Construction method and application of zebrafish reln gene knockout model

Abstract

The invention relates to a construction method and application of a zebrafish reln gene knockout model. According to the method, a sixth exon of a reln gene of the zebra fish is targeted through a CRISPR / Cas9 technology, and a stable passage reln mutant zebra fish model is generated. The method comprises the steps of gene knockout, mutant identification, expression pattern analysis, microscopic morphology observation and nerve electrophysiology testing. The model can be used for researching neurodevelopment abnormality caused by Reelin protein deletion, such as neuron migration defect and epilepsy phenotype, and an efficient and stable platform is provided for nervous system disease mechanism research and drug screening. The method integrates multi-dimensional verification means, is easy and convenient to operate and high in repeatability, and has important application value.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering and animal models, and in particular to a method for constructing and applying a zebrafish reln gene knockout model. Background Technology

[0002] The RELN gene is located on the long arm of human chromosome 7, region 2, band 2, subband 1 (7q22.1), and consists of 65 exons. Its encoded protein, Reelin, is an extracellular matrix glycoprotein secreted by Cajal-Retzius cells, playing a crucial regulatory role in the migration and localization of neurons in the embryonic brain. During cerebellar development, Reelin influences the formation of normal cerebellar structures. After birth, Reelin is primarily secreted by GABAergic neurons, participating in dendritic and axonal growth, synapse formation, and plasticity regulation, thus affecting learning and cognition. Zebrafish (Danio rerio), as a tropical vertebrate model, has a genome highly homologous to humans (approximately 70%), a similar nervous system, and its genome has been fully sequenced. There is a temporal correspondence between zebrafish development and human development: 3 days after fertilization (3 dpf) corresponds to human birth, while 4 dpf, 5 dpf, 6 dpf, and 7 dpf correspond to 3 months, 6 months, 9 months, and 12 months of infant age, respectively. This developmental timing advantage, combined with efficient gene manipulation technologies such as CRISPR / Cas9 and in vivo imaging capabilities, makes zebrafish an ideal platform for studying neurological diseases. While zebrafish gene editing tools are mature, systematic knockout models for the reln gene and methods for multidimensional phenotypic validation are still incomplete, particularly regarding the lack of comprehensive data on neurodevelopmental abnormalities caused by Reelin deficiency (such as brain structural changes and epileptiform activity).

[0003] Therefore, this invention proposes a method for constructing and applying a zebrafish reln gene knockout model. Summary of the Invention

[0004] The purpose of this invention is to address the shortcomings of existing technologies by proposing a method for constructing and applying a zebrafish reln gene knockout model.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: A method for constructing a zebrafish reln gene knockout model includes the following steps: S1: Using CRISPR / Cas9 technology, the 6th exon of the zebrafish reln gene was targeted to generate a reln chimeric mutant; S2: A stable, homozygous reln mutant is obtained through hybridization and self-pollination.

[0006] Preferably, the target of the CRISPR / Cas9 technology is located in the 6th exon sequence of the reln gene.

[0007] Preferably, the mutant identification includes gene extraction, PCR amplification and agarose gel electrophoresis analysis, and Sanger sequencing.

[0008] A method for detecting the expression pattern of the reln gene in zebrafish is proposed, which uses whole embryo in situ hybridization technology and digoxigenin-labeled antisense probes to detect the expression of reln during the embryonic development stage of zebrafish.

[0009] A method for observing the neuromorphology of zebrafish was developed. The Tg[huc:GFP] strain of zebrafish was used, and microscopic imaging was performed at 5 dpf. The brain area, interocular distance, and body length parameters were measured using ImageJ software.

[0010] A method for testing the neurophysiology of zebrafish is proposed, which uses glass microelectrodes to record local field potentials from the optic tectum at a sampling frequency of 10 kHz and detects epilepsy-related signals.

[0011] Preferably, the electrophysiological tests are performed on 5-6 dpf zebrafish juveniles, and the data are analyzed using DClamp software.

[0012] A zebrafish reln gene knockout mutant was constructed using a zebrafish gene knockout model and mutation verification method. Its phenotype includes a reduction in hindbrain area and total brain area.

[0013] The application of a zebrafish reln gene knockout mutant in neurodevelopmental disease research, used to mimic neuronal migration abnormalities or epileptic phenotypes caused by Reelin protein deficiency.

[0014] A kit for constructing a zebrafish reln gene knockout model, comprising a CRISPR / Cas9 targeting component and gene identification primers.

[0015] The beneficial effects of this invention are as follows: 1. The model of this invention can simulate the phenotype of human RELN-related diseases, providing a platform for mechanism research and drug development. Attached Figure Description

[0016] Figure 1 The image shows representative electrophysiological signals in zebrafish, illustrating an example of epilepsy-related signals in the mutant group. Figure 2 This invention used whole-embryo in situ hybridization to detect the expression pattern of reln in zebrafish, the target site for knockout model construction, and the growth mutant reln. -10 / -10 A schematic diagram of base deletions and predicted protein truncation combinations; Figure 3This is a schematic diagram of the microscopic morphological observation results of the present invention. Detailed Implementation

[0017] The technical solution of the present invention will be further described in detail below with reference to specific embodiments.

[0018] In the description of this invention, it should be noted that, unless otherwise explicitly specified and limited, the terms "installation," "connection," "linking," and "setting" should be interpreted broadly. For example, they can refer to a fixed connection or setting, a detachable connection or setting, or an integral connection or setting. Those skilled in the art can understand the specific meaning of the above terms in this invention according to the specific circumstances.

[0019] Example 1: Laboratory Animals and Culture Tg[huc:GFP] and AB background zebrafish were selected and reared at 28.5±0.5℃ with a light / dark cycle of 14 / 10 hours. Embryos were cultured in E3 medium to 5 dpf before being used in experiments.

[0020] reln gene knockout and mutant construction CRISPR / Cas9 technology was used to target exon 6 of reln to obtain the F0 generation chimeric mutant. F0 mutants were laterally crossed with Tg[huc:GFP] to obtain the F1 generation heterozygotes, and self-crossing yielded the F2 generation homozygous mutant reln- / -. Gene identification was achieved through DNA extraction from tail fin tissue: 50 mM NaOH was added and the mixture was lysed at 95°C for 20-30 minutes, followed by neutralization with Tris-HCl. PCR amplification was then performed (system: 19.25 μl ddH2O, 2.5 μl 10X buffer, 1 μl dNTP, 0.5 μl forward and reverse primers, 0.125 μl rTaq enzyme). The products were verified by 2% agarose gel electrophoresis and Sanger sequencing.

[0021] Expression pattern analysis Reln expression was detected by whole embryo in situ hybridization: the probe template was amplified using primers (forward: 5'-CTCTGAACACCAGCACAGCCTCAGTCC-3', reverse: 5'-GTGTGTGGAGTCTGCAGCTCAGTGG-3'), cloned into the pGEMT-easy vector, and hybridized after digoxigenin labeling. Results showed that reln was expressed in the forebrain, midbrain, and hindbrain after 24 hpf.

[0022] Microscopic morphological observation Five-day-of-fly (5dpf) Tg[huc:GFP] zebrafish were imaged using a Nikon SMZ800N microscope (2x bright field, 4x fluorescence). Eye distance, body length, and brain area were measured using ImageJ. Results: The hindbrain area (p=0.00093) and total brain area (p=0.0499) of the reln- / - mutant group were significantly smaller than those of the control group; other parameters showed no difference.

[0023] Neurophysiological testing The optic tectum field potential of 5-6 dpf zebrafish was recorded using glass microelectrodes. The amplifier sampling frequency was 10 kHz, the filter was 1 Hz-5 kHz, and the duration was 15 minutes. In the control group, 30 / 30 samples were normal, while in the mutant group, 4 / 32 samples showed epileptic signals, but the difference was not statistically significant (p=0.1136).

[0024] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for constructing a zebrafish reln gene knockout model, characterized in that, Includes the following steps: S1: Using CRISPR / Cas9 technology, the 6th exon of the zebrafish reln gene was targeted to generate a reln chimeric mutant; S2: A stable, homozygous reln mutant is obtained through hybridization and self-pollination.

2. The method for constructing a zebrafish reln gene knockout model according to claim 1, characterized in that, The target of the CRISPR / Cas9 technology is located in the 6th exon sequence of the reln gene.

3. The method for constructing a zebrafish reln gene knockout model according to claim 1, characterized in that, The mutant identification process includes gene extraction, PCR amplification, agarose gel electrophoresis analysis, and Sanger sequencing.

4. A method for detecting the expression pattern of the reln gene in zebrafish, characterized in that, The expression of reln during the embryonic development stage in zebrafish was detected using whole embryo in situ hybridization with digoxigenin-labeled antisense probes.

5. A method for observing the neural morphology of zebrafish, characterized in that, Tg[huc:GFP] strain zebrafish were selected, and images were taken under a microscope at 5 dpf. Brain area, interocular distance, and body length parameters were measured using ImageJ software.

6. A method for testing the neurophysiological function of zebrafish, characterized in that, Local field potentials were recorded from the top cover using glass microelectrodes at a sampling frequency of 10 kHz to detect epilepsy-related signals.

7. The zebrafish neuroelectrophysiological testing method according to claim 6, characterized in that: The electrophysiological tests were performed on 5-6 dpf zebrafish juveniles, and the data were analyzed using DClamp software.

8. A zebrafish reln gene knockout mutant, characterized in that, The zebrafish gene knockout model and mutation verification method described in any one of claims 1-3 is used to construct the model, and its phenotype includes a reduction in hindbrain area and total brain area.

9. The application of the zebrafish reln gene knockout mutant as described in claim 8 in the study of neurodevelopmental diseases, characterized in that, Used to mimic abnormal neuronal migration or epileptic phenotypes caused by Reelin protein deficiency.

10. A kit for constructing a zebrafish reln gene knockout model, characterized in that, It includes CRISPR / Cas9 targeting components and gene identification primers.

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