CN114908098A - Preparation method and application of zebra fish hoxb1a gene deletion mutant - Google Patents
Preparation method and application of zebra fish hoxb1a gene deletion mutant Download PDFInfo
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Abstract
The invention discloses a preparation method and application of a zebra fish hoxb1a gene deletion mutant, which comprises the following steps: determining that a knockout target of the hoxb1a gene is located on the 1 st exon of the hoxb1a gene to design a gRNA sequence, carrying out PCR amplification by taking a gRNA skeleton plasmid as a template, purifying, carrying out in vitro transcription to obtain a gRNA, mixing the gRNA of the hoxb1a and a Cas9 protein, introducing the mixture into a unicellular embryo of zebra fish, and culturing to obtain the stably inherited hoxb1a gene deletion mutant. According to the invention, the gene hoxb1a is knocked out on the zebra fish genome by using the CRISPR technology for the first time, so that the specific knock-out of the hoxb1a gene in the zebra fish is realized, and the method can be used as an animal model for researching the biological function of the hoxb1a gene and diseases related to hoxb1a gene deletion.
Description
Technical Field
The invention belongs to the field of molecular biology, and particularly relates to a preparation method and application of a zebra fish hoxb1a gene deletion mutant.
Background
In recent years, the ability of the CRISPR/Cas9 system to edit genome has greatly revolutionized the field of genome engineering, and this system was first discovered in the acquired immune system of bacteria, recognized by exogenous DNA such as phage or plasmid, integrated into the CRISPR locus, and concatenated with repeats to form short spacer sequences, and then this CRISPR locus is transcribed into mature small RNAs called crRNA, each of which comprises a repeat sequence and a separate spacer sequence. These crrnas are exactly reverse complementary to the corresponding spacer sequence and can help to recognize the spacer sequence, and if the crRNA binds to the Cas protein to form a ribonucleoprotein complex, the crRNA will be cleaved by the Cas protein at the target site. The CRISPR/Cas9 system which is most widely applied at present belongs to Type II in the CRISPR/Cas system, crRNA and tracRNA are improved into a gRNA from two teams of Doudna and charpietier in 2012, and gene editing in organisms is successfully realized with Cas 9. However, in organisms, once Cas9 and sgRNA function, it results in double-stranded DNA breaks (DSB), and the organism self-repairs.
The Hox gene is a family of transcription factors that encode homeobox-containing genes, which are widely present in metazoans, play a key role in determining the formation of body morphology, and are involved in regulating organ development and maturation. In 2015, Zaffran laboratory found that HOXB1 mutant had the phenotype of abnormal cardiac development, such as damaged ventricular septum, abnormal vascular location, and shortened outflow tract, and that these abnormalities were caused by disturbed proliferation and differentiation of cardiac progenitors from the second cardiogenic region. In addition, the mutation of the candidate gene HOXB1 found by Vahidi Mehrjardi MY and the like may cause the occurrence of rare congenital facial nerve diseases, such as Hereditary Congenital Facial Paralysis (HCFP).
Hereditary Congenital Facial Palsy (HCFP) is a rare congenital cranial nerve disorder disease manifested as non-progressive solitary facial palsy (cranial nerve VII) caused by abnormal development of the facial nuclei and nerves thereof. Congenital Facial Paralysis (CFP) is classified as acquired or developed, unilateral or bilateral, complete (paralysis) or incomplete (paralysis). In order to determine the cause, careful diagnostic examination and differential diagnosis are important, since prognosis and treatment vary according to the underlying pathophysiology. According to previous studies, the disease is thought to have genetic heterogeneity and different genetic patterns. Two gene sites, HCFP1 (MIM% 601471) and HCFP2 (MIM% 604185), were defined in 2 autosomal dominant hereditary HCFP families, respectively, but none were identified. The only known autosomal recessive HCFP (HCFP3) is caused by a homoallelic mutation of HOXB1 (MIM% 142968) in chr17q21.32(MIM 614744). Recently, 4 mutations (3 missense and 1 nonsense) in HOXB1 gene were found in 12 patients from 5 unrelated closely related families of HCFP3, and a new homozygous truncation mutation in HOXB1 was found by genetic screening of currently known candidate genes for the disease. The relevant experimental data support the conclusion that: loss of HOXB1 function can lead to facial weakness and developmental deformities.
