CN114868707B - Zebra fish model for metabolic encephalopathy and arrhythmia diseases and application thereof - Google Patents
Zebra fish model for metabolic encephalopathy and arrhythmia diseases and application thereof Download PDFInfo
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Abstract
The invention provides a zebra fish model for screening and improving metabolic encephalopathy and arrhythmia TRMEA disease drugs and a construction method thereof, and the zebra fish model takes zebra fish tango2 as a target gene, utilizes a genome editing technology, microinjects tango2 specific sgRNA and Cas9mRNA synthesized in vitro into fertilized eggs of wild zebra fish, and utilizes specific primers to screen generation by generation to obtain pure zygote mutant zebra fish with tango2 gene specificity knocked out. The invention can obtain genetically stable tan go2 deletion mutant zebra fish, and the mutant can be used for evaluating the treatment effect of various medicaments on TRMEA diseases by utilizing the transparency of the zebra fish embryo and the similar symptoms of death, arrhythmia, muscle damage, bradykinesia and the like of the mutant zebra fish juvenile fish to TRMEA diseases.
Description
Technical Field
The invention belongs to the technical field of animal model construction, and particularly relates to a drug screening zebra fish model, a construction method and application thereof, which can be used for screening drugs for treating metabolic encephalopathy and arrhythmia diseases.
Background
Metabolic encephalopathy and arrhythmic disease (TRMEA) are a new human genetic disease discovered in recent years, first reported in 2016. The initial onset time of the patient is between 2 months and 8 years, the main clinical symptoms comprise metabolic syndrome, encephalopathy, arrhythmia, hypoevolutism, rhabdomyolysis and the like, and the clinical symptoms are complex and various.
At present, no medicine specially used for preventing and treating metabolic encephalopathy and arrhythmia diseases exists, and the main reason is that the genetic disease is discovered in recent years, and relevant disease animal models and medicine screening methods are lacked. In order to develop a drug capable of accurately treating the metabolic encephalopathy and the arrhythmia diseases, the establishment of a stable, easy-to-operate and high-flux specific animal model is urgent, and the model is very important for promoting drug screening and subsequent drug effect inspection.
The zebra fish, as a common model animal, has a gene similarity as high as about 85% with human beings, has the advantages of convenient culture, short growth cycle, strong reproductive capacity, transparent embryo, convenient observation, simple administration mode and the like, and is widely used for biomedical scientific research of organ development, cardiovascular diseases, drug screening and the like. A metabolic encephalopathy and arrhythmia disease model is established by mutating zebra fish related genes, and the method has important value and clinical significance for screening TRMEA and related disease treatment drugs.
Disclosure of Invention
The invention aims to provide a zebra fish model for screening medicines for improving and treating metabolic encephalopathy and arrhythmia TRMEA (true Reynamic amplification) disease and a construction method thereof; thereby making up for the deficiencies of the prior art.
The invention firstly provides an application of the tan go2 gene, which is an application in preparing a metabolic encephalopathy and arrhythmia disease (TRMEA) zebra fish model;
the invention provides a method for constructing a zebra fish model with metabolic encephalopathy and arrhythmia diseases (TRMEA), which is characterized in that after mutation is carried out on the tan go2 gene of the fertilized egg of the zebra fish, the mutated fertilized egg is incubated to obtain the zebra fish model;
furthermore, the zebrafish model is a tan go2 gene homozygous mutant obtained by selfing.
Preferably, the mutation of the tan go2 gene is a gene editing operation performed on the second exon of the tan go2 gene;
the gene editing is specifically described as an embodiment, and is performed by a CRISPR-Cas9 system;
furthermore, when the CRISPR-Cas9 system is used for gene editing, a specific sgRNA is used, and the corresponding nucleotide sequence of the gRNA is GGAGCTACTAATGTACCTGT;
the invention also provides a method for screening a medicine for treating TRMEA (TRMEA), which is to screen the medicine by using the constructed tan go2 gene mutant zebra fish as a model animal.
The invention takes the zebra fish tango2 as a target gene, utilizes a genome editing technology, microinjects the in vitro synthesized tango2 specific sgRNA and Cas9mRNA in the fertilized eggs of wild zebra fish, and utilizes specific primers to screen generation by generation to obtain the pure mutant zebra fish with the tango2 gene specificity knocked out. The invention can obtain genetically stable tan go2 deletion mutant zebra fish, and the mutant can be used for evaluating the treatment effect of various medicaments on TRMEA diseases by utilizing the transparency of zebra fish embryos and the similar symptoms of death, arrhythmia, muscle damage, bradykinesia and the like of the mutant zebra fish juvenile fish to patients with the TRMEA diseases. The construction method of the zebra fish animal model has specificity and important physiological significance for screening TRMEA disease treatment drugs.
