CN114480497B - Construction and application method of ep400 gene knockout zebra fish heart failure model - Google Patents
Construction and application method of ep400 gene knockout zebra fish heart failure model Download PDFInfo
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Abstract
The invention relates to the technical field of gene knockout, in particular to a gene knockout geneep400Construction and application methods of gene knockout zebra fish heart failure model. The invention utilizes CRISPR/Cas9 gene editing technology, designs 2 unique targeting sites at the 11 th and 12 th exons, knocks out 162bp fragments between the two targeting sites, breeds outep400Knocking out homozygotes, so as to construct a zebra fish model for heart failure of zebra fish; in addition, the invention also disclosesep400The mutant phenotype of zebra fish is knocked out. One aspect of the invention can be used for exploringep400The biological function of the gene in vivo is disclosed for the first time on the other handep400Genes have relevance to heart failure for future investigationep400Leading to heart failure mechanism research and screening of related drugs to provide an ideal animal experimental model.
Description
Technical Field
The invention relates to the field of gene knockout, in particular to a method for preparing a gene expression vectorep400Construction and application methods of gene knockout zebra fish heart failure model.
Background
ep400The (E1A binding protein p) gene is located on the No. 8 chromosome of zebra fish and contains 52 exons, the full length of cDNA is 9126bp, EP400 is an SWR1 ATP-dependent chromatin remodelling protein, and consists of a histone acetylation Tip60/p400 complex with Tip60, and participates in transcription regulation of various genes, and plays a role in inhibiting apoptosis or aging in cell process, but no role exists so farep400And reports related to the occurrence and development of heart diseases.
The zebra fish has high conservation with the genes and signal paths in the heart development process of human beings; the zebra fish and mammal have the advantages of similarity of heart, small individual, easy breeding, rapid development, strong reproductive capacity, in vitro fertilization, in vitro embryo development, transparency and the like, and meanwhile, the physiological and pathological characteristics of the zebra fish are very similar to those of human beings, so that the zebra fish can be widely applied to the fields of disease research, genetic development, drug screening and the like.
The gene editing technology is one of the most main technologies in the current animal model establishment, gene function and disease treatment research. The CRISPR/Cas9 technology is used as a novel gene editing technology, has the advantages of simple operation, low cost, high efficiency and the like, and can be widely applied to multiple species, so that the research on gene functions and disease models is greatly promoted. The technology mainly changes the genetic information of organisms by means of deletion mutation, gene inactivation, chromosome large fragment deletion, exogenous gene introduction and the like, and expresses mutation characters after stable inheritance in a germline, and has wide application value in researching the action of specific genes in organisms in the growth and development process and constructing disease models, so the technology has become a research hot spot of modern molecular biology.
The CRISPR/Cas9 gene editing system consists of guide RNA (called gRNA for short) and Cas9 protein, wherein the gRNA sequence comprises a section of 20bp fragment complementarily matched with a DNA fragment target sequence and another section of sgRNA sequence for recruiting the Cas9 protein to perform a shearing function; cas9 protein is an RNA-mediated nuclease that cleaves a specific DNA sequence of a gene of interest through the mediation of gRNA; the point to be added is that when the Cas9 protein plays a shearing role, it needs to identify a PAM sequence (i.e., NGG) of a triplet base, then shear the upstream 3-8 bases of the PAM sequence, the organism itself will excite the DNA damage repair mechanism of the organism itself, and during the repair process, the target sequence position will cause insertion or deletion of the base, so as to realize editing of the target gene.
Disclosure of Invention
The invention knocks out zebra fish through specific gRNA combination based on CRISPR/Cas9 gene editing technologyep400Construction of a 162bp fragment between exons 11 and 12 of the Gene (wherein the exon is 76bp and the intron is 86 bp)ep400A knockout line; deep investigation of in vivo using fluorescence signal changes in homozygotes of cardiac transgenic fluorescent linesep400Biological function of the Gene, the mutant phenotype of heart failure was determined by observing and analyzing changes in the morphology, heart beat and cellular tissue structure of the zebra fish, thereby constructingep400Gene knockout zebra fish heart failure model. This is also the first time proposedep400The gene has correlation with the occurrence of heart failure diseases, and has important significance on the pathogenesis and clinical medication of heart failure diseases.
The technical scheme for solving the technical problems is as follows:
1) CRISPR/Cas9 technologyep400Synthesis of gRNA at Gene targeting site
(1) Analysis by NCBI and Ensembl on-line softwareep400Gene exon 11 and 12 sequencesDesigning a primer for synthesizing a target site according to a CRISPR/Cas9 technology target site design principle;
(2) To linearize plasmidsp42250 is template, and the designed specific target site PCR primer is used for PCR amplification;
(3) After DNA fragment purification and recovery, in vitro transcription and RNA purification are carried out to obtain gRNA (1F and 2F)
2) Microinjection is carried out on an injection system composed of Cas9 protein and gRNA
(1) Premixing Cas9 protein and gRNA system, and debugging a microinjection device for later use;
(2) Selecting normal wild zebra fish embryos which develop to a single cell stage, placing the normal wild zebra fish embryos on an injection plate configured by agarose gel, and injecting a certain amount of premix into single cells of the embryos by a microinjection instrument;
(3) Culturing the injected embryo for subsequent experiments
3) Zebra fishep400Gene targeting site gRNA effectiveness detection
(1) Randomly selecting 3 tubes of the injected embryo and the uninjected embryo of the control group after the injected embryo grows for about 48 hours, adding a proper amount of lysate into 8 embryos of each tube, and performing overnight pyrolysis at 65 ℃ to obtain genome DNA;
(2) The genome DNA is used as a template,ep400PCR amplification with detection-F/R as primer, analysis of the target fragment by agarose gel electrophoresis after the reaction, and detection of the target site availability by Sanger sequencing of the PCR product
4) Zebra fishep400Gene F0 generation chimera screening
Culturing the rest injected embryo to sexual maturity, genotyping, screening F0 generation chimeric
5) Zebra fishep400Gene F1 generation gene knockout heterozygote screening
Hybridizing the obtained F0 generation chimeric with wild zebra fish of different sexes, detecting hereditary property of the obtained offspring embryo, and breeding hereditary offspring until adult fishep400Gene F1 generationGene knockout heterozygous mutant
6) Zebra fishep400Gene F2 generation gene knockout homozygote screening
Mating F1 generation heterozygote adult fish with the same genotype (the ratio of male to female is 1:1) to obtain F2 generation embryo, and breeding the embryo until adult fish, and then carrying out genotype identification to obtainep400Gene F1 generation gene knockout homozygote
7) Zebra fishep400Gene knockout homozygote centrality failure mutation phenotype observation and analysis
Mating F1 heterozygote adult fish with zebra fish heart transgene marker strain (such as Tg (cmlc 2: EGFP), tg (cytoer: EGFP), etc.), to obtain stable genetic carrier fluorescent markerep400The heterozygote of F1 generation is then mated with the heterozygote of F1 generation without fluorescent mark, the cultured embryo is collected and developed to a certain period, and the heart development of the embryo is observed and photographed under a confocal microscope.
The beneficial effects of the invention are as follows:
the invention knocks out zebra fish through specific gRNA combination based on CRISPR/Cas9 gene editing technologyep400Construction of a 162bp fragment between exons 11 and 12 of the Gene (wherein the exon is 76bp and the intron is 86 bp)ep400The gene knockout strain is used for observing and analyzing the variation of the fluorescence signal of the heart transgenic fluorescent strain in homozygote to determine the mutation phenotype of heart failure, thereby constructingep400Gene knockout zebra fish heart failure model. The invention can help to explore in vivoep400The biological function of the gene is also proposed for the first timeep400The gene has correlation with the occurrence of heart failure diseases, which has profound significance for heart failure pathogenesis and clinical medication.
Drawings
FIG. 1 is a zebra fishep400Construction of a gene targeting site structure;
FIG. 2 is a zebra fishep400A gene knockout strain genotype identification result diagram;
FIG. 3 is a wild typeControl group WT and zebra fishep400Aligning gene sequences of gene homozygotes;
FIG. 4 is a zebra fishep400Gene homozygote heart mutation phenotype result graph
Detailed Description
The invention will now be described in detail with reference to the drawings and the accompanying specific examples.
Example 1:
1) Respectively designing CRISPR/Cas9 gene knockout target site and detection primer
Querying zebra fish on National Center for Biotechnology Information (NCBI)ep400Genomic DNA sequences of genes were designed on the website The ZiFiT Targeter (http:// ZiFiT. Org/ZiFiT /) according to CRISPR/Cas9 knockout principleep400Targeting sites of the gene (1 targeting site is selected in each of the 11 th and 12 th exon regions);
specific target site PCR primers were as follows:
f1 (Forward primer)
tgtaatacgactcactataggacctcgctgtcagatgtgggttttagagctagaaatagc
F2 (Forward primer)
tgtaatacgactcactataggtacaatagaagagcagggttttagagctagaaatagc
R (reverse primer): aagcaccgactcggtgccact
PCR detection primer:
F(5’- gcttgcctctggaaattctgct -3’)
R(5’- tcgaagccctcagcgtaagcc -3’)
2) Specific gRNA in vitro Synthesis
a. Using BsaI restriction enzymep42250 linearization to linearizep42250 vector as template, PCR amplification was performed by the specific primers (F1/R and F2/R) of 1) under the following reaction conditions: pre-denaturation at 95℃for 5min, (denaturation at 95℃for 30s, annealing at 60℃for 30s, extension at 72℃for 15 s) for 35 cycles, followed by a further 8min at 72 ℃; after the reaction is finished, 1 mu L of sample is spotted on 1.6% agarose gel, electrophoresis detection is carried out, and after the strip detection is correct, agarose gel DNA fragment recovery is carried out;
The PCR reaction system is as follows:
ddH 2 O 15 μL
2× Buffer 25 μL
dNTPs(10 mM) 1 μL
Primer F1/F2(10 uM) 2 μL
Primer R(10 uM) 2 μL
template 4. Mu.L
High-fidelity enzyme 1 mu L
Total volume of 50. Mu.L
b. Measuring the concentration of purified DNA, and using the DNA as a template to carry out in vitro transcription by using a 20 mu L system to synthesize specific gRNA;
in vitro transcription reaction system:
template DNA 12. Mu.L
10×Buffer 2 μL
rATP(10 mM) 1 μL
rUTP(10 mM) 1 μL
rCTP(10 mM) 1 μL
rGTP(10 mM) 1 μL
T7 RNA polymerase 2. Mu.L
Total volume of 20. Mu.L
The whole reaction was added to an EP tube of 1.5 mL RNase-Free, mixed well and then put in a water bath at 37℃for 2.5. 2.5 h; then adding 1 mu L of DNase into the transcription system, and placing the mixture in a water bath kettle at 37 ℃ to react for 40-50 min so as to digest the DNA template; then taking 1 mu L of transcription end product, namely gRNA, and carrying out agarose gel electrophoresis to detect the transcription efficiency;
c. purification of specific gRNA
Purifying the successfully transcribed gRNA by using an RNeasy Mini kit, and storing the gRNA at-20 ℃; sucking 1 mu L of the purified gRNA solution for concentration detection;
3) Microinjection of zebra fish embryos
Transferring embryo to special microinjection culture dish made of agarose by sucking with straw within 30 min after fertilization, premixing Cas9 protein and gRNA (1F and 2F) injection system (wherein Cas9 protein is purchased from thermo fisher company) in advance, wherein Cas9 protein concentration is 5 μg/μl, gRNA final concentration is 80-100ng/μl, and injecting about 1.8 nL Cas9 protein and gRNA mixed solution into fertilized ovum of one cell stage; placing the injected fertilized eggs in E3 water, and incubating at 28 ℃;
4) Sanger sequencing detects the availability of target sites
After microinjection is carried out on zebra fish embryos, 3 tubes of embryos are randomly selected, 8 embryos are taken out from each tube, genome is extracted by cracking,
a. extraction of zebra fish genome
(1) After fertilization of zebra fish embryos at 48hpf, wild type (control group) and post-injection embryos were collected in 1.5 mL Ep tubes, 8 embryos per tube, 100. Mu.L of cell lysate was added to the EP tube containing the embryos, 1. Mu.L of proteinase K, and the mixture was placed in a 55℃incubator for lysis overnight;
(2) Placing the EP tube on an oscillator for full oscillation, adding equal volume of pre-cooled isopropanol, fully reversing and uniformly mixing, centrifuging at 12000 rpm for 10min at 4 ℃, and pouring out supernatant;
(3) Adding 200 μl of 75% ethanol, centrifuging at 12000 rpm for 5min at 4deg.C, removing supernatant, and air drying in a super clean bench for 8-10min;
(4) Adding 10 mu L of deionized water, and fully blowing and uniformly mixing to obtain the zebra fish genome DNA;
b. PCR amplification of target sequences
After extracting the genome DNA, designing a Primer sequence according to a genome region of about 100-300 bp at the upstream and downstream of a CRISPR target site by utilizing Primer Premier 3.0 software to amplify a target DNA fragment;
the PCR reaction system is as follows:
2×Es Taq MasterMix 10 μL
Primer F (10 μM) 0.6 μL
Primer R (10 μM) 0.6 μL
template 1. Mu.L
ddH 2 O 7.8 μL
Total volume of 20. Mu.L
After shaking and mixing evenly, centrifuging at 4 ℃, and carrying out amplification reaction on a PCR instrument under the following reaction conditions: pre-denaturation at 95℃for 5min, (denaturation at 95℃for 30s, annealing at 62℃for 30s, extension at 72℃for 40 s) 30 cycles, followed by 8min at 72 ℃;
c. detecting PCR products by using 1.6% agarose gel electrophoresis, wherein the size of the PCR products is 498bp, the difference between target sites 1F and 2F is 101bp, if the designed target sites are all effective, 1 band smaller than 498bp can be obtained in the agarose gel electrophoresis, for the reliability of the result, sanger sequencing is carried out on the part of the PCR products, and the effectiveness of the set target sites 1F and 2F is further detected by using a sequenced peak diagram;
5) Zebra fishep400Gene F0 generation chimera screening
Culturing the embryo injected in the step 3) to be mature, collecting partial fish tail tissue of the fish, carrying out genotype identification, and screening zebra fish by the genotype identification stepep400Gene F0 generation chimeras;
6) Zebra fishep400Gene F1 generation heterozygote screening
The obtained F0 generation chimera is hybridized with wild zebra fish to obtain F1 generation embryo, and the embryo is cultured at 28 ℃ to observe the survival rate of F1 generation in the initial stage; after the embryo develops for 48 hours, taking 3 tubes of embryos for each F1 generation, and carrying out mutation hereditary identification on 8 embryos per tube; extracting genome of each embryo, performing PCR amplification reaction, detecting PCR fragment size by agarose gel electrophoresis, and screening to obtain stable inheritance zebra fishep400Gene F1 generation heterozygotes (specific methods are described above);
6) Zebra fishep400Gene F2 generation homozygote selection
Mating the F1 generation heterozygotes with the same genotype with male and female heterozygotes at a ratio of 1:1, and culturing the F2 generation embryo in a constant temperature oven at 28 ℃ to obtain the zebra fishep400Homozygote of F2 generation of gene
7) Zebra fishep400Gene knockout homozygote centrality failure mutation phenotype observation and analysis
Mating F1 heterozygote adult fish with zebra fish heart transgene marker strain (such as Tg (cmlc 2: EGFP), tg (cytoer: EGFP), etc.), to obtain stable genetic carrier fluorescent markerep400The heterozygote of F1 generation is then mated with the heterozygote of F1 generation without fluorescent mark, the cultured embryo is collected and developed to a certain period, and the heart development of the embryo is observed and photographed under a confocal microscope.
FIG. 1 is a zebra fishep400Construction of a gene targeting site structure; FIG. 2 is a zebra fishep400Gene knockout strain genotype identification result diagram, diagram A is a zebra fish F2 generation embryo genotype detection electrophoresis result diagram, and diagram B is WT and WTep400Sequencing peak diagram of PCR product of gene knockout strain, from the figure, it can be known that zebra fish has been successfully constructedep400A knockout line; FIG. 3 shows wild type control WT and zebra fishep400As can be seen from the graphs obtained by comparing gene sequences of homozygotes of the genes, the result obtained by comparing the gene sequences of the zebra fishep400A162 bp fragment (86 bp for the intron and 76bp for the exon) was deleted from the knockout line. FIG. 4 is a zebra fishep400Gene homozygote heart mutation phenotype result diagram, wherein the diagram A is a diagram of the transgenic zebra fish Tg [ (]cmlc2EGFP) based phenotype results, as seen in the graph, when compared to wild type control WTep400 -/- Group embryo development to day four (4 d), abnormal heart cyclization, and development to day five (5 d), heart development more malformation, formation of linearized heart, and simultaneous chamber reduction during development; panel B shows that the transgenic zebra fish Tg is%cypherEGFP) based phenotype results, from which it can be seen that, compared to the wild type control WT,ep400 -/- the embryos developed in the group until day four and day five developed mutant phenotypes with loose myofiber distribution, abnormal heart z-line arrangement, and the like, which were highly consistent with the reported heart failure phenotype.
The invention is based on CRISPR/Cas9 gene editing technology through specificitygRNA combinatorial knockdownep400Gene, first constructionep400Gene knockout zebra fish heart failure model. The heart failure model provided by the invention is helpful for deep research in vivoep400The biological function of the gene provides an ideal animal model for screening related drugs of heart failure in future, which has profound significance for heart failure pathogenesis and clinical medication.
The foregoing description is only a preferred embodiment of the present invention, and is not intended to limit the invention in any way, but any simple modification or equivalent variation of the above embodiment according to the technology of the present invention falls within the scope of the present invention.
Sequence listing
<110> Hunan university of teachers and students
<120> construction and application method of ep400 gene knockout zebra fish heart failure model
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 60
<212> DNA
<213> zebra fish (Danio rerio)
<400> 1
tgtaatacga ctcactatag gacctcgctg tcagatgtgg gttttagagc tagaaatagc 60
<210> 2
<211> 58
<212> DNA
<213> zebra fish (Danio rerio)
<400> 2
tgtaatacga ctcactatag gtacaataga agagcagggt tttagagcta gaaatagc 58
<210> 3
<211> 21
<212> DNA
<213> zebra fish (Danio rerio)
<400> 3
aagcaccgac tcggtgccac t 21
<210> 4
<211> 22
<212> DNA
<213> zebra fish (Danio rerio)
<400> 4
gcttgcctct ggaaattctg ct 22
<210> 5
<211> 21
<212> DNA
<213> zebra fish (Danio rerio)
<400> 5
tcgaagccct cagcgtaagc c 21
Claims (6)
1. The method comprises the following steps ofep400The method for constructing the gene knockout zebra fish heart failure model is characterized by comprising the following steps of:
step 1: CRISPR/Cas9 technologyep400Synthesizing gene targeting sites gRNA-1F and gRNA-2F;
step 2: forming a premixing system by the Cas9 protein and the gRNA, and performing embryo microinjection;
step 3: zebra fishep400Detecting the effectiveness of the gene targeting sites gRNA-1F and gRNA-2F;
step 4: zebra fishep400Screening a gene F0 generation chimeric;
step 5: zebra fishep400Screening gene F1 generation gene knockout heterozygotes;
step 6: zebra fishep400Screening the gene F2 generation gene knockout homozygote;
step 7: zebra fishep400Gene knockout homozygote central force failure mutation phenotype observation and analysis.
2. The method according to claim 1ep400The method for constructing the gene knockout zebra fish heart failure model is characterized by comprising the following steps of: in step 1ep400The design and synthesis of the gene targeting site gRNA are carried out according to the following steps: online soft via NCBI and EnsemblPiece, in zebra fishep400The unique 2 targeting sites gRNA are designed and synthesized at the sequences of the 11 th and 12 th exons of the genes, and the primer sequences are as follows: gRNA-1F: tgtaatacgactcactataggacctcgctgtcagatgtgggttttagagctagaaatagc, gRNA-2F: tgtaatacgactcactataggtacaatagaagagcagggttttagagctagaaatagc.
3. The method according to claim 1ep400The method for constructing the gene knockout zebra fish heart failure model is characterized by comprising the following steps of: the injection system in the step 2 comprises 2 targeting sites and Cas9 protein, wherein the concentration of the Cas9 protein is 5 mug/mu L, and the final concentration of gRNA is 80-100 ng/mu L.
4. The method according to claim 1ep400The method for constructing the gene knockout zebra fish heart failure model is characterized by comprising the following steps of: the detection and screening described in steps 3 and 4 was performed by analysis of fragments between two targeting sites using Sanger sequencing to detect the availability and genotype of 2 targeting sites, gRNA-1F and gRNA-2F.
5. The method according to claim 1ep400The method for constructing the gene knockout zebra fish heart failure model is characterized by comprising the following steps of: co-injection of gRNA-1F and gRNA-2F at 2 effective targeting sites described in Steps 5 and 6 knocked out zebra fishep400Construction of a 162bp fragment between exons 11 and 12 of the Gene to construct Zebra fishep400Gene F1 generation gene knockout heterozygote and F2 generation gene knockout homozygote.
6. The method according to claim 1ep400The method for constructing the gene knockout zebra fish heart failure model is characterized by comprising the following steps of: step 7, zebra fish was observed and analyzedep400The homozygous heart failure mutant phenotype after gene knockout was performed as follows: transgenic fluorescent labeling strain utilizing zebra fish heart tissueep400Hybridization of knockout lines to construct cardiac tissue strips with green fluorescent markersep400Gene knockout lines, then observed under confocal microscopy, were analyzed by changes to fluorescent signalsep400Gene knockout resulted in a change in heart morphology, heart beat and histiocyte structure phenotype, defining a heart failure mutant phenotype.
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