CN-122012626-A - Construction method and application of zebra fish reln gene knockout model

Abstract

The invention relates to a construction method and application of a zebra fish reln gene knockout model. The method targets the 6 th exon of zebra fish reln genes through CRISPR/Cas9 technology to generate a reln mutant zebra fish model with stable passage. The method comprises gene knockout, mutant identification, expression pattern analysis, microscopic morphology observation and neuro-electrophysiologic test. The model can be used for researching the abnormal nerve development caused by Reelin protein deficiency, such as neuron migration defect and epileptic phenotype, and provides a high-efficiency and stable platform for researching nervous system disease mechanism and screening medicines. The invention integrates a multi-dimensional verification means, is simple and convenient to operate, has strong repeatability and has important application value.

Inventors

• HU YUE
• CHEN XIAOQI

Assignees

• 重庆医科大学附属儿童医院

Dates

Publication Date

20260512

Application Date

20260227

Claims (10)

1. 1. The construction method of the zebra fish reln gene knockout model is characterized by comprising the following steps of: S1, targeting the 6 th exon of zebra fish reln gene by using CRISPR/Cas9 technology to generate reln chimeric mutant; S2, obtaining a reln homozygous mutant with stable passage through hybridization and selfing.
2. 2. The method of claim 1, wherein the CRISPR/Cas9 technology targets the 6 th exon sequence of reln gene.
3. 3. The method of claim 1, wherein the mutant identification comprises gene extraction, PCR amplification and agarose gel electrophoresis analysis, sanger sequencing.
4. 4. A detection method of zebra fish reln gene expression pattern is characterized in that the whole embryo in situ hybridization technology is used, and digoxin marked antisense probe is used for detecting reln expression in the embryo development stage of zebra fish.
5. 5. A method for observing the nerve morphology of zebra fish features that the zebra fish strain Tg huc is chosen, the image is made by microscope at 5dpf, and the image J software is used to measure the brain area, eye distance and length parameters.
6. 6. A zebra fish nerve electrophysiological test method is characterized in that a glass microelectrode is used for recording local field potential from a visual top cover, sampling frequency is 10kHz, and epileptic related signals are detected.
7. 7. The method of claim 6, wherein the electrophysiological testing is performed on young fish of the zebra fish at 5-6dpf, and the data is analyzed by DClamp software.
8. 8. A zebra fish reln gene knockout mutant, characterized in that the phenotype comprises a reduction in hindbrain area and whole brain area, which is constructed by the zebra fish gene knockout model and mutation verification construction method according to any one of claims 1-3.
9. 9. Use of the zebra fish reln gene knockout mutant of claim 8 in the study of neurological diseases to simulate abnormal neuronal migration or epileptic phenotypes caused by Reelin protein loss.
10. 10. The kit for constructing the zebra fish reln gene knockout model is characterized by comprising a CRISPR/Cas9 targeting component and a gene identification primer.

Description

Construction method and application of zebra fish reln gene knockout model Technical Field The invention relates to the field of genetic engineering and animal models, in particular to a construction method and application of a zebra fish reln gene knockout model. Background The RELN gene is located in the 2 region 2 band 1 sub-band (7q22.1) of the long arm of human chromosome 7, consisting of 65 exons. The coded protein Reelin is extracellular matrix glycoprotein secreted by Cajal-Retzius cells, has important regulation function on migration and localization of embryonic brain neurons, and affects formation of normal structures of cerebellum in cerebellum development by Reelin. After birth, reelin proteins are mainly secreted by GABA neurons and participate in dendritic and axon growth, and the formation and plasticity of synapses are regulated, so that learning and cognition are affected. Zebra fish (Danio rerio) is a tropical vertebrate model whose genome is highly homologous (about 70%) to humans, the nervous system is similar to humans, and the genome has been completely sequenced. Zebra fish development process corresponds to human presence time, 3 days after fertilization (3 dpf) corresponds to human birth time, and 4 dpf, 5 dpf, 6 dpf and 7 dpf correspond to 3 months, 6 months, 9 months and 12 months of age of infants, respectively. The development time sequence advantage combines with CRISPR/Cas9 and other efficient gene operation technologies and living body imaging capability, so that the zebra fish becomes an ideal platform for researching nervous system diseases. In the prior art, zebra fish gene editing tools are mature, but a systematic knockout model and a multidimensional phenotype verification method aiming at reln genes are not perfect, and particularly, comprehensive data is lacking in research on nerve dysplasia (such as brain structural change and epileptic-like activity) caused by Reelin deletion. Therefore, the invention provides a construction method and application of a zebra fish reln gene knockout model. Disclosure of Invention The invention aims to solve the defects in the prior art, and provides a construction method and application of a zebra fish reln gene knockout model. In order to achieve the above purpose, the present invention adopts the following technical scheme: A construction method of a zebra fish reln gene knockout model comprises the following steps: S1, targeting the 6 th exon of zebra fish reln gene by using CRISPR/Cas9 technology to generate reln chimeric mutant; S2, obtaining a reln homozygous mutant with stable passage through hybridization and selfing. Preferably, the CRISPR/Cas9 technology targets the 6 th exon sequence of reln genes. Preferably, the mutant identification includes gene extraction, PCR amplification and agarose gel electrophoresis analysis, sanger sequencing. A detection method of zebra fish reln gene expression pattern uses whole embryo in situ hybridization technology to detect reln expression in zebra fish embryo development stage with digoxin marked antisense probe. A method for observing the nerve morphology of zebra fish includes such steps as choosing the strain of zebra fish with Tg huc (GFP), imaging at 5dpf by microscope, and measuring the brain area, eye distance and body length by imageJ software. A zebra fish nerve electrophysiological test method uses a glass microelectrode to record local field potential from a visual top cover, the sampling frequency is 10kHz, and epileptic related signals are detected. Preferably, the electrophysiological test is performed on young fish of 5-6dpf zebra fish, and the data is analyzed by DClamp software. A zebra fish reln gene knockout mutant is constructed by a zebra fish gene knockout model and a mutation verification construction method, and the phenotype of the zebra fish reln gene knockout mutant comprises a reduction of hindbrain area and whole brain area. The application of zebra fish reln gene knockout mutant in research of nerve development diseases is used for simulating abnormal neuron migration or epileptic phenotype caused by Reelin protein deletion. A kit for constructing a zebra fish reln gene knockout model comprises a CRISPR/Cas9 targeting component and a gene identification primer. The beneficial effects of the invention are as follows: 1. the model of the invention can simulate the phenotype of human RELN related diseases and provides a platform for mechanism research and drug development. Drawings FIG. 1 is a graph of representative electrophysiological signals of zebra fish showing examples of mutant group epileptic related signals; FIG. 2 is a schematic diagram of a predicted protein truncated combination of a whole embryo in situ hybridization detection reln of the expression pattern result in zebra fish, a target point constructed by a knockout model, and a base deletion of a growth mutant reln -10/-10; FIG. 3 is a schematic diagram of the microscopic morphology observa