Drawings
FIG. 1: and (3) a phenotype map of 12-day-old young wild zebra fish and the tan go2 mutant zebra fish.
FIG. 2: and (4) a result graph of survival curves of the wild zebra fish and the tan go2 mutant zebra fish.
FIG. 3: behavioral result graphs of 12-day-old young wild zebra fish and the tan go2 mutant zebra fish.
FIG. 4: heart rate results and arrhythmia results of 12-day-old young zebra fish of wild zebra fish and the tan go2 mutant zebra fish are shown.
FIG. 5: results of muscle injury of 12-day-old young wild zebra fish and the tan go2 mutant zebra fish are shown.
Detailed Description
The construction method of the tan go2 gene mutant zebra fish model provided by the invention is to construct the tan go2 mutant zebra fish model through a CRISPR-Cas9 system; however, other conventional gene editing methods can be adopted to obtain the zebra fish tango2 gene mutant.
The invention relates to a specific method for constructing a zebra fish tango2 gene mutant, which comprises the following steps:
1) Construction of Tango2 mutant zebra fish
A target site gRNA sequence is designed and synthesized near a second exon by taking the zebra fish tango2 gene as a target sequence. And constructing an in vitro transcription template by using a pUC53 vector, and carrying out in vitro transcription, purification and recovery to obtain the sgRNA.
Wherein the zebra fish tango2 gene has a specific NCBI number of 445324 disclosed in the prior art, and contains 9 exons and 8 introns;
in the method of the present invention, it is also possible to mutate the tan go2 gene and cause a disease by performing gene editing in other exons or introns of the tan go2 gene.
The sgRNA and Cas9mRNA were co-microinjected into zebrafish zygotes for gene editing.
One specific sequence of the sgrnas is GGAGCTACTAATGTACCTGT, but other sgrnas can also have similar effects.
Culturing the fertilized eggs after injection, extracting genome DNA as a template, designing a primer according to the sequence of the tan go2, identifying and confirming the genotype of the zebra fish after injection, and screening to obtain the P0 generation of tan go2 mutant zebra fish.
Mating the P0 generation of the tan go2 mutant zebra fish with the wild zebra fish, collecting embryos, obtaining genome DNA, carrying out genotype identification, and screening to obtain stably inherited F1 generation of tan go2 heterozygous mutant zebra fish; the specific tango2 mutation site obtained was analyzed by sequencing genomic DNA.
Selfing the F1 generation of the tan go2 heterozygous mutant zebra fish, and screening to obtain the homozygous mutant F2 generation of the tan go2 mutant zebra fish. Selfing the F2 generation homozygous mutant zebra fish to obtain F3 generation homozygous zebra fish, and using the obtained F3 generation tango2 mutant zebra fish as a disease model for TRMEA disease drug screening.
the pure zebra fish obtained by selfing the tan go2 heterozygous mutant zebra fish can normally develop, but the progeny of the pure zebra fish selfing all die in the early development stage, so the disease model is preserved and maintained by the tan go2 heterozygous mutant.
The tango2 gene mutation zebra fish is used as an animal model for drug screening of metabolic encephalopathy and arrhythmia diseases.
The present invention will be described in detail below with reference to examples and the accompanying drawings.
Example 1 construction of a Tango2 mutant Zebra fish model
Comparing a human TANGO2 gene sequence on NCBI to obtain a zebra fish TANGO2 gene sequence, and designing and synthesizing a target gRNA sequence on a second exon of TANGO2 according to a CRISPR/Cas9 knockout method principle as follows: GGAGCTACTAATGTACCTGT.
And (3) pairing by using equivalent gRNAs to obtain gRNA primer double chains, constructing an in vitro transcription template by using a pUC57 vector, carrying out sgRNA in vitro transcription by using T7 RNA polymerase, further purifying and recovering to obtain the sgRNA.
The in vitro transcription system is: linearized template 2. Mu.g, 5 Xtranscription buffer 8. Mu.l, 10 Xdithionitol solution 4. Mu.l, 10 Xribonucleotide buffer 4. Mu.l, RNase inhibitor 2. Mu.l, T7 RNA polymerase 4. Mu.l, DEPC to 40. Mu.l system. The reaction was carried out at 37 ℃ for 4 hours.
And preparing a mixed working solution of the sgRNA and the Cas9mRNA, and injecting the mixed working solution into a zebra fish fertilized egg one-cell stage animal grade by microinjection, wherein the using ratio of the sgRNA to the Cas9mRNA is 1:5.
