CN114934069A - Construction method and application of double-mutant zebra fish line with kctd10 and tbx5a genes - Google Patents

Construction method and application of double-mutant zebra fish line with kctd10 and tbx5a genes Download PDF

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CN114934069A
CN114934069A CN202210760659.1A CN202210760659A CN114934069A CN 114934069 A CN114934069 A CN 114934069A CN 202210760659 A CN202210760659 A CN 202210760659A CN 114934069 A CN114934069 A CN 114934069A
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tbx5a
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祖尧
芮宇梦
赵霞
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Shanghai Ocean University
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Abstract

The invention discloses a construction method and application of a double-mutant zebra fish line with kctd10 and tbx5a genes, wherein a gRNA sequence is designed in the first exon of a tbx5a gene by using a CRISPR technology, and the gRNA sequence and a Cas9 protein are injected into a unicellular embryo of a spontaneous mutant of the zebra fish kctd10 gene, so that stably inherited double-mutant zebra fish with kctd10 and tbx5a genes are obtained by identification, screening and culture. the tbx5a gene mutant shows the symptoms like heart malformation of human psychotropic syndrome, and the double mutant zebra fish of kctd10 and tbx5a partially relieves the phenotype of abnormal cardiac cyclization and saves the ectopic expression of the atrioventricular compartment marker gene.

Description

Construction method and application of double-mutant zebra fish line with kctd10 and tbx5a genes
Technical Field
The invention belongs to the field of molecular biology, and particularly relates to a construction method and application of a kctd10 and tbx5a gene double-mutant zebra fish system.
Background
Congenital heart disease is the most common congenital defect, accounting for 30% of fetal fatality, and severe cardiac malformations in one child per 1000 newborns (Moore-Morris, 2018), still a major health problem. Hand syndrome is one of congenital heart diseases, the disorders of which are upper limb defects and congenital dysplasia of the heart (Baban, 2014), early studies found that hand syndrome is caused by mutations in the exon of TBX5 (Alessandro d. mori, 2004), Holt-Oram syndrome (HOS) is characterized by upper limb defects, congenital heart malformations and heart conduction diseases, approximately 85% of individuals with congenital heart diseases are caused by mutations that are pathogenic to TBX5, while diseased parents have a 50% probability of transmitting pathogenic mutations to offspring (McDermott, Deborah a.2004).
TBX5 has a wide role in the development of morphology and conduction systems of the heart as the first related mutant gene found in human hand-heart syndrome, and studies have shown that TBX5 is a core transcription factor of heart development and interacts with heart core transcription factors such as GATA4 and NKX2-5 to regulate the expression of other factors (McCulley DJ, Black BL.2012). In zebrafish, the tbx5 gene doubles to become the tbx5a/tbx5b gene, the effect of tbx5a in regulating the heart is dose sensitive, Albalat R et al demonstrate that tbx5a/tbx5b is functionally redundant, tbx5a is centrally distributed in the retina, heart and the pectoral fin of zebrafish, while tbx5b is distributed mainly in the retina, fin (Ricard Albalat, 2010). Deborah m.garrity et al constructed a disease model of the cardiopulmonary syndrome zebrafish by knocking out tbx5a to phenotypically enlarge the pericardial cavity, elongate the heart, malformation or deletion of the development of the upper limbs or pectoral fins of the mutant zebrafish (Deborah m.garrity, 2002). In 2014, researchers of Dazuo et al, the first author of the same, discovered a mutant of cardiac developmental deformity, and identified that the affected gene was kctd 10. The main mutant phenotypes of the traditional Chinese medicine are pericardial cavity swelling, abnormal heart contraction, abnormal formation of intervals between atria and ventricles, failure of torsion cyclization of the heart, heart malformation, and death of blood circulation stopping, and the fact that the kctd10 and tbx5a genes have an interaction relationship in cardiac development is found. The cardiac development of the zebra fish is regulated by multiple genes, and the kctd10 and the tbx5a play a vital role in the cardiac development, so that the zebra fish with the kctd10 and the tbx5a gene mutation is not only beneficial to researching the gene regulation network of the heart, but also beneficial to further understanding the pathogenesis of the heart-hand syndrome.
At present, CRISPR/Cas9 technology is commonly used for constructing single-gene mutants in zebra fish, and researches on constructing double-mutant and multigene mutant zebra fish by using the technology are few. The regulation network of the heart is complex, and the pathogenic cause of congenital heart diseases such as the heart-hand syndrome and the like is probably formed by multigene complex regulation, so that the establishment of a double-gene mutant by utilizing the CRISPR gene editing technology is necessary to research the pathogenic mechanism of the heart-hand syndrome. The CRISPR/Cas9 technology is used for carrying out gene knockout on zebra fish disease-related genes to construct a zebra fish disease model, and medicines related to diseases can be screened out more quickly. Drug screening of zebrafish is usually performed in live zebrafish embryos or larvae which exhibit multiple biological processes and possess an intact vertebrate organ system, and zebrafish can detect a broader phenotype than cultured cells (MacRae CA, Peterson RT, 2015).
