestion products, the band corresponding to the size of the target plasmid was cut out, placed in a 1.5 mL centrifuge tube, and weighed. Three times the volume of the gel block was added to the centrifuge tube, and the mixture was heated at 55°C until the gel block dissolved. The resulting liquid was transferred to an adsorption column, centrifuged at 12000 rpm for 1 min, 200 µL of Wash Buffer was added, and the mixture was centrifuged at 12000 rpm for 1 min. The waste liquid was discarded, and the process was repeated once to remove residual liquid.

[0056] After centrifugation at 12000 rpm for 1 min, the adsorption column was placed in a new 1.5 mL centrifuge tube, the ethanol was dried by opening the cap, 15 µL of preheated ddH2O at 65 °C was added, the column was allowed to stand for 5 min, and then centrifuged at 12000 rpm for 2 min. The concentration and purity of the recovered product were determined using a micro spectrophotometer.

[0057] S1.3. Overlap extension PCR technology was used to construct the cds region of the target gene lhfpl5b and the sequence containing the fluorescent protein DsRed. The cds region sequence of the lhfpl5b gene was downloaded from a database, and the primers were designed as follows:

[0058] lhfpl5b-F: 5'-GACTACAAAGACGACGACGACAAAACCGGTATGGCAAAAGAAAAGATGCT-3';

[0059] lhfpl5b-R: 5'-TTGGAGGAGCGCACCATGGTGGCTGCGTTGTTTTCTCCCTCCACCTC-3';

[0060] The reaction system is shown in the table below:

[0061] Components volume 2×KOD Buffer 25µL 2mM dNTPs 10µL KOD Enzyme 1µL lhfpl5b-F 1.5µL lhfpl5b-R 1.5µL zebrafish cDNA ≤50ng <![CDATA[ddH2O]]> XµL Total 50µL

[0062] The reaction conditions are shown in the table below:

[0063] temperature time Cycle number 94℃ 5 min 98℃ 10s 35 cycles 55℃ 30s 35 cycles 68℃ 50s 35 cycles 68℃ 5min 12℃ ∞ ;

[0065] S1.4. Using the myo6b-tbx1-DsRed original plasmid as a template, extract the sequence containing the DsRed fluorescent protein, and purify the band containing the DsRed fluorescent protein. The primers are designed as follows:

[0066] dsred-F: 5'

[0067] -GAGGTGGAGGGAGAAAACAACGCAGCCACCATGGTGCGCTCCTCCAA-3';

[0068] dsred-R: 5'

[0069] -GTATCTTATCATGTCTGGATCATCATCGATGTGTACCGTAAAACGACGGCC-3';

[0070] The reaction system is shown in the table below:

[0071] Components volume 2×KOD Buffer 25 µL 2mM dNTPs 10µL KOD Enzyme 1µL dsred-F 1.5µL dsred-R 1.5µL myo6b-tbx1-DsRed plasmid ≤50ng <![CDATA[ddH2O]]> X µL Total 50µL

[0072] The reaction conditions are shown in the table below:

[0073] temperature time Cycle number 94℃ 5 min 98℃ 10s 35 cycles 55℃ 30s 35 cycles 68℃ 1 minute 10 seconds 35 cycles 68℃ 5min 4℃ ∞ ;

[0075] S1.5. Using overlap extension PCR technology, the target gene lhfpl5b and DsRed fluorescent protein were seamlessly linked using complementary sequences between the two fragments, resulting in a fusion expression fragment of lhfpl5b linked to DsRed fluorescent protein. The band of the fusion expression fragment was then purified. The reaction system is shown in the table below:

[0076] Components volume 2×KOD Buffer 25µL 2mM dNTPs 10µL KOD Enzyme 1µL lhfpl5b-F 1.5µL dsred-R 1.5µL lhfpl5b cds fragment 100ng desred fragment 100ng <![CDATA[ddH2O]]> XµL Total 50µL

[0077] The reaction conditions are shown in the table below:

[0078] temperature time Cycle number 94℃ 5 min 98℃ 10s 30 cycles 55℃ 30s 30 cycles 68℃ 2min 30 cycles 68℃ 5min 4℃ ∞ ;

[0080] S1.6. The linearized myo6b-tbx1-DsRed plasmid is overlapped and extended with the lhfpl5b-DsRed fragment to construct the myo6b-lhfpl5b-DsRed plasmid. The reaction system is shown in the table below:

[0081] Components volume Linearization of myo6b-tbx1-DsRed plasmid 200ng lhfpl5b-DsRed fragment 70ng 2× CE Mix 1µL <![CDATA[ddH2O]]> XµL Total 10µL

[0082] The reaction conditions were 50℃ for 5 minutes.

[0083] In this embodiment, the preparation of Tol2 mRNA in step S2 includes the following steps:

[0084] S2.1 Linearize pCS2-transposase by single-enzyme digestion, and purify the bands cut from the single-enzyme digested plasmid. The digestion system is shown in the table below:

[0085] Components volume 10×Cutsmart Buffer 5µL Not I 2µL pCS2-transposase 2µg <![CDATA[ddH2O]]> XµL Total 50µL

[0086] The reaction conditions are 37℃ for ≥2 h;

[0087] S2.2 Transcribe Tol2 mRNA using the MAXIscript™ SP6 transcription kit. The transcription system is shown in the table below:

[0088] Components volume 10×Reaction Buffer 2µL 2×NTP / CAP 10µL Linearized pCS2-transposase plasmid 1µg SP6 Enzyme 2µL <![CDATA[ddH2O]]> XµL Total 20µL

[0089] The reaction conditions were 37 °C for ≥2 h, followed by the addition of 1 µL DNase and incubation at 37 °C for 15 min to remove the DNA template.

[0090] S2.3. The transcript obtained in step S2.2 is purified using the LiCl purification method, including the following steps:

[0091] The transcription product obtained in step S2.2 was added to 20 µL of LiCl and 100 µL of isopropanol pre-cooled at -20℃ and precipitated overnight at -20℃. After complete precipitation, the product was removed, centrifuged at 13000 rpm for 30 min at 4℃, and the supernatant was discarded. The precipitate was gently washed with 80% ethanol, centrifuged at 13000 rpm for 15 min at 4℃, and the supernatant was discarded. After drying, 20 µL of ddH2O was added to dissolve and remove impurities. The product concentration was determined using a micro spectrophotometer before aliquoting and use.

[0092] In this embodiment, the microinjection in step S3 includes the following steps:

[0093] S3.1. The night before injection, place AB wild-type zebrafish in the hybridization tank and separate the males and females with a baffle. The next morning, remove the baffle to allow them to lay eggs externally and allow fertilization. After obtaining a certain number of embryos, remove the embryos with a filter and rinse them with system water onto the injection plate.

[0094] The injection needle is made by fixing a borosilicate glass capillary with an outer diameter of 1 micrometer and an inner diameter of 0.7 micrometers in a needle-drawing device, and then drawing it into an injection glass needle through ultra-high temperature heating.

[0095] The preparation process of the injection plate is as follows: Weigh 0.8g of agarose on an electronic balance and place it in an Erlenmeyer flask. Add 45mL of E3 culture medium and heat. Boil several times until the liquid is clear. After cooling slightly, pour it into a culture dish and gently place it into an injection mold. Wait for it to solidify to make an injection plate.

[0096] S3.2. The myo6b-lhfpl5b-DsRed plasmid obtained in step S1 and the Tol2 transposase mRNA obtained in step S2 are mixed and injected into zebrafish embryos. The preparation system is shown in the table below:

[0097] Components Dosage Tol2 mRNA 1µL myo6b-lhfpl5b-DsRed plasmid 80ng <![CDATA[ddH2O]]> XµL Total 5µL ;

[0099] S3.3. Using a micro-sampler, inject the injection system into a capillary glass needle until it reaches the appropriate diameter. Inject at the 1-cell stage of the embryo (to maximize transposition efficiency). Plasmid injection is as follows: Figure 3 As shown; after injection, it is immersed in E3 culture medium and placed in a constant temperature incubator at 28.5℃.

[0100] In this embodiment, step S4 involves screening for stable genetically inherited transgenic fish strains, such as... Figure 4 As shown, the process includes the following steps:

[0101] S4.1. Take 4 dpf juvenile fish injected with myo6b-lhfpl5b-DsRed plasmid and observe them under a confocal microscope for the presence of hair cells marked with DsRed fluorescent protein. Select the fish with marked hair cells and raise them. The process of selecting G0 generation transgenic fish under a confocal microscope is as follows:

[0102] Juvenile fish injected with myo6b-lhfpl5b-DsRed plasmid at 4dpf were placed in a culture dish. An appropriate amount of anesthetic was added until swimming stopped and the heart rate slowed down but the fish were still visible. 0.75% low-melting-point agarose was prepared with E3, melted at 65℃, and kept warm at 37–40℃. 10–15 juvenile fish were aspirated into a small dish. The residual liquid at the bottom of the dish was removed, and 40–60 µL of warm agarose was added. The juvenile fish were then quickly moved to their side using a needle. A 40× objective lens was immersed in water, and the 561 nm channel was opened to observe whether there were hair cells marked with DsRed fluorescent protein. Juvenile fish with marked hair cells were gently picked out with a needle and continued to be fed.

[0103] S4.2. Diplomatic screening of F1 generation transgenic fish: After the G0 generation is raised to adulthood, the G0 generation fish are mated with AB wild-type zebrafish to produce eggs and obtain F1 generation embryos. At 4 dpf, they are observed under a confocal microscope to screen for juvenile fish that can be labeled with zebrafish inner ear and neurothalamic hair cells and continue to be raised.

[0104] S4.3. Diplomatic selection to obtain F2 generation of transgenic fish with stable inheritance: F1 generation is raised to adulthood, and then diplomacy is continued to obtain F2 generation of transgenic fish with stable inheritance of lhfpl5b marker hair cells.

[0105] Example 2: Transgenic expression and fluorescence tracing in transgenic zebrafish

[0106] 1. Expression of lhfpl5b

[0107] By combining single-cell sequencing data, the Seurat package in R language was used to perform data visualization analysis to determine the expression of lhfpl5b in zebrafish hair cells.

[0108] 2. Zebrafish Culture

[0109] Zebrafish juveniles were cultured in E3 medium at a constant temperature of 28.5℃ to 6 dpf, then fed with paramecium to 16 dpf, and then transferred to a zebrafish culture system. The culture system was fed brine shrimp twice daily, with the water temperature maintained at approximately 28.5℃, a conductivity of 500-550 μS / cm, a pH of 6-7, and simulated light hours of 8:30-22:30 (daytime) / 22:30-8:30 (darkness).

[0110] 3. In vivo fluorescence tracing of Tg(myo6b:lhfpl5b-DsRed) transgenic zebrafish

[0111] After stable F2 generation transgenic zebrafish were cultured to 4 dpf, they were anesthetized, fixed, and observed under a confocal microscope to examine the labeling of inner ear hair cells and lateral line nerve thalamic hair cells. The results are as follows: Figure 6 As shown. Figure 6 A. Whole-fish imaging under a 20× objective lens revealed that DsRed fluorescence signals were mainly distributed in the inner ear sensory region of the head and in punctate structures along the lateral line of the trunk / tail, exhibiting a regular arrangement consistent with the anatomical distribution characteristics of the lateral line neural mounds; other tissues of the fish showed low background signal. For example... Figure 6 B. Figure 6 C Figure 6 D and Figure 6 D', further verification of the local area under a 40× objective lens revealed clusters of strongly positive cells in the inner ear, and typical hair cell clusters composed of multiple strongly positive cells were observed in the lateral line nerve thalamus.

[0112] Crossbreeding of Tg(myo6b:lhfpl5b-DsRed) and Tg(pou4f3:EGFP) fish strains

[0113] Stable genetically inherited Tg(myo6b:lhfpl5b-DsRed) females were placed in a hybridization tank with Tg(pou4f3:EGFP) males (or vice versa). The resulting embryos were removed using a filter screen and cultured. At 4 days post-fertilization (dpf), chimeric marking was observed under a confocal microscope. The results are as follows: Figure 7 As shown, the two fluorescent signals are mainly distributed in hair cell-rich areas such as the inner ear and lateral line nerve mounds; EGFP and DsRed can be observed to co-localize significantly in the same cell cluster in multiple nerve mounds, and under high magnification of a single nerve mound, it can be confirmed that most cells are double positive.

[0114] In summary, this invention addresses the challenge of stable, specific, and reproducible visualization and tracing of key MET channel components in hair cells under in vivo conditions, as demonstrated in existing technologies. It proposes a method for constructing a myo6b-lhfpl5b-DsRed transgenic fish strain based on the Tol2 transposon system. The strain selected using this method exhibits directed expression of the fluorescent fusion protein driven by the myo6b promoter, yielding clear and reproducible in vivo signals across multiple developmental stages and visually demonstrating the intracellular localization and temporal changes of lhfpl5b. This method can be used to elucidate protein distribution and regulatory mechanisms in hair cell development, stereocilia assembly, and damage repair. Combined with deafness gene mutants such as tmc1 / 2 and tmie, it can elucidate deafness-causing molecular pathways and provide an in vivo platform for drug screening and intervention strategies. Furthermore, it can be used in conjunction with transgenic fish strains such as Tg(pou4f3:EGFP) to achieve dual-channel co-localization and quantitative analysis, thus providing a stable and efficient genetic tool for MET channel research.

