Visual myocardial specific gene knockout zebrafish model and construction method and application thereof
By constructing a Tg transgenic line in a zebrafish model and using the cmlc2 promoter to drive the expression of Cas9 protein and mCherry fluorescent protein, in vivo visualization of myocardial-specific gene knockout was achieved. This solves the problems of complex construction and unstable efficiency in existing technologies, improves the reliability of experiments, and simplifies the operation process.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-31
- Publication Date
- 2026-03-20
AI Technical Summary
Existing methods for constructing tissue-specific gene knockout zebrafish models rely on traditional gene editing tools or early CRISPR/Cas9 strategies. These methods are complex, have lengthy design cycles, unstable editing efficiency, and lack direct reporting systems, resulting in poor reliability and reproducibility of experimental results. They also fail to confirm the expression of Cas9 protein in target cardiomyocytes in live embryos in real time, and are cumbersome and costly.
A Tg transgenic zebrafish strain was constructed, and the expression of Cas9 protein and red fluorescent protein mCherry was driven by the cmlc2 promoter, which is highly specific to cardiomyocytes. The Cas9 protein was coupled with the fluorescent signal by linking the P2A self-cleaving peptide sequence. The Tol2 transposon system was used to stably integrate the gene into the genome, thereby achieving cardiomyocyte-specific gene knockout and in vivo visualization observation through co-expression of fluorescent proteins.
This method enables strictly specific expression of Cas9 protein in cardiomyocytes and real-time visualization of gene editing, simplifies model identification and experimental screening processes, avoids lethality or tissue abnormalities caused by systemic knockout, and improves the accuracy and efficiency of experiments.
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Figure CN121694283A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the fields of genetic engineering and model animal technology, and in particular to a visualized myocardial-specific gene knockout zebrafish model, its construction method, and its application. Background Technology
[0002] Heart disease is one of the leading causes of death worldwide. Its pathogenesis is complex and involves abnormal regulation of many genes. Constructing gene knockout models using animal models is an important means of studying the function of specific genes in heart development, homeostasis maintenance and disease occurrence.
[0003] Zebrafish have become an ideal vertebrate model for studying heart development and diseases due to their advantages such as high genetic conservation with humans, transparent embryos, rapid development, high reproductive capacity, and relatively low experimental costs. The application of traditional gene knockout technology in zebrafish is relatively mature, but it generally suffers from problems such as long construction cycle, unstable efficiency, or off-target risk. The emergence of CRISPR / Cas9 technology has greatly improved the efficiency and convenience of gene editing.
[0004] Existing methods for constructing tissue-specific gene knockout zebrafish models mainly rely on traditional gene editing tools or early-generation CRISPR / Cas9 strategies. In practical applications, while traditional technologies such as TALENs or ZFNs can achieve gene editing, the construction process is complex, the design cycle is lengthy, the editing efficiency fluctuates greatly between different experimental batches, and there are unpredictable off-target effects, which challenge the reliability and reproducibility of experimental results. At the same time, existing myocardial-specific CRISPR / Cas9 models usually only drive the expression of Cas9 protein without a direct reporter system. Therefore, researchers cannot confirm in real time whether the Cas9 protein is successfully expressed in the target cardiomyocytes and its expression pattern in live embryos. After each experiment, cumbersome follow-up operations must be performed, such as extracting the genome for PCR identification, preparing slides for immunohistochemical staining, or indirectly inferring the editing efficiency through molecular detection. This is not only time-consuming, labor-intensive, and costly, but may also introduce errors due to sample processing, which is particularly unfavorable for rapid screening of a large number of embryos.