In recent years, CRISPR makes it easy to obtain a mutant, and also causes a gene compensation effect on a knocked-out gene, and further, a phenomenon of a gene mutation phenotype cannot be observed. In 2015, the d.stainier group reported that no mutant phenotype was observed in the CRISPR gene knockout egfl7, but significant abnormalities in vascular defects were shown after knockdown of egfl7 morpholino, mainly due to activation of a compensatory network in the body to buffer deleterious mutations, which was not observed after translational or transcriptional knockout (Rossi, a., 2015). Therefore, besides CRISPR construction of mutants, MO knockdown genes are used for constructing animal models, and the study on gene dose effect is also significant.
At present, in a plurality of existing zebra fish models, a zebra fish double-mutant model about congenital heart diseases does not exist, and it is necessary to efficiently and accurately construct a disease model. The double mutant zebra fish constructed by the method not only shows the phenotype of heart and upper limb defects similar to those of HOS (human cardio-palmus) syndrome, but also has influence on the expression of important genes in the development of the heart. Therefore, the double mutant zebra fish of kctd10 and tbx5a constructed in the invention can further study the mechanism caused by HOS human beginner syndrome in the heart complex signal pathway, and can be used for screening related drugs of HOS diseases.
Disclosure of Invention
The invention mainly aims to provide a construction method of a Kctd10 and tbx5a gene double-mutant zebra fish line.
The invention also aims to provide application of the double mutant zebra fish line with the kctd10 and the tbx5a genes in constructing animal models and medicament screening related to the heart-hand syndrome of congenital heart disease.
In order to achieve the purpose, the invention adopts the technical scheme that:
the invention provides a construction method of a kctd10 and tbx5a gene double mutant zebra fish line, which comprises the following steps:
(1) designing a gRNA sequence shown as SEQ ID NO. 1 on a first exon of the tbx5a gene;
(2) tbx5a gRNA primers are designed and synthesized, and the sequences are shown as SEQ ID NO. 2 and SEQ ID NO. 3;
(3) carrying out PCR reaction by taking the gRNA primers and the gRNA-plasmid as templates, and purifying after detecting a PCR product by electrophoresis;
(4) the PCR purified product is subjected to in vitro transcription under the RNase-Free condition, and gRNA is obtained after purification;
(5) microinjecting the purified gRNA and Cas9 protein into a unicellular-stage embryo of the inside cross of the kctd10 mutant zebra fish obtained by spontaneous mutation, culturing the fish with successful knockout to an adult, detecting the knockout efficiency of tbx5a by using TAKARA T7E1 enzyme, detecting the mutation condition of the kctd10 by PCR, and culturing and growing the zebra fish with successful knockout as F0;
(6) when F0 zebra fish is in sexual maturity, hybridizing the mutant F0 zebra fish with WT wild zebra fish, and sequencing to obtain double mutant zebra fish which is bred and grown as F1;
(7) f1 pairing male and female zebra fish with double mutations, and selecting zebra fish with mutations in kctd10 and tbx5 a.
Preferably, in step (3), the conditions of the PCR reaction are: pre-denaturation at 94 ℃ for 3min, and entering three-step circulation: 34 cycles of 94-30 s, 65-30 s and 72-40 s, then 72-5 min, and finally keeping the temperature at 12 ℃.
Preferably, in step (5), the method for detecting the knockout efficiency of tbx5a comprises: and after 24h, taking 3 groups and 5 embryos in one group, putting the group into a PCR tube, extracting DNA by an alkaline cracking method, designing primers with sequences shown as SEQ ID NO. 4 and SEQ ID NO. 5 to carry out PCR amplification on the knockout sites to detect tbx5a knockout efficiency, and simultaneously designing primers with sequences shown as SEQ ID NO. 6 and SEQ ID NO. 7 to carry out PCR amplification to detect the mutation condition of kctd 10.
Preferably, in step (5), the conditions of the PCR reaction are: pre-denaturation at 94 ℃ for 3min, entering three-step circulation: 34 cycles of 94-30 s, 60-30 s and 72-40 s, then 72-5 min, and finally keeping the temperature at 12 ℃.
Preferably, in the step (7), the F1 double-mutated male and female zebra fish is confirmed by sexual maturity of F1 zebra fish after 3-4 months, cutting off the tail parts of the female fish and the male fish to extract tail fin DNA, carrying out PCR and T7E1 enzyme digestion to confirm whether mutation exists, and sequencing to confirm the mutation type.
kctd10 -/- Mutant zebrafish exhibit pericardial swelling, abnormal cardiac cyclization, head reduction and body length reduction; tbx5a -/- The mutant zebrafish has the phenotype that the pericardial cavity is abnormally swollen, the heart is elongated into thin lines, the normal cyclization is not realized, and a pair of pectoral fins are deleted, and the mutant zebrafish shows the symptoms of heart malformation similar to human hand syndrome. The double-mutant zebra fish with kctd10 and tbx5a obtained by the construction method partially relieves the phenotype of abnormal cardiac cyclization and simultaneously saves the ectopic expression of the atrioventricular compartment marker gene. In addition, occurrence is associated with kctd10 -/- Mutant zebrafish reduced head and body length phenotypes and tbx5a -/- Mutant zebrafish lack the same phenotype of a pair of fins.