It has been reported that mouse HoxB1 mutation affects the proliferation and differentiation of progenitor cells in the secondary cardiogenic region, suggesting that the HoxB1 gene plays a crucial role in embryonic development. The gene plays an important role in cardiovascular diseases, but the specific action mechanism and the affected downstream signal pathway of the gene need to be studied deeply. In experimental research, the mouse mutant has certain defects in heart function gene research, the living body can not be observed in detail in real time, and the zebra fish is transparent in embryo when being young, so that the mutant is a good model animal for researching cardiovascular diseases.
Disclosure of Invention
The invention aims to provide a preparation method of a zebra fish hoxb1a gene deletion mutant, which is characterized in that a CRISPR/Cas9 technology is used for carrying out hoxb1a gene knockout on a zebra fish genome for the first time, so that the hoxb1a gene in zebra fish is specifically knocked out, and other gene functions cannot be interfered, and the zebra fish with the hoxb1a knocked out is obtained.
The invention also aims to provide a zebra fish hoxb1a gene deletion mutant as an animal model for providing a theoretical basis for the exploration of heart development mechanisms and human HCFP related diseases.
The purpose of the invention is realized by the following technical scheme:
the preparation method of the zebra fish hoxb1a gene deletion mutant provided by the invention is used for preparing the zebra fish hoxb1a gene deletion mutant by a CRISPR/Cas9 technology, and comprises the following steps:
s1, determining that a hoxb1a gene knockout target designs a gRNA sequence on the 1 st exon of a zebra fish hoxb1a gene;
s2, designing an upstream primer T7-hoxb1a-sfd and a downstream gRNA reverse primer required for synthesizing hoxb1a gRNA;
s3, performing PCR amplification by using an upstream primer T7-hoxb1a-sfd and a downstream gRNA reverse primer by using a gRNA framework plasmid as a template;
s4, carrying out in vitro transcription on the PCR product obtained in the step S3 to obtain gRNA;
s5, introducing the gRNA and the Cas9 protein into zebrafish;
s6, culturing to obtain the zebra fish hoxb1a gene mutant with stable inheritance.
In step S1, the target gRNA sequence is GGAACTGGGACAACAAGTTA (SEQ ID NO: 1).
In step S2, the upstream primer F1(T7+Target site+
) Namely, the sequence of the primer T7-hoxb1a-sfd is as follows:
the sequence of the downstream primer R1(trans reverse), i.e. the gRNA reverse primer, is:
AAAAAAAGCACCGACTCGGTGCCAC(SEQ ID NO:3)。
in step S4, the sequence of the gRNA is TAATACGACTCACTATAGGAACTGGGACAACAAGTTAGTTTTAGAGCTAGAAATAGCGGACAGATTCATGTCCTGGACGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT (SEQ ID NO: 6).
Preferably, in step S5, the introduction of the gRNA and Cas9 protein into zebrafish specifically includes: mixing the gRNA with the Cas9 protein, and microinjecting into a zebra fish one-cell stage embryo; wherein the final concentration of gRNA is 100 ng/mu L, and the final concentration of Cas9 protein is 800 ng/mu L; each fertilized egg was injected with 1 nL.
Preferably, step S6 specifically includes the following steps:
a1, respectively taking zebra fish introduced with gRNA and Cas9 protein and wild type uninjected zebra fish embryos for hoxb1a gene knockout detection, and determining positive F of hoxb1a gene knockout 0 Culturing to adult fish;
a2, knock-out positive F of hoxb1a gene 0 Carrying out heritability and effective mutation detection on the adult fish and wild zebra fish outcrossing, and screening heritability effective mutation F 1 Feeding to adult fish; the hoxb1a F is obtained by genotyping 1 Mutant zebrafish;
a3, same mutant hoxb1a F 1 Carrying out internal crossing on the mutant zebra fish to obtain hoxb1a F 2 Mutant zebrafish;
a4, identified as F 2 The homozygote of the hoxb1a gene knockout in the generation is the zebra fish hoxb1a gene mutant with stable inheritance.
More preferably, in step A1, the primer sequences used for the hoxb1a gene knockout assay include:
the upstream primer hoxb1 a-F: ACGCTGATGGACGACTTTACG (SEQ ID NO: 4);
the downstream primer hoxb1 a-R: CAATCCACCTGTTTTGGGGG (SEQ ID NO: 5).