Culturing the fertilized eggs after injection, collecting more than 30 embryos after 48 hours, and extracting genome DNA. Designing genotype identifying primers according to the sequence of the tan go2, wherein the primers are respectively a forward primer: TAACTCACTGTCACATCTCTTC; reverse primer: GCATGATTCAGCATCCACTGGAC. Screening by PCR amplification and 2% agarose gel electrophoresis, and feeding to adult fish.
The genome DNA extraction steps are as follows: 1.0M Tris-HCl (pH 8.8), 0.5M EDTA (pH 8.0) and 20% Tween-20 tissue lysates were prepared as described for proteinase K: proteinase K was added to the lysate (V: V =1: 200), and 20. Mu.l of the extraction mixture was added to each Ep tube, and after lysis at 65 ℃ for 30 minutes, proteinase K was inactivated by treatment at 95 ℃ for 10 minutes.
The PCR amplification system is as follows: taking 95 deg.C 5min,95 deg.C 30s, 58 deg.C 30s, and 72 deg.C 1min as a cycle, performing 30 cycles, and finally 72 deg.C 10min.
And (3) extracting genome DNA by tail shearing, and carrying out PCR amplification screening to obtain a P0 generation mutant for successfully knocking out the tan go2 gene. Mating the P0 generation of the tan go2 mutant zebra fish with the wild zebra fish, collecting embryos, obtaining genome DNA, carrying out genotype identification, and screening to obtain the stably inherited F1 generation of the tan go2 mutant zebra fish.
Screening to obtain a heritable F1 generation mutant, extracting embryo RNA and carrying out reverse transcription to obtain cDNA, and designing a specific primer on a first exon, wherein:
a forward primer: 5'-ATGTGCATCATCTTCTTGAAGTTCGACC-3';
reverse primer: 5'-GGGGTGTTTAGCTCCTCGTTATTG-3';
and identifying whether the sgRNA works or not by using an RT-PCR amplification and sequencing method, and analyzing to obtain a specific tan go2 mutation site.
Selfing the F1-generation tan go2 mutant zebra fish, shearing the tail to extract genome DNA, and screening to obtain homozygous mutant F2-generation tan go2 mutant zebra fish, namely the tan go2 mutant zebra fish.
And reserving and feeding the heterozygous F2 generation of the tan go2 mutant zebra fish for breed conservation and obtaining the homozygous tan go2 mutant zebra fish.
Example 2 phenotypic analysis of Tango2 mutant Zebra fish
The method comprises the steps of enabling wild zebra fish and first-generation tan go2 homozygous zebra fish to be selfed respectively, collecting fertilized eggs, observing physiological characteristics of the zebra fish in multiple aspects such as development cycle, body shape, activity, righting reflection, swim bladder size and predation capacity, detecting the heart beat rhythm of the zebra fish 7 days after fertilization, detecting the heart beat frequency, movement tracks, muscle damage conditions and brain pathological forms of the zebra fish 12 days after fertilization, and verifying effectiveness of a TANGO2 gene mutation disease drug screening model.
The specific phenotypes are as follows:
1) Morphological observations of disease models: with the progress of growth and development, the tan go2 mutant zebra fish gradually has morbidity symptoms such as death and weakened righting reaction capability. The zebra fish is placed under a stereoscopic microscope through a groove glass slide and a 3% methyl cellulose solution, the posture is adjusted to enable the zebra fish to lie on the side, and the head, the body and the tail of the zebra fish are in the same horizontal plane, so that the zebra fish is suitable for observation and recording. Under the bright field condition, the appearance of tan go2 mutant zebra fish swim bladder atrophy and pericardial edema phenotype can be seen, and the phenotype result is shown in fig. 1.
2) Survival curve: after fertilization, 50 embryos are randomly selected from each group, cultured in an incubator at the constant temperature of 28.5 ℃ for 5 days, transferred to a juvenile fish culture box, and fed twice a day after transfer. The number of zebrafish deaths was recorded daily. The result shows that the tan go2 mutant zebra fish die from 5 days after fertilization and almost all the zebra fish die by 15 days, and the statistical result is shown in figure 2.
3) And (3) behavioral analysis: behavioral analysis was performed on zebrafish on day 12 post fertilization. Placing one zebra fish in each hole by using a 24-hole plate, stimulating the zebra fish under the light condition for 10 minutes after the zebra fish is in the dark condition for 10 minutes, circulating for 40 minutes in total twice, displaying the motion track condition of the zebra fish by infrared tracing, recording and analyzing the motion track and the activity of the zebra fish, repeating 8 juvenile fishes in each group, and analyzing three groups in total. The result shows that the motion capability of the tan go2 mutant zebra fish is obviously weakened, the trace tracing density and the activity are obviously reduced, and particularly the activity of the tan go2 mutant zebra fish is far lower than that of the wild zebra fish under the dark condition. The statistical results are shown in fig. 3.
4) Analysis of cardiac phenotype: and shooting 12-day-old zebra fish for 1 minute for video recording and counting the heartbeat times of the zebra fish per minute by using the phenotypic method of the disease model. The method comprises the steps of inhibiting melanin formation of the zebra fish by using phenylthiourea, shooting the position of the chest of the 7-day-old zebra fish for 30s of video, analyzing the arrhythmia index of the zebra fish by using Semi-automatic Optical Heart Analysis software, and analyzing the ventricular diastolic area of the zebra fish by using image-J software. The results show that the number of heart beats per minute of the tango2 mutant zebra fish is reduced, the arrhythmia index is increased, the ventricular diastole time is prolonged, the area is increased, and the statistical result is shown in fig. 4.
5) Muscle damage analysis: 12-day-old zebrafish were anesthetized with 1 × anesthetic for 1 minute, and after adjusting the posture with 3% methylcellulose, a polarizer on an Olympus IX83 microscope was adjusted so that only the muscle refracted the light and photographed. Meanwhile, the zebra fish is fixed for 24 hours by using 4% paraformaldehyde, dehydrated by 50%, 70%, 90% and 100% gradient ethanol, transparent by xylene, and embedded by paraffin for slicing after wax immersion. And selecting qualified paraffin sections for HE staining. The zebra fish fixed by 4% paraformaldehyde is treated by proteinase K, stained by 0.5% phalloidin for 30 minutes, and photographed by a super-resolution confocal laser scanning nano microscope. The results show that the muscle fiber morphology of the tan go2 mutant zebrafish is disrupted (fig. 5).
Example 3: application of tango2 mutant zebra fish
When the tango2 gene mutant zebra fish constructed in the example 1 is used for screening medicines for treating TRMEA diseases, medicines capable of improving or rescuing phenotypes such as death, arrhythmia, abnormal morphology and the like of young tango2 mutant zebra fish are target medicines.
Setting a wild type control group, a tan go2 mutant control group and a tan go2 mutant drug treatment group, adopting a 24-hole cell plate, placing 10 zebra fishes in each hole, and culturing at 28.5 ℃. The zebra fish breeding water is used for preparing a 5mM 3-pyridine carboxylic acid amide solution. The administration was carried out at a final concentration of 5 μ M at 1 day of age of zebrafish embryo, and changes in phenotype such as morphology, survival, behavior, heartbeat, etc. of the tango2 mutant zebrafish were observed by the method used in example 2 and recorded for 12 days. The results show that the time of the death symptoms of the tango2 mutant zebra fish is obviously delayed under the treatment of 3-pyridine carboxylic acid amide with the concentration of 5 mu M, and the 3-pyridine carboxylic acid amide solution has a certain relieving effect on the symptoms of the zebra fish model.
The described embodiments are preferred embodiments of the present invention, and alterations and modifications may be effected thereto by those skilled in the art, and it is intended that the appended claims be construed to include preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
Variations and modifications to the embodiments may occur to those skilled in the art without departing from the spirit and scope of the invention. Such changes and modifications are within the scope of the appended claims and their equivalents, and the present invention also includes these changes and modifications.
Claims (9)
1. The application of the tan go2 gene is characterized in that the application is the application in preparing a zebra fish model with metabolic encephalopathy and arrhythmia diseases.
2. A method for constructing a zebra fish model with metabolic encephalopathy and arrhythmia diseases is characterized in that the method comprises the steps of mutating a tan go2 gene of a fertilized egg of the zebra fish, and hatching the mutated fertilized egg to obtain the zebra fish model.
3. The method of claim 2, wherein said method is a homozygous mutation in the tango2 gene.
4. The method of claim 3, wherein said homozygous mutation is a homozygous mutant of the tan 2 gene obtained by selfing a tan 2 heterozygous mutant zebrafish.
5. The method of claim 2, wherein said mutation in the tan go2 gene is a gene editing in an exon or an intron of the tan go2 gene.
6. The method of claim 5, wherein said mutation in the tan go2 gene is a gene editing operation performed on the second exon of the tan go2 gene.
7. The method of claim 5, wherein the gene editing is gene editing using a CRISPR-Cas9 system.
8. The method as claimed in claim 7, wherein the CRISPR-Cas9 system uses sgRNA whose nucleotide sequence corresponding to the gRNA is GGAGCTACTAATGTACCTGT for gene editing.
9. A method for screening a drug for treating a TRMEA disease, which comprises screening a drug having a therapeutic effect using the tango2 gene mutant zebrafish constructed according to the method of claim 2 as a model animal.
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