The invention also provides application of the double mutant zebra fish line with the kctd10 and the tbx5a genes in constructing animal models and medicament screening related to the congenital heart disease hand-heart syndrome.
Compared with the prior art, the invention has the beneficial effects that:
(1) according to the invention, a Kctd10 and tbx5a gene double-mutant zebra fish system is constructed by a CRISPR/Cas9 technology, a gRNA sequence is designed on the first exon of tbx5a, tbx5a is knocked out by a microinjection method, a mature Kctd10 deletion mutant is mated, a purified gRNA of tbx5a and a Cas9 protein are microinjected into a zebra fish single-cell embryo of the Kctd10, a Kctd10 and tbx5a gene double-mutant zebra fish system obtained by screening offspring and a Kctd10 and tbx5a gene double-mutant zebra fish system obtained by internal crossing are constructed. the tbx5a gene mutant shows the symptoms like heart malformation of human psychohand syndrome, and the double mutant zebra fish of kctd10 and tbx5a partially relieves the phenotype of abnormal heart cyclization, and simultaneously saves the ectopic expression of the compartment interval marker gene, so that the double mutant can be used for researching the interaction relationship of kctd10 and tbx5a, the pathogenesis of congenital heart disease psychohand syndrome and screening the medicines related to the disease.
(2) Compared with the traditional gene editing technology, the CRISPR/Cas9 has the characteristics of low toxicity, high accuracy, high efficiency, short success period and the like, and has important significance in treatment of the cardiovascular and cerebrovascular syndromes and research of small-molecule medicines by successfully knocking out tbx5a genes on the spontaneous mutant of the zebra fish kctd10 gene to construct a double-mutant zebra fish animal model.
(3) According to the invention, morphholino is used for knocking down the kctd10 and tbx5a genes, and the interaction of the two genes in the early heart development process is verified, so that not only is the dose relationship between kctd10 and tbx5a found, but also the expression regions and expression amounts of important genes nppa, nppb and tbx2b in the early heart development regulation network are also influenced, and the fact that the double-mutant model has strong practicability and reliability in the research of pathogenesis of congenital heart-hand syndrome is shown.
(4) The invention screens out the micromolecule medicine which has relieving effect on mutant phenotype by using the double mutant zebra fish model of kctd10 and tbx5a genes, and the double mutant zebra fish model has unique advantages in medicine screening: the zebra fish egg-laying drug can be used as an effective animal model of the zebra fish with the psychotropic syndrome of human, the drug can be dissolved in culture water and effectively absorbed through the body surface and the gill part, the culture is convenient and fast, the high-throughput drug screening can be carried out on most eggs, and the drug is beneficial to screening the related drugs of the disease.
Drawings
FIG. 1 is a graph showing the knock-out pattern of tbx5a gene and the results of F0 and F1 knock-out assays after tbx5a knock-out; wherein, A is WT and tbx5a +/- Sum of hybrid Zebra Fish tbx5a -/- Sequencing of pure and zebrafish; panel B is a schematic of the first exon editing of the gene at tbx5a using CRISPR/Cas 9; panel C shows the results of the electrophoretic knockout, where M is marker, tbx5a F 0 1, 2, 3, 4, 5 and 6 in the KO electrophoresis band mean that tbx5a genotype is detected +/- And tbx5a F 1 Genotypes of 1, 3, 4 and 6 in the Screen electrophoresis band were WT, and genotypes of 2 and 5 were tbx5a -/- 。
FIG. 2 is a schematic flow chart of constructing double mutant zebra fish with kctd10 and tbx5a genes on the basis of spontaneously mutated kctd10 zebra fish.
FIG. 3 shows the heart phenotype results of the double mutant zebra fish remitting single mutant with kctd10 and tbx5a genes; in the figures (A-D) are photographs of the heart in bright field, where H refers to the heart and PC refers to the pericardial space; (E-H) is a fluorescence image of the heart of the zebra fish with four genotypes, and the zebra fish heart is drawn by lines; the (I-L) picture is a fluorescence picture of the blood vessels of the zebra fish, corresponding to the body segment of the zebra fish, and the (M-P) picture is a three-dimensional structural plan picture of the heart of the zebra fish, which is shot under a confocal microscope, wherein the green fluorescence marks are heart cells, and the red fluorescence marks are vascular endothelial cells; in the figure, V denotes the ventricle, AVC denotes the atrioventricular septum, and A denotes the atrium.