More preferably, step S6 includes the steps of:
(1) designing primers around the target point, so that the distance between the primers and the two sides of the target point is greater than 100bp, and the absolute value of the difference of the distances between the primers and the target point is greater than 100 bp; selecting a pair of healthy WT zebra fishes as parents, cutting tails to perform PCR, directly sending PCR products to sequence, detecting whether the gene to be knocked out of parent fishes is homozygote, requiring that target sequences of adult fishes to be injected are homozygote, and reselecting the adult fishes to be injected if the sequencing result shows that the target sequences are heterozygote;
(2) microinjecting gRNA and Cas9 protein into zebra fish, and mixing the gRNA with the final concentration of 100 ng/mu L of an injection system; cas9 protein: 800 ng/. mu.L; the total volume V is 1 nL;
(3) dead eggs were picked out at night on the day of injection,simultaneously changing half of new water, changing water once every morning and evening, 48h after fertilization, performing double-outer primer PCR detection, and knocking out F successfully 0 Feeding zebra fish;
(4) after 3-4 months of sexual maturity of zebra fish, the mutated F is removed 0 Hybridizing zebra fish with wild zebra fish to obtain heterozygote with certain probability, collecting embryo, extracting genome, performing PCR with detection primer, TA cloning, sequencing to determine genotype, and determining F capable of being inherited and having frame shift mutation 1 Feeding zebra fish;
(5) after sexual maturity of 3-4 months, F 1 And (3) cutting the tail of the adult male fish and female fish of the mutant zebra fish again, carrying out genotype identification and screening, and mating the mutant zebra fish again to obtain the mutant zebra fish homozygous for hoxb1a gene deletion.
More preferably, the zebrafish hoxb1a gene mutant has a cardiac malformation phenomenon that pericardial edema and cardiac cyclization are abnormal at 4.5dpf, and the chambers of atria and ventricles of the in situ hybridization mutant have no obvious abnormal development.
The invention also provides application of the zebra fish hoxb1a gene deletion mutant as an animal model in researching biological functions of a hoxb1a gene and diseases related to hoxb1a gene deletion, wherein the zebra fish hoxb1a gene deletion mutant is prepared by any one of the preparation methods of the zebra fish hoxb1a gene deletion mutant.
The invention constructs the hoxb1a gene deletion mutant on the zebra fish by using the CRISPR/Cas9 technology, has the technical advantages of fast growth and large quantity, has obvious phenotype, can better simulate diseases such as abnormal cardiac development, abnormal craniofacial development and the like, fills in the blank of related research, simulates the disease caused by the hoxb1a gene deletion, is convenient for observing the disease phenotype caused by the hoxb1a gene deletion on the zebra fish, and provides a mode method for the subsequent hox gene function research by using the model to carry out a drug screening experiment, and has the following beneficial effects:
1) a section of specific targeting site is designed by using a CRISPR/Cas9 technology for the first time, so that the specific knockout of the hoxb1a gene in the zebra fish is realized, other genes are not influenced while the hoxb1a gene is knocked out, the zebra fish with the hoxb1a specific knockout is obtained, and a valuable thought and method are provided for the knockout of related single sites.
2) The hoxb1a gene codes 316 amino acids, while the mutant with 13bp deletion codes 214 amino acids, wherein the first 97 amino acids are coded correctly, and the coded amino acids are not expressed because of the appearance of a terminator.
3) The hoxb1a gene mutation can be stably inherited, so that the subsequent functional mechanism of the hoxb1a gene can be conveniently and deeply researched.
4)hoxb1a -/- The mutant zebrafish have a severe phenotype, and significant cardiac malformations such as pericardial edema and abnormal cardiac cyclization were observed at 4.5dpf (days post fertilization), accompanied by malformation of facial development.
5) By using zebra fish hoxb1a -/- Compared with the mouse HoxB1 mutant, the mutant has the advantages that the embryo is transparent, so that the heart deformity can be observed conveniently, about 300 offspring can be produced by one pair of parents, and the sample size of experimental analysis and drug screening is increased.
Drawings
FIG. 1 is a CRISPR/Cas 9-mediated knockout pattern diagram of zebrafish hoxb1a gene, and sequencing and T7E1 experimental detection after hoxb1a gene knockout show that the hoxb1a gene knockout is successful.
FIG. 2 shows hoxb1a F 0 A germline transmission detection result; 5 tail hoxb1a F is taken 0 Carrying out outcrossing on adult fish successfully knocked out by gene detection and wild zebra fish to obtain F 1 And (3) taking 5 embryos in one tube, carrying out T7E1 enzyme digestion identification on 3-4 tubes, and transmitting the mutation to the offspring of the 5-tailed zebra fish as shown by the enzyme digestion result.