[0005] To address the aforementioned issues, this approach involves constructing a Tg transgenic zebrafish strain. Utilizing the cardiomyocyte-specific cmlc2 promoter, it simultaneously drives the expression of zebrafish codon-optimized Cas9 protein and the red fluorescent protein mCherry. These two proteins are linked via a self-cleaving peptide sequence of P2A, ensuring independent and complete functional protein production after transcription. This allows for complete coupling between Cas9 protein expression and the mCherry fluorescence signal. Furthermore, a transgenic vector containing these elements is constructed using molecular cloning technology, and stably integrated into the zebrafish genome using the Tol2 transposon system. After passage selection, homozygous or heterozygous stable genetic strains are obtained. In use, researchers only need to microinject a single s gene designed to target a specific gene into the early embryos of this zebrafish strain. gRNA can achieve specific knockout of the gene in cardiomyocytes. Co-expression of the fluorescent protein mCherry enables non-invasive, real-time, and in vivo visualization of Cas9-positive cardiomyocytes. Researchers can directly screen individuals with successful transgene integration and confirm the expression location of Cas9 under a fluorescence microscope, completely eliminating the tedious and destructive genotype verification steps, greatly simplifying the model identification and experimental screening process. Furthermore, the cmlc2 promoter is used to achieve strict cardiomyocyte-specific expression of the Cas9 protein. Combined with the injection of targeted sgRNA, the gene editing event is ensured to be highly confined to the heart tissue, avoiding early embryonic lethality or other tissue developmental abnormalities that may be caused by systemic knockout. This allows researchers to study the specific function of the gene in the heart more precisely. Summary of the Invention
[0006] To overcome the limitations of existing tissue-specific gene knockout zebrafish models, which primarily rely on traditional gene editing tools or early-generation CRISPR / Cas9 strategies, traditional technologies such as TALENs or ZFNs, while capable of gene editing, suffer from complex construction processes, lengthy design cycles, significant fluctuations in editing efficiency across different experimental batches, and unpredictable off-target effects. These challenges compromise the reliability and reproducibility of experimental results. Furthermore, existing myocardial-specific CRISPR / Cas9 models typically only drive Cas9 protein expression without a direct reporter system. Consequently, researchers cannot confirm in real-time whether Cas9 protein is successfully expressed in target cardiomyocytes and its expression pattern in live embryos. After each experiment, cumbersome follow-up procedures are required, such as extracting the genome for PCR identification, preparing slides for immunohistochemical staining, or indirectly inferring editing efficiency through molecular detection. This process is not only time-consuming, labor-intensive, and costly but may also introduce errors due to sample processing, making it particularly unsuitable for rapid screening of large numbers of embryos.
[0007] The technical solution of the present invention is: a visual zebrafish model of myocardial specific gene knockout, comprising a model, the model being a transgenic zebrafish, whose genome contains a Cas9 protein coding sequence driven by a myocardial specific promoter.
[0008] Preferably, the myocardium-specific promoter is the cmlc2 promoter. This promoter ensures that the Cas9 protein is expressed only in cardiomyocytes, avoiding unnecessary editing or toxicity in other tissues.
[0009] Preferably, the cmlc2 promoter drives the expression of a fusion protein or co-expression unit of Cas9 protein and reporter protein. The reporter protein is preferably a fluorescent protein, specifically the red fluorescent protein mCherry. The co-expression of the fluorescent protein causes zebrafish cardiomyocytes that have successfully integrated and expressed the transgene to fluoresce, enabling the visual tracking of Cas9-expressing cells.
[0010] Preferably, the Cas9 protein-coding sequence and the reporter protein-coding sequence are linked by a self-cleaving peptide sequence, specifically the P2A peptide sequence, which can generate two independent, fully functional Cas9 proteins and mCherry proteins after translation.
[0011] As a preferred choice, the transgenic zebrafish strain is Tg.
[0012] A method for constructing a visual zebrafish model of myocardial-specific gene knockout includes the following steps:
[0013] S11: Construct a transgenic expression vector containing a myocardial-specific promoter, a Cas9 protein-coding sequence, and an optional reporter protein-coding sequence;
[0014] S12: Transgenic expression vector was introduced into zebrafish embryos, and transgenic zebrafish that stably integrated and expressed Cas9 protein were screened for. generation;
[0015] S13: Will Transgenic zebrafish are bred to sexual maturity, and homozygous or heterozygous strains that can stably inherit the transgene are obtained through passaging and screening.
[0016] As a preferred option, sgRNA targeting specific genes is injected into the 1-cell stage embryos of a visualized myocardial-specific gene knockout zebrafish model. Target genes include, but are not limited to, heart-related genes such as nkx2.5, tbx5, and flnc.
[0017] As a preferred option, the visualized myocardial-specific gene knockout zebrafish model is used in the preparation of research models for heart diseases, including congenital heart disease, dilated cardiomyopathy, myocardial hypertrophy, heart failure, or arrhythmia. The visualized myocardial-specific gene knockout zebrafish model is also used in the screening or evaluation of drugs for the treatment of heart diseases.
[0018] The beneficial effects of this invention are:
[0019] Compared to existing technologies, constructing tissue-specific gene knockout zebrafish models mainly relies on traditional gene editing tools or early-generation CRISPR / Cas9 strategies. While traditional technologies such as TALENs or ZFNs can achieve gene editing in practical applications, the construction process is complex, the design cycle is lengthy, the editing efficiency fluctuates significantly between different experimental batches, and unpredictable off-target effects exist, challenging the reliability and reproducibility of experimental results. Furthermore, existing myocardial-specific CRISPR / Cas9 models typically only drive Cas9 protein expression without a direct reporter system, thus researchers cannot confirm the results in real-time in living embryos. Determining whether Cas9 protein is successfully expressed in target cardiomyocytes and its expression pattern requires tedious follow-up procedures after each experiment, such as extracting the genome for PCR identification, preparing slides for immunohistochemical staining, or indirectly inferring editing efficiency through molecular detection. This process is not only time-consuming, labor-intensive, and costly, but may also introduce errors due to sample processing, making it particularly unsuitable for rapid screening of large numbers of embryos. This approach constructs a Tg transgenic zebrafish strain and utilizes the cardiomyocyte-specific cmlc2 promoter to simultaneously drive the expression of zebrafish codon-optimized Cas9 protein and the red fluorescent protein mCherry, both expressed via P2A self-cleavage. Peptide sequence linkage ensures the production of independent and complete functional proteins after transcription and translation, enabling complete coupling between Cas9 protein expression and mCherry fluorescence signal. Simultaneously, transgenic vectors containing these elements are constructed using molecular cloning technology and stably integrated into the zebrafish genome using the Tol2 transposon system. After passage selection, homozygous or heterozygous stable genetic lines are obtained. In use, researchers only need to microinject a single sgRNA designed to target a specific gene into the early embryos of zebrafish of this line to achieve specific knockout of that gene in cardiomyocytes. Co-expression of the fluorescent protein mCherry enables the control of Cas9 protein expression. Non-invasive, real-time, and in vivo visualization of Cas9-positive cardiomyocytes allows researchers to directly screen individuals with successful transgene integration and confirm the expression location of Cas9 under a fluorescence microscope. This completely eliminates the tedious and destructive genotype verification steps, greatly simplifying model identification and initial screening processes. Furthermore, the use of the cmlc2 promoter enables strictly myocardial-specific expression of the Cas9 protein. Combined with the injection of targeted sgRNA, this ensures that the gene editing event is highly confined to the heart tissue, avoiding early embryonic lethality or other tissue developmental abnormalities that may result from systemic knockout. This allows researchers to more accurately study the specific functions of genes in the heart. Attached Figure Description
[0020] Figure 1 The diagram shown is a flowchart of a method for constructing a visual myocardial-specific gene knockout zebrafish model according to the present invention.
[0021] Figure 2The diagram illustrates a visual method for drug screening using a zebrafish model with a myocardial-specific gene knockout.
[0022] Figure 3 The illustration shown is a visual representation of the mCherry red fluorescent protein coding sequence of a myocardial-specific gene knockout zebrafish model according to the present invention. Detailed Implementation
[0023] The present invention will be further described below with reference to the accompanying drawings and embodiments.
[0024] Please see Figure 1-2 The present invention provides an embodiment of a visual myocardial-specific gene knockout zebrafish model, comprising a model, the model being a transgenic zebrafish whose genome contains a Cas9 protein coding sequence driven by a myocardial-specific promoter.
[0025] Preferably, the myocardium-specific promoter is the cmlc2 promoter. This promoter ensures that the Cas9 protein is expressed only in cardiomyocytes, avoiding unnecessary editing or toxicity in other tissues.
[0026] Preferably, the cmlc2 promoter drives the expression of a fusion protein or co-expression unit of Cas9 protein and reporter protein. The reporter protein is preferably a fluorescent protein, specifically the red fluorescent protein mCherry. The co-expression of the fluorescent protein causes zebrafish cardiomyocytes that have successfully integrated and expressed the transgene to fluoresce, enabling the visual tracking of Cas9-expressing cells.
[0027] Preferably, the Cas9 protein-coding sequence and the reporter protein-coding sequence are linked by a self-cleaving peptide sequence, specifically the P2A peptide sequence, which can generate two independent, fully functional Cas9 proteins and mCherry proteins after translation.
[0028] As a preferred choice, the transgenic zebrafish strain is Tg.
[0029] A method for constructing a visual zebrafish model of myocardial-specific gene knockout includes the following steps:
[0030] S11: Construct a transgenic expression vector containing a myocardial-specific promoter, a Cas9 protein-coding sequence, and an optional reporter protein-coding sequence;
[0031] S12: Transgenic expression vector was introduced into zebrafish embryos, and transgenic zebrafish that stably integrated and expressed Cas9 protein were screened for. generation;
[0032] S13: Will Transgenic zebrafish are bred to sexual maturity, and homozygous or heterozygous strains that can stably inherit the transgene are obtained through passaging and screening.
[0033] As a preferred option, sgRNA targeting specific genes is injected into the 1-cell stage embryos of a visualized myocardial-specific gene knockout zebrafish model. Target genes include, but are not limited to, heart-related genes such as nkx2.5, tbx5, and flnc.
[0034] As a preferred option, the visualized myocardial-specific gene knockout zebrafish model is used in the preparation of research models for heart diseases, including congenital heart disease, dilated cardiomyopathy, myocardial hypertrophy, heart failure, or arrhythmia. The visualized myocardial-specific gene knockout zebrafish model is also used in the screening or evaluation of drugs for the treatment of heart diseases.
[0035] Example 1: Construction of a Tg(cmlc2:Cas9-P2A-mCherry) transgenic zebrafish model
[0036] Using Tol2 as the backbone vector, the zebrafish cmlc2 promoter sequence, the zebrafish codon-optimized Streptococcus pyogenes Cas9 gene coding sequence, the P2A self-cleaving peptide sequence, the mCherry red fluorescent protein coding sequence, and the SV40 polyA signal sequence were sequentially cloned to construct the transgenic vector pDestTol2-cmlc2:Cas9-P2A-mCherry. The mCherry red fluorescent protein coding sequence can be converted to GFP green fluorescence or BFP blue fluorescence. See details... Figure 3 .
[0037] The recombinant plasmid (concentration of 25-100 ng / μL) was mixed with Tol2 transposase mRNA transcribed in vitro (concentration of 25-50 ng / μL) and injected into the cytoplasm of 1-cell stage embryos of wild-type AB strain zebrafish.
[0038] The injected embryos were placed in a 28.5℃ incubator for development. After fertilization (hpf) for 48-72 hours, embryos exhibiting obvious red fluorescence in the heart region were selected under a fluorescence stereomicroscope and cultured separately until adult fish. These are the embryos that were selected. Founder Fish.
[0039] Fluorescent positive Adult fish were mated with wild-type AB fish, and their offspring were collected. Embryos were observed for fluorescence at 72 hpf, from each In a pedigree screening, approximately 50% of the embryos expressed red fluorescence in their hearts (indicating that the embryos in this pedigree...). The fish are reproductive chimeras, and the transgene has been integrated into the gametes. Fluorescently positive samples from this family were collected. The juvenile fish were raised to adulthood, and the integration of the transgene was verified by PCR and sequencing. Positive results were obtained. Individuals mate with each other to obtain From these generations, families with 100% embryonic heart expression of red fluorescence were selected, thus obtaining the homozygous Tg(cmlc2:Cas9-P2A-mCherry) transgenic zebrafish strain. This strain exhibits stable and bright expression of myocardial-specific red fluorescence.
[0040] Example 2: Achieving myocardial-specific gene knockout using the Tg(cmlc2:Cas9-P2A-mCherry model
[0041] A specific sgRNA was designed and synthesized targeting an exon region of the zebrafish heart flnc.
[0042] Chemically synthesized sgRNA-flnc (concentration of 100-250 ng / μL) was injected into 1-cell stage embryos of Tg(cmlc2:Cas9-P2A-mCherry) zebrafish, and wild-type embryos injected with only sgRNA were set up as negative controls.
[0043] The injected embryos were cultured at 28.5℃ to 72 hpf. Under a microscope, red fluorescence was clearly visible in the heart region of the experimental group embryos, indicating that the model was effective. Compared with the control group, most of the experimental group embryos showed significant cardiac phenotypic abnormalities, including cardiac edema, ventricular dilation, and thinning of the ventricular wall, which is consistent with the known flnc knockout phenotype.
[0044] Example 3: Preliminary Exploration of Drug Screening Using the Tg(cmlc2:Cas9-P2A-mCherry) Model
[0045] Following the method in Example 2, a batch of zebrafish embryos with myocardial-specific flnc knockout were prepared.
[0046] After embryos developed to 24 hpf (after sgRNA injection), model embryos exhibiting the ventricular dilation phenotype were randomly divided into groups and cultured in embryo culture media containing different concentrations of potential therapeutic drugs. A model embryo group containing only DMSO solvent was established as the disease control group, and wild-type embryos were established as the normal control group.
[0047] Embryos were cultured to 72-96 hpf, and their survival rate, cardiac edema, cardiac contraction frequency and rhythm were observed and recorded under a fluorescence microscope. By comparing the improvement of cardiac phenotype in the drug-treated group and the disease control group, the therapeutic effect of the drug on the ventricular dilation model can be evaluated.
[0048] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention.
Claims
1. A visual zebrafish model of myocardial-specific gene knockout; characterized in that: This includes a model, which is a transgenic zebrafish, whose genome contains a Cas9 protein-coding sequence driven by a myocardial-specific promoter.
2. The visual myocardial-specific gene knockout zebrafish model and its construction method and application as described in claim 1, characterized in that: The myocardial-specific promoter is the cmlc2 promoter.
3. The visual myocardial-specific gene knockout zebrafish model according to claim 2, characterized in that: The cmlc2 promoter drives the expression of fusion proteins or co-expression units of Cas9 protein and reporter protein.
4. The visual myocardial-specific gene knockout zebrafish model according to claim 3, characterized in that: The reporter protein is a fluorescent protein, specifically the red fluorescent protein mCherry.
5. The visual myocardial-specific gene knockout zebrafish model according to claim 1, characterized in that: The Cas9 protein-coding sequence and the reporter protein-coding sequence are linked by a self-cleaving peptide sequence, specifically the P2A peptide sequence.
6. A visual zebrafish model for knocking out myocardial specific genes according to any one of claims 1-5, characterized in that: The genetically modified zebrafish strain is Tg.
7. The visual myocardial-specific gene knockout zebrafish model according to claim 1, characterized in that: The visualization of myocardial-specific gene knockout zebrafish models has been applied in the preparation of research models for heart diseases, including congenital heart disease, dilated cardiomyopathy, myocardial hypertrophy, heart failure, or arrhythmia.
8. The visual myocardial-specific gene knockout zebrafish model according to claim 1, characterized in that: Visualizing myocardial-specific gene knockout zebrafish models can also be used in screening or evaluating drugs for treating heart diseases.
9. A visual zebrafish model for knocking out myocardial specific genes according to any one of claims 1-6, characterized in that: A method for constructing a visual zebrafish model of myocardial-specific gene knockout includes the following steps: S11: Construct a transgenic expression vector containing a myocardial-specific promoter, a Cas9 protein-coding sequence, and an optional reporter protein-coding sequence; S12: The transgenic expression vector is introduced into zebrafish embryos, and transgenic zebrafish that stably integrate and express Cas9 protein are screened to obtain transgenic zebrafish. generation; S13: The above Transgenic zebrafish are bred to sexual maturity, and homozygous or heterozygous strains that can stably inherit the transgene are obtained through passaging and screening.
10. The method for constructing a visual myocardial-specific gene knockout zebrafish model according to claim 7, characterized in that: sgRNA targeting a specific gene, which is related to cardiac development, function, or disease, was injected into 1-cell stage embryos of a visual myocardial-specific gene knockout zebrafish model.