FIG. 4 shows the result that the expression levels of important heart genes nppa and nppb in the double mutant zebra fish with kctd10 and tbx5a genes are restored to normal levels; panels a and E are normal expression of nppa and nppb in wild-type WT zebrafish; in B and F, it can be seen that the two AVC marker genes are overexpressed throughout the heart of the kctd10 mutant; in the C and G plots, the expression of nppa and nppb is absent at AVC (AVC positions are indicated by black triangle arrows); in panels D and H, marker expression for two AVC reverts to wild-type-like expression, where black arrows in the panels indicate normal expression of markers nppa and nppb, white triangular arrows indicate at cardiac AVC where nppa and nppb are overexpressed, and black three-piece arrows indicate that expression of nppa and nppb is absent at AVC.
FIG. 5 is a graph of the dual morpholinos remitting single morpholinos heart abnormality phenotype results of zebrafish; wherein (A-D) is a picture of the lateral phenotype of WT and mo treated zebrafish in bright field; (E-H) is a lateral fluorogram of the four zebrafish hearts; (I-L) is the presence or absence of pectoral fins in a picture of the dorsal surface of the zebrafish under bright field conditions; (M-P) is a plan view of the three-dimensional structure of the zebra fish heart taken under a confocal microscope; (Q-X) promiscuous expression of nppb and tbx2b in mo.
Fig. 6 shows the effect of different concentrations of resveratrol on tbx5a mutant heart and myocardial wall thickness of double mutant zebrafish with kctd10 and tbx5a genes, wherein a is atrial, V is ventricular, and the myocardial wall thickness of heart has significant changes.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the drawings of the embodiments of the present invention. It is to be understood that the embodiments described are only a few embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the described embodiments of the invention without any inventive step, are within the scope of protection of the invention.
The primers used in the following examples were synthesized by Shanghai Biometrics, T7 vector was obtained from Beijing Urujin laboratories, Cas9 was obtained from Nanjing Kingsrey Biometrics, enzymes for reaction in PCR were obtained from Beijing Quanjin Biometrics, morpholino was obtained from Beijing Sizhengbo Biotech, resveratrol was obtained from Shanghai Biometrics, T7E1 enzyme for enzyme digestion detection was produced by TARAKA, Agarose was produced by Spanish Agarose Regula Agarose G-10, 100gram per bottle, NBT/BCIP was produced by Roche, fetal bovine serum FBS was obtained from Gibco brand. The rest inorganic (NaOH, Tris-HCl, etc.) and organic reagents (ethanol, dimethyl sulfoxide DMSO, etc.) are purchased from chemical reagent limited of national drug group, and the used zebra fish is a wild zebra fish AB strain from the zebra fish platform of Shanghai ocean university aquatic product and Life institute.
The technical route adopted in the following examples is as follows:
(1) designing tbx5a gRNA locus, PCR-purifying, in vitro transcription-purifying, injecting into a single-cell embryo of a kctd10 heterozygote in a microscopic way, detecting knockout efficiency, feeding into an adult, carrying out outcrossing with wild type zebra fish, detecting whether genetic mutation can be carried out, feeding offspring into adult fish, shearing tail and extracting genome, identifying whether mutation exists and mutation type, carrying out internal crossing on double-heterozygote mutant zebra fish to obtain the homozygous zebra fish of the kctd10 and the tbx5a double mutants, homozygous lethal-obtaining heterozygous tbx5a mutant zebra fish, heterozygous kctd10 mutant zebra fish and double mutant zebra fish of kctd10 and tbx5 a-offspring is raised to adult fish-tailed to identify whether the zebra fish is double mutant zebra fish-screening the double mutant zebra fish of kctd10 and tbx5 a-to obtain the double mutant zebra fish of kctd10 and tbx5a which can be stably inherited.
The double-mutant zebra fish with the stable inheritance, namely the kctd10 and the tbx5a, are subjected to internal crossing, the zebra fish with different genotypes is selected according to the phenotype, and the pictures and nppa and nppb in-situ hybridization experiments are observed under a fluorescent microscope.
(2) Preparing a microinjection glue injection needle, preparing tbx5a mo and kctd10mo reagents, microinjecting, obtaining the knockdown zebrafish of kctd10 and tbx5aMO, phenotype observation and in situ hybridization experiments.
(3) Grouping the acquired stably inherited double-mutant zebra fish internal crossing-embryos of kctd10 and tbx5a, preparing resveratrol solutions and DMSO (dimethyl sulfoxide) solutions with different concentrations, soaking in resveratrol concentration gradient drugs, observing phenotypes, and acquiring data such as heart rate, heart phenotype, cyclization angle and the like of an optimal concentration-recording statistical experimental group and a control group.
The following examples are methods for obtaining double mutant zebrafish with kctd10 and tbx5a genes by CRISPR/Cas9 technology (fig. 1 and 2), and the steps are as follows:
(1) the genomic sequence of zebrafish tbx5a was downloaded at NCBI and the gRNA sequence was designed at the first exon of the tbx5a gene with a target sequence of P1: 5'-TTCGGCTCCAAAACTCTCCC-3' (SEQ ID NO: 1).