FIG. 3 is F 1 -hoxb1a adult zebra fish genotype detection T7E1 endonuclease enzyme digestion identification gel electrophoresis result; and (3) detecting 13 zebra fish hoxb1a genes obtained by carrying out outcrossing on 13 tails by tail shearing, and detecting by T7E1 to obtain 13 positive zebra fishes.
FIG. 4 shows the sequencing results of the genotypes of the hoxb1a wild-type and homozygous mutant; (a) sequencing peak plots for wild type and mutant; (b) comparing the amino acid sequences encoded by the wild type and the mutant; (c) wild type and mutant Hoxb1a protein secondary structure pattern diagrams.
FIG. 5 shows cardiac malformations of pericardial edema in zebrafish after hoxb1a gene deletion, indicated by arrows (a, b); (a ', b') enlarged picture of the heart region, the dashed line delineating the pericardial cavity.
FIG. 6 shows that the absence of hoxb1a results in an abnormal zebrafish heart. Pictures taken with a light microscope show enlargement of the hoxb1a mutant pericardial cavity (fig. 6a, c); hoxb1a mutant compared to wild-type cardiac cyclization abnormality (fig. 6b, d, e, f); in situ hybridization results of heart chamber specific expression gene anf showed that hoxb1a mutant anf signal was ectopically expressed at the atrioventricular septum of the heart as indicated by the arrow, whereas wild type was only expressed in the ventricles and atria, not in the atrioventricular septum (fig. 6g, h).
FIG. 7 shows the malformation of zebrafish facial development after hoxb1a gene deletion (a, b); (a ', b') facial area enlarged, dotted line depicting the difference compared to wild type for the mutant, and arrow indicating that the mandibular area mutant is thicker than wild type.
Detailed Description
The present invention will be described in detail with reference to examples. The following examples will assist those skilled in the art in further understanding the invention, but are not intended to limit the invention in any way. It should be noted that it would be apparent to those skilled in the art that several modifications and improvements can be made without departing from the inventive concept. All falling within the scope of the present invention.
Examples
1 materials and apparatus
1.1 Zebra fish and plasmids used in the experiment
The zebra fish used in the experiment is derived from a zebra fish platform of the aquatic product and life college of Shanghai ocean university.
All gRNA backbone plasmids were derived from 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-.
1.2 Primary reagents
Table 1: experimental reagent
1.3 Main instruments
Table 2: laboratory apparatus
2 method of experiment
2.1gRNA Synthesis
1) Target design
(1) Sequence and structural information of hoxb1a gene (ENSDARG00000039077 and ENSDARG00000024771) were obtained from zebra fish genome database (http:// www.ensembl.org/Danio _ rerio/Info/Index)
(2) Target sites were designed on the online site http:// ZiFiT.
Designing the target spot:
a. the length is preferably 20bp, if no 20bp, the length is 19bp, and the 5' end is started by GG, so that the synthesis efficiency of the T7 promoter is improved;
b. the target site with high GC content was selected as much as possible without having a continuous AT sequence, and the 3 bases immediately 3' to the target site were NGG (N is an arbitrary base, called PAM region).
(3) After the target site is designed, the specificity of the target site is detected at the NCBI website, and if the same sequence exists in other positions on the whole genome, the same sequence is discarded.
(4) Designing a detection primer, wherein T7E1 enzyme is required to be used for detecting mutation, in order to make a detected gel picture more definite, the designed detection primer requires that the distance difference between an upstream primer and a downstream primer and a target point is more than 100bp, and the respective distances between the upstream primer and the downstream primer and the target point are more than 100 bp.
(5) The WT zebra fish used for gene knockout is trimmed and subjected to alkaline lysis to obtain genome DNA, and a segment of sequence near a target point is amplified by PCR.
(6) The WT zebra fish is detected by using the endonuclease T7E1, whether the amplified fragment can be cut by the enzyme T7E1 is determined, and if the amplified fragment cannot be cut, the WT zebra fish can be used for subsequent knockout detection; when the cleavage is performed, it is necessary to select a specific enzyme based on the amplified sequence information and perform the enzyme cleavage detection.
(7) Sequencing and identifying: and (3) sequencing the PCR product, comparing a peak diagram with a sequence, and confirming that the parent is a homozygote without natural mutation, thereby ensuring that the subsequently prepared mutant is caused by gene knockout.