[0115] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for constructing transgenic fish lines based on the Tol2 transposon system, characterized in that, Using microinjection, a myo6b-lhfpl5b-DsRed plasmid was constructed with myo6b as the promoter, lhfpl5b as the target gene, and DsRed as the red fluorescent protein tag. The plasmid containing the Tol2 transposition site and Tol2 transposase mRNA were co-injected into fertilized eggs of the AB zebrafish strain. Through successive generations of screening, a transgenic fish strain with stably inherited MET channel gene lhfpl5b-tagged hair cells was obtained. The process included the following steps: S1. Construction of myo6b-lhfpl5b-DsRed plasmid: Using myo6b promoter, lhfpl5b as the target gene, and DsRed as the red fluorescent protein tag, the myo6b-tbx1-DsRed original plasmid was linearized and purified, and then myo6b-lhfpl5b-DsRed plasmid was obtained by homologous recombination. S2. Preparation of Tol2 mRNA: The Tol2 transposase plasmid was linearized, transcribed, and purified to obtain Tol2 transposase mRNA; S3. Microinjection: The myo6b-lhfpl5b-DsRed plasmid obtained in step S1 and the Tol2 transposase mRNA obtained in step S2 were mixed and injected into the fertilized eggs of the AB zebrafish strain by microinjection, and cultured in E3 culture medium and incubated in a constant temperature incubator at 28.5℃. S4. Screening for stable genetically inherited transgenic fish lines: Select juvenile fish with hair cells marked with DsRed fluorescent protein, and after diplomatic screening, obtain transgenic fish lines with stable genetically inherited lhfpl5b-marked hair cells.

2. The method for constructing transgenic fish lines based on the Tol2 transposon system according to claim 1, characterized in that, The construction of the myo6b-lhfpl5b-DsRed plasmid in step S1 includes the following steps: S1.1 Linearize the original plasmid myo6b-tbx1-DsRed by double enzyme digestion; S1.2, The linearized product of myo6b-tbx1-DsRed double digestion was subjected to electrophoresis, and the bands of the size of the double digestion were purified by gel extraction. S1.3, Overlap extension PCR technology was used to construct the target gene lhfpl5b cds region and the sequence containing the fluorescent protein DsRed; S1.4 Using the myo6b-tbx1-DsRed original plasmid as a template, extract the sequence containing DsRed fluorescent protein and purify the band containing DsRed fluorescent protein; S1.

5. By using overlapping extension PCR to achieve seamless ligation of the target gene lhfpl5b and DsRed fluorescent protein using complementary sequences between the two fragments, a fusion expression fragment of the target gene lhfpl5b and DsRed fluorescent protein is obtained. The linearized myo6b-tbx1-DsRed plasmid is then overlapped and extended with the lhfpl5b-DsRed fragment to construct the myo6b-lhfpl5b-DsRed plasmid.

3. The method for constructing transgenic fish lines based on the Tol2 transposon system according to claim 1, characterized in that, The preparation of Tol2 mRNA in step S2 includes the following steps: S2.1 Linearize pCS2-transposase by single enzyme digestion, and purify the bands cut from the single enzyme digestion plasmid; S2.2, Transcribe Tol2 mRNA; S2.

3. The transcript obtained in step S2.2 is purified using the LiCl purification method. The microinjection in step S3 includes the following steps: S3.

1. The night before the injection, place the AB wild-type zebrafish in the hybridization tank and separate the males and females with a baffle. The next morning, remove the baffle to allow them to lay eggs and fertilize externally. After obtaining a certain number of embryos, use a filter to remove the embryos and rinse them with system water onto the injection plate. S3.

2. The myo6b-lhfpl5b-DsRed plasmid obtained in step S1 and the Tol2 transposase mRNA obtained in step S2 are mixed and injected into zebrafish embryos. S3.

3. The injection system is injected into a capillary glass needle using a micro-sample needle until it reaches the appropriate diameter, and then injected at the 1-cell stage of the embryo. After injection, the needle is immersed in E3 culture medium and placed in a constant temperature incubator at 28.5℃ for culture.

4. The method for constructing transgenic fish lines based on the Tol2 transposon system according to claim 3, characterized in that, In step S3.2, the mRNA concentrations of myo6b-lhfpl5b-DsRed plasmid and Tol2 transposase were both 16 ng / µL, with a total volume of 5 µL.

5. The method for constructing transgenic fish lines based on the Tol2 transposon system according to claim 1, characterized in that, Step S4, screening for stable genetically inherited transgenic fish lines, includes the following steps: S4.1 Take 4 dpf injections of myo6b-lhfpl5b-DsRed plasmid and place them under a confocal microscope to observe whether there are hair cells marked with DsRed fluorescent protein. Select the fish with marked hair cells and raise them. S4.

2. Diplomatic screening of F1 generation transgenic fish: After the G0 generation is raised to adulthood, the G0 generation fish are mated with AB wild-type zebrafish to produce eggs and obtain F1 generation embryos. At 4 dpf, they are observed under a confocal microscope to screen for juvenile fish that can be labeled with zebrafish inner ear and neurothalamic hair cells and continue to be raised. S4.

3. Diplomatic selection to obtain F2 generation stable genetically inherited transgenic fish lines: F1 generation fish are raised to adulthood, and then diplomatic selection is continued to obtain F2 generation stable genetically inherited lhfpl5b marker hair cells transgenic fish lines.

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