(2) After the gRNA target was found, the BLAST function of NCBI (https:// www.ncbi.nlm.nih.gov) was used to test the specificity of the target, primers for the target were synthesized, tbx5a gRNA primers were designed and synthesized using Primer5.0 software on NCBI with the primer sequence F1: 5 'TAATAC GACTCACTATATTCGGCTCCAAAACTCTGTTTTAGAGCTAGAAATAGC-3' (SEQ ID NO:2), R1: 5'-AAAAAAAGCACCGACTCGGTGCCAC-3' (SEQ ID NO:3), where the target sequence would be in reverse, http:// www.bioinformatics.org/sms/rev _ comp.htmL could be used to change the reverse sequence to forward.
(3) The primers and the gRNA-plasmid are used as templates for carrying out PCR reaction, and the reaction system is as follows:
Figure BDA0003724201240000061
wherein the gRNA-plasmid is obtained from the literature: chang N, Sun C, Gao L, Zhu D, Xu X, Zhu X, Xiong JW, Xi JJ. genome editing with RNA-guided Cas9 nucleic in zebrafish embryo, Cell Res, 2013, 23(4): 465-. The PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 3min, and entering three-step circulation: 34 cycles of 94-30 s, 65-30 s and 72-40 s, then 72-5 min, and finally keeping the temperature at 12 ℃; after detecting the PCR product by electrophoresis, the PCR product is purified by using a DNA purification kit and eluted and dissolved by DEPC water.
(4) Under the RNase-Free condition, the PCR purified product is subjected to in vitro transcription to obtain gRNA, and the transcription system is as follows:
Figure BDA0003724201240000071
the reaction conditions are as follows: after 37-1 h, 1 mu L DNase is added for 37-15 min. gRNA was purified by LiCl precipitation after in vitro transcription: gRNA was precipitated by adding 2.5. mu.L of 4M LiCl, and then 100. mu.L of 100% ethanol was added. Incubating in a freezer at-80 ℃ for at least 2 hours (overnight treatment is also possible); centrifuge at 12000rpm/min for 15min at 4 ℃. Removing supernatant, and washing with precooled 70% ethanol twice by turning upside down in order to remove impurity interference; ventilating at room temperature in a super clean bench, and air drying for 5 min. Then adding 15 mu L of RNase-free water for dissolving, and detecting the concentration by using the Nanodrop.
(5) The purified gRNA and the Cas9 protein are mixed and then injected into zebra fish embryos at the single cell stage of kctd10 in a micro-injection mode, and the final concentration of the Cas9 protein is 800ng/uL, the final concentration of the gRNA is 100ng/uL and 1 nl/piece is injected. And (3) taking 3 groups after 24 hours, putting a group of 5 embryos into a PCR (polymerase chain reaction) tubule, and extracting DNA by an alkaline cleavage method: adding 30 μ L of 50mM NaOH solution, taking out after 95-10 min, fully shaking and breaking the tissue on a shaking instrument, continuing to shake and break the tissue for 95-10 min, adding 3 μ L of 1M Tris-HCl (pH 8), and centrifuging at 10000rpm/min-5 min.
Design primer F2: 5'-CGGGCGTCTTGAAGTGATG-3' (SEQ ID NO:4), R2: 5'-TCGAGGCACTAG TTGACGTT-3' (SEQ ID NO:5), and performing PCR amplification on the knockout site by using the primers, wherein the reaction system is as follows:
Figure BDA0003724201240000072
the PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 3min, and entering three-step circulation: 34 cycles of 94-30 s, 58-30 s and 72-40 s, then 72-5 min, and finally keeping the temperature at 12 ℃. Then TAKARA T7E1 enzyme is used for carrying out knockout efficiency detection, the batch of injected group small fishes which are successfully knocked out are fed to grow up and used as F0, and the T7E1 enzyme digestion detection reaction system is as follows:
Figure BDA0003724201240000073
Figure BDA0003724201240000081
the reaction conditions are as follows: 95-30 s, 85-5 min, 4 ℃ infinity, adding 0.25uL of T7E1 enzyme after reaction, and carrying out water bath at 37 ℃ for 45 min.
(6) And F0 sexual maturity of the zebra fish after 3-4 months, hybridizing the mutated zebra fish with WT wild zebra fish to obtain heterozygote F1 with a certain probability, taking embryos, extracting DNA according to the alkaline cracking method, carrying out PCR amplification on the knock-out sites by using the primers, detecting the mutation efficiency by using T7E1 enzyme, sending sequencing confirmation, and culturing and growing the mutated zebra fish.
(7) F1 zebra fish sexual maturity after 3-4 months, cutting the fish tails of female fish and male fish to extract tail fin DNA, carrying out PCR and T7E1 enzyme digestion to confirm whether mutation exists, and confirming the mutation type through sequencing. And (3) pairing the mutated male and female zebra fish to obtain a tbx5a knockout mutant, a kctd10 homozygous mutant. The tbx5a homozygous mutant is observed to die 6-7 days after fertilization, and the kctd10 heterozygous mutant and tbx5a and kctd10 double-mutation homozygous offspring die at 5dpf, finally obtaining tbx5a and kctd10 heterozygous mutant.
(8) And (3) mating male and female mutants of tbx5a and kctd10 after obtaining the tbx5a heterozygous mutant to obtain the double mutant heterozygous zebra fish F3 of the kctd10 and the tbx5a with certain probability. After 3-4 months, tail fin DNA extraction was performed on the tail excision of F3 zebra fish using primers F2: 5'-CGGGCGTCTTGAAGTGATG-3' (SEQ ID NO:4), R2: 5'-TCGAGGCACTAG TTGACGTT-3' (SEQ ID NO:5) the PCR procedure described above was followed to detect mutations in tbx5a and primer F3: 5'-TGTGTGCTTCTGCAACATCCT-3' (SEQ ID NO:6), R3: 5'-GACACTCATCTACCAGGCCC-3' (SEQ ID NO:7) was used to detect mutations in kctd10 by PCR in the following reaction system:
Figure BDA0003724201240000082
the reaction procedure is as follows: pre-denaturation at 94 ℃ for 3min, entering three-step circulation: 34 cycles of 94-30 s, 60-30 s and 72-40 s, then 72-5 min, and finally keeping the temperature at 12 ℃. The PCR product is then sent to sanger sequencing for detection of specific mutation conditions. Zebrafish with mutations generated both in kctd10 and tbx5a were selected, i.e., zebrafish heterozygous for the double mutants, kctd10 and tbx5 a. Further, selfing the double-mutant heterozygous zebra fish of the kctd10 and the tbx5a to obtain F4, observing that the homozygous mutant offspring has death phenomenon, and finally obtaining the double-mutant heterozygous zebra fish of the kctd10 and the tbx5 a.
In situ hybridization experiments: firstly, in embryos obtained by internal crossing of double mutant zebra fish embryos of kctd10 and tbx5a, 24 hours after fertilization, a prepared PTU solution is added to inhibit pigment generation of zebra fish, the zebra fish embryos for the time required by the experiment are collected in a 2mL centrifuge tube, the collected embryos are rinsed by PBST, the embryos are fixed by 4% paraformaldehyde, and then the embryos are fixed on a shaker at 4 ℃ overnight. The embryo is taken out for dehydration the next day, and the dehydration steps are as follows: PBST 5min, 25% ethanol 5min, 50% ethanol 5min, 75% ethanol 5min, and 100% ethanol 5 min.
The first day: dehydrated embryos were removed and rehydrated from 100% ethanol to PBST. The rehydration steps are as follows: 5min of 75% ethanol, 5min of 50% ethanol, 5min of 25% ethanol and 5min of PBST, preparing proteinase k solution to digest embryos, and selecting different digestion times according to the development time of the embryos.
After digestion, adding 4% paraformaldehyde to fix the embryo, and placing in a shaking table at room temperature for 20 min; after fixation, the solution is rinsed for 5min by PBST for 5 times; adding 400 μ L of prehybridization solution preheated at 65 deg.C, and prehybridization in a prehybridization furnace at 65 deg.C for 2-3 hr; the prehybridization solution HYB was aspirated, the probe solution preheated to 65 ℃ was added, and hybridization was carried out overnight in a prehybridization oven.
The following day: sucking away the probe solution, preparing the prehybridization solution and the 2XSSC solution into a gradient washing solution, and rinsing the embryo at 65 ℃ for 15min each time; rinsing twice by preparing 0.2XSSC after the final gradient is rinsed, wherein the rinsing time is half an hour each time; the embryos are then rinsed using a gradient wash formulated with 0.2xSSC and PBST at room temperature. Adding fetal bovine serum BSA and lamb serum into PBST to prepare a blocking solution, and blocking the embryos for 3-4 hours at room temperature after adding the blocking solution. Antibody solutions were prepared by adding digoxin antibody to the blocking solution, added to the embryos, and incubated with antibody overnight (>12h) on a shaker at 4 ℃.
And on the third day: and (3) taking out a sample, rinsing for 5 times by using a PBST solution, wherein the rinsing time is 15min each time, preparing a BCIP/NBT chromogenic solution, performing signal chromogenic on the embryo at room temperature, and finally stopping the chromogenic reaction by using PBST.
Example 2
In this embodiment, the accuracy of double-mutant zebrafish construction and the dose regulation relationship between the double-mutant zebrafish and the double-mutant zebrafish are verified by knocking down kctd10 and tbx5a genes through morpholino, which comprises the following specific steps:
preparing 1% agarose gel for injection and injection needle one day ahead, placing WT-male and female zebra fish in fish tank, dissolving morpholino in water, and using RNase Free ddH 2 O A1 mM morpholino solution was prepared and stored at room temperature under light-shielding and sealing conditions. Removing the baffle plate in the morning, taking a part of embryos as a control group within half an hour after the zebra fish spawns, injecting a prepared morpholino solution into the other part of embryonic animals, wherein three combinations of injected morpholino concentration are favorable for the generation of phenotype, namely injecting 4ng of kctd10mo to generate and generate a control group similar to kctd10 -/- Similar phenotype, 5ng tbx5amo produced tbx5a -/- Similar phenotype, and 6ng kctd10mo and 3ng tbx5amo appeared similar to that of the double mutant. And placing the injected embryos and the control group embryos in an incubator at 28.5 ℃ for culture, picking out dead eggs after 12h, replacing culture water, observing the phenotype of the zebra fish after 3dpf, and carrying out in-situ hybridization experiments on important genes nppb and tbx2b related to cardiac development.
Example 3
In the embodiment, the effect of resveratrol on double-mutant zebra fish with kctd10 and tbx5a is used for verifying the drug screening effect, and the specific steps are as follows:
mating the female and male zebra fish of the kctd10 and the tbx5a double mutant finally obtained in example 1, dividing the embryos in 24h of fertilization into one group by 30, setting three groups of experiments of the resveratrol experimental group and the DMSO control group, and adding 30mL of resveratrol solution and DMSO solution with different concentrations into each plate.
(1) Preparing a resveratrol solution: the mass (mg) to be weighed by a precision weighing balance (concentration (mM) × volume (mL) × molecular weight (g/mol)) can be calculated from the formula according to the volume and concentration of the resveratrol required. For example, 100mL of a 100. mu. mol resveratrol solution was prepared, 2.28mg of resveratrol was weighed based on the molecular weight 228 using a precision weighing balance and dissolved in 1mL of the resveratrol solution, and 99mL of E3 solution was added. The gradient solution of resveratrol with concentration of 10 mu mol, 100 mu mol, 1mmol and 10mmol is also calculated according to the formula, and the resveratrol with lower concentration can be diluted and prepared with the resveratrol with high concentration.
Preparing a DMSO solution: 1mL of DMSO was dissolved in the same volume of E3 solution.
E3 solution preparation: 5mM NaCl, 0.17mM KCl, 0.33mM CaCl 2 0.33mM MgSO 4 。
(2) Embryos soaked in resveratrol and DMSO solutions for three days after fertilization are picked out under a visual microscope according to phenotypes of three mutants, namely a kctd10 homozygous mutant, a tbx5a homozygous mutant and a kctd10 and tbx5a homozygous mutant, and data such as zebrafish heart morphology, zebrafish heart rate, cyclization angle and death time of each mutant of an experimental group and a control group are observed and analyzed.
(3) After analyzing the data, each group obtained different types of mutants according to the phenotype, each mutant was subjected to DNA extraction using tbx5a primer F2: 5'-CGGGCGTCTTGAAGTGATG-3' (SEQ ID NO:4), R2: 5'-TCGAGGCACTAG TTGACGTT-3' (SEQ ID NO:5) and kctd10 primer F3: 5'-TGTGTGCTTCTGCAACATCCT-3' (SEQ ID NO:6), R3: 5'-GACACTCATCTACCAGGCCC-3' (SEQ ID NO:7) PCR was performed to detect mutations in each fish, using the following specific protocol: pre-denaturation at 94 ℃ for 3min, entering three-step circulation: 34 cycles of 94-30 s, 60-30 s and 72-40 s, then 72-5 min, finally preserving the temperature at 12 ℃, sending the PCR product to sanger sequencing for detecting the specific mutation condition. According to the sequencing result, juvenile fish with more phenotype selection corresponds to genotype mutation conditions, and the influence of resveratrol on the myocardial wall thickness, cyclization angle and the like of the tbx5a mutant can be known, so that the kctd10 and tbx5a double mutants have the function of screening small molecule drugs related to the hand syndrome.
Results
The kctd10 and tbx5a double mutants can relieve the heart abnormality caused by tbx5a or kctd10 mutation, compared with the heart abnormality elongation caused by tbx5a mutation and the heart cyclization abnormality caused by kctd10 mutation, the hearts of the kctd10 and tbx5a double mutants can make up for the defects of the single mutants, and the heart cyclization angle and elongation are restored (figure 3). In situ hybridization experiments show that the expression of some important marker genes of cardiac development is under the same regulation and control effect as the phenotype of the heart, and the double mutant zebra fish of kctd10 and tbx5a partially relieves the phenotype of abnormal cardiac cyclization and saves the ectopic expression of the atrioventricular interval marker genes (figure 4). Similar results were obtained by morpholinos experiments, and the double mutant morpholinos of kctd10 and tbx5a can relieve the heart abnormality caused by tbx5a or kctd10 morpholinos, and knock-down is performed by using mo with different doses, which indicates that the tbx5a or kctd10 gene regulates the heart development network through expression of different doses, thereby influencing the development of the heart (fig. 5). The obtained double mutant model was treated with resveratrol at different concentrations, and found to have an effect on tbx5a gene-mutated zebra fish, and treatment of tbx5a mutant zebra fish with 10 μmol of resveratrol partially relieved the abnormal heart phenotype, increased myocardial wall thickness and restored cardiac cyclization (fig. 6).
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Sequence listing
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Construction method and application of <120> kctd10 and tbx5a gene double-mutant zebra fish line
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Claims (8)

  1. The method for constructing the double-mutant zebra fish line with the kctd10 and the tbx5a genes is characterized by comprising the following steps of:
    (1) designing a gRNA sequence shown as SEQ ID NO. 1 on a first exon of a tbx5a gene;
    (2) tbx5a gRNA primers are designed and synthesized, and the sequences are shown as SEQ ID NO. 2 and SEQ ID NO. 3;
    (3) carrying out PCR reaction by taking the gRNA primers and the gRNA-plasmid as templates, and purifying after detecting a PCR product by electrophoresis;
    (4) the purified PCR product is subjected to in vitro transcription under the RNase-Free condition, and is purified to obtain gRNA;
    (5) injecting the purified gRNA and Cas9 protein into a unicellular embryo of zebra fish kctd10 gene spontaneous mutation in vivo crossing by a micro-injection method, knocking out the knocking-out efficiency of tbx5a by T7E1 enzyme digestion detection after successful fish culture to adult, detecting the mutation condition of kctd10 by PCR, and feeding and growing the zebra fish with successful knocking-out as F0;
    (6) f0 hybridizing the matured zebra fish with wild zebra fish, and determining that the zebra fish with double mutations is bred and grown up as F1 by sequencing;
    (7) and (3) matching the F1 double-mutation male and female zebra fish, and selecting the Kctd10 and tbx5a double-gene mutation zebra fish to obtain the double-mutation zebra fish.
  2. 2. The method for constructing the double mutant zebrafish line with the kctd10 and tbx5a genes as claimed in claim 1, wherein in steps (3) and (5), the PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 3min, and entering three-step circulation: 34 cycles of 94-30 s, 65-30 s and 72-40 s, then 72-5 min, and finally keeping the temperature at 12 ℃.
  3. 3. The method for constructing a double mutant zebrafish line with kctd10 and tbx5a genes according to claim 1, wherein the method for detecting tbx5a knockout efficiency in step (5) comprises: extracting DNA of zebra fish embryos by an alkaline lysis method, designing primers with sequences shown as SEQ ID NO. 4 and SEQ ID NO. 5 to perform PCR amplification on a knockout site to detect tbx5a knockout efficiency, and designing primers with sequences shown as SEQ ID NO. 6 and SEQ ID NO. 7 to perform PCR amplification to detect the mutation condition of kctd 10.
  4. 4. The method for constructing the double mutant zebra fish line with the kctd10 and the tbx5a genes as claimed in claim 1, wherein in step (7), after the sexual maturity of the F1 zebra fish, the tails of the female fish and the male fish are partially cut off, tail fin DNA extraction is carried out, PCR and T7E1 enzyme digestion are carried out to confirm whether the mutation occurs, and sequencing is carried out to confirm the mutation type.
  5. 5. The method for constructing the double mutant zebra fish line with the kctd10 and the tbx5a genes according to any one of claims 1-4, wherein the double mutant zebra fish with the kctd10 and the tbx5a genes shows partial alleviation of the phenomena of lumen swelling in the center of single gene mutation and abnormal heart cyclization degree.
  6. 6. Use of a double mutant zebrafish line having kctd10 and tbx5a genes, obtained by the method for constructing the double mutant zebrafish line having kctd10 and tbx5a genes according to any one of claims 1 to 5, for constructing an animal model associated with congenital heart disease hand-heart syndrome.
  7. 7. The application of the double-mutant zebra fish line with the kctd10 gene and the tbx5a gene in drug screening is obtained by the construction method of the double-mutant zebra fish line with the kctd10 gene and the tbx5a gene disclosed by any one of claims 1 to 5.
  8. 8. The use of claim 7, wherein the drug is a potential small molecule drug for the treatment of hand-heart syndrome.
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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110484549A (en) * 2018-04-20 2019-11-22 北京大学 Genome targeting modification method

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110484549A (en) * 2018-04-20 2019-11-22 北京大学 Genome targeting modification method

Non-Patent Citations (3)

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Title
XIANGJUN TONG等: "Kctd10 regulates heart morphogenesis by repressing the transcriptional activity of Tbx5a in zebrafish", NATURE COMMUNICATIONS, pages 5 *
丰昀;张敏;彭文婷;李映蓉;杨倩;蒋乐龙;王光伟;: "KCTD10基因干扰RNA慢病毒载体的构建", 湖南师范大学学报(医学版), no. 02 *
白金尊: "斑马鱼tbx5a基因在心脏发育过程中与kctd10及nkx2.5之间作用机制的初步研究", 中国优秀硕士学位论文全文数据库 医药卫生科技辑(月刊), no. 06, pages 20 - 23 *

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