Table 3: hoxb1a target site sequence
2) Designing a detection primer: the designed primers should ensure that the distance between the two sides of the target is more than 100bp, and the difference between the distance from the upstream primer to the target and the distance from the downstream primer to the target is more than 100bp and at least 50 bp. The primer amplification should have specificity, and the amplified fragment is about 500 bp. The primers were synthesized by Shanghai Bioengineering Co., Ltd. (Table 4).
Table 4: primer information for experiments
3) gRNA product synthesis: fragments were amplified using primer T7-hoxb1a-sfd, gRNA reverse primer and 2 × EasyTaq PCR SuperMix (+ dye) using gRNA backbone plasmid as template and purified with kit.
4) In vitro transcription:
table 5: in vitro transcription reaction system
| Nuclease-free Water | to 20μL |
| DNA template | 1μg |
| 10×Transcription Buffer | 2μL |
| 10mM ATP/CTP/GTP/UTP | 1μL/1μL/1μL/1μL |
| T7Enzyme Mix | 2μL |
Note that: finally, 10 × Transmission Buffer and T7Enzyme mix were added
Mixing, centrifuging for a short time, and incubating at 37 deg.C for 80 min; then, 1. mu.L of TURBO DNase was added to the system and mixed well, centrifuged briefly and incubated at 37 ℃ for 15 min.
5) Purifying gRNA:
(1) to 20. mu.L of the in vitro transcription system, 2.5. mu.L of 4M LiCl and 100. mu.L of absolute ethanol were added, gently mixed and centrifuged briefly and then placed in a-80 ℃ freezer for at least 1 h.
(2) The sample was removed from the freezer, centrifuged at 12000rmp at 4 ℃ for 15 min. The supernatant was discarded and the precipitate was washed with 70% ethanol. Centrifuge at 12000rmp for 5min at 4 ℃. After discarding the supernatant, the centrifuge tube was placed in a fume hood to volatilize the ethanol.
(3) And adding an appropriate amount of DEPC water to dissolve the gRNA precipitate according to the size of the precipitate. The concentration and OD values were then determined by Nanodrop and electrophoresis, where the sequence of gRNA was TAATACGACTCACTATAGGAACTGGGACAACAAGTTAGTTTTAGAGCTAGAAATAGCGGACAGATTCATGTCCTGGACGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT (SEQ ID NO: 6).
2.2 microinjection
The gRNA was mixed with Cas9 protein (purchased from gencriispr NLS-Cas9-NLS (chrysosper, Z03389-25)), and the mixed material was injected into zebrafish one-cell stage embryos using a microinjection instrument, leaving a batch of uninjected embryos as a control for each injection. Final concentration of mixed injection: gRNA 100 ng/. mu.L, Cas9 protein 800 ng/. mu.L.
2.3 detection of successful knockout and knockout efficiency (T7E1 enzyme digestion detection)
(1) Extraction of fish egg genome
Each group of 5 eggs was incubated with 35. mu.L of 50mM NaOH at 95 ℃ for 20min, shaken out, and centrifuged once for a while. Then, 3.5. mu.L of 1M Tris-HCl (pH. apprxeq.8.0) was added thereto, followed by vigorous shaking and centrifugation.
(2) PCR amplification of fragments of interest
And amplifying the target fragment according to the primer designed near the target point.
Table 6: PCR reaction system
| H 2 O | to 25μL |
| Mix Taq Enzyme | 12.5μL |
| F/R | 0.5μL/0.5μL |
| Form panel | 10ng |
And (3) PCR reaction conditions:
pre-denaturation at 98 ℃ for 2 sec; denaturation at 98 ℃ for 10sec, annealing at 55 ℃ for 30sec, and extension at 72 ℃ for 1min for 36 cycles; further extension at 72 deg.C for 5 min; storing at 4 ℃.
2% agarose gel 120V electrophoresis for 25 min.
(3) T7E1 endonuclease enzyme digestion detection
Table 7: T7E1 enzyme digestion pre-reaction system
| Buffer | 1.1μL |
| PCR product | 5μL |
| H 2 O | 4μL |
| Total | 11.1μL |
Incubate at 95 ℃ for 5min, cool to room temperature, add 0.5. mu. L T7E1 enzyme and ddH 2 The O1: 1 mixture was incubated at 37 ℃ for 45 min.
(4) Electrophoretic detection
And imaging the electrophoresed agarose gel by using a gel electrophoresis imager after electrophoresis, observing a target band, and judging whether the knockout is successful.
2.4 Hoxb1a homozygous mutant zebrafish genotype identification
And carrying out genotype screening and identification on different deletion types.
3 results of the experiment
3.1 construction of the hoxb1a mutant
3.1.1 hoxb1a F 0 Results of Gene knockout assay
The T7E1 enzyme cutting result shows that the hoxb1a gene knockout is successful (FIG. 1).
3.1.2 hoxb1a F 0 germline Transmission test results
5 tail hoxb1a F 0 Carrying out outcrossing on adult fish successfully knocked out by gene detection and wild zebra fish to obtain F 1 And (3) carrying out T7E1 enzyme digestion identification on 5 embryos in one tube, wherein the enzyme digestion result shows that the 5 zebra fish transmit the mutation to the offspring (figure 2).
3.1.3 hoxb1a F 1 Genotype identification of heterozygous mutant zebra fish
The method comprises the steps of detecting 13-tail-out-crossing zebra fish hoxb1a genes through a tail shearing test, obtaining 13 positive zebra fishes through T7E1 detection, carrying out TA cloning, and determining that the 6 fishes have effective mutation, wherein the mutation type is-13 bp (figure 3 and figure 4).
3.1.4 hoxb1aF 2 Mutant zebra fish phenotype observation photographing
The hoxb1a heterozygous mutant was in vivo crossed, harvested and cultured after egg laying for early embryo development observations, and cardiac pericardial cavity edema and phenomena of failure of the heart to cyclize normally were observed at 4.5dpf (fig. 5, fig. 6 a-d). In situ hybridization experiments expression profiles of myl7, anf and vmhc cardiac marker genes were detected in 4.5dpf hoxb1a mutant and control. Myl7 gene is specifically expressed in cardiac myocardium, vmhc gene is specifically expressed in ventricular myocardium, anf gene is specifically expressed in heart chamber, and atrioventricular space (AVC) is not expressed in normal wild type zebrafish; in situ hybridization results of heart chamber-specific expressed gene anf showed that hoxb1a mutant anf signal was ectopically expressed at the atrioventricular septum of the heart as indicated by the arrow, whereas wild type was only expressed in ventricles and atria, not in the atrioventricular septum (fig. 6g, h), indicating hoxb1a -/- The atrial and ventricular chamber morphology was not abnormal, but affected the development of the atrioventricular septum (fig. 6e, f, i, j), while the wild type developed normally in the face, while the homozygous mutant developed abnormally in the face at 4.5dpf (fig. 7).
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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Claims (7)
1. The preparation method of the zebra fish hoxb1a gene deletion mutant is characterized in that the zebra fish hoxb1a gene deletion mutant is prepared by a CRISPR/Cas9 technology, and comprises the following steps:
s1, determining that a hoxb1a gene knockout target point designs a gRNA sequence on the 1 st exon of a zebra fish hoxb1a gene, wherein the sequence is shown as SEQ ID NO. 1;
s2, designing an upstream primer T7-hoxb1a-sfd and a downstream gRNA reverse primer required for synthesizing hoxb1a gRNA, wherein the sequences are respectively shown as SEQ ID NO. 2 and SEQ ID NO. 3;
s3, performing PCR amplification by using a gRNA framework plasmid as a template and an upstream primer T7-hoxb1a-sfd and a downstream gRNA reverse primer;
s4, carrying out in vitro transcription on the PCR product of the step S3 to obtain gRNA, wherein the sequence of the gRNA is shown as SEQ ID NO. 6;
s5, introducing the gRNA and the Cas9 protein into zebrafish;
s6, culturing to obtain the zebra fish hoxb1a gene mutant with stable inheritance.
2. The method for preparing the zebrafish hoxb1a gene deletion mutant according to claim 1, wherein in step S5, gRNA and Cas9 protein are mixed and microinjected into the zebrafish embryo at the one-cell stage, wherein the final concentration of gRNA is 100ng/μ L, the final concentration of Cas9 protein is 800ng/μ L, and 1nL is injected into each fertilized egg.
3. The method for preparing the zebrafish hoxb1a gene deletion mutant according to claim 1, wherein the step S6 specifically comprises the following steps:
a1, respectively taking zebra fish introduced with gRNA and Cas9 protein and wild type uninjected zebra fish embryos for hoxb1a gene knockout detection, and determining positive F of hoxb1a gene knockout 0 Culturing to adult fish;
a2, knock-out positive F of hoxb1a gene 0 Carrying out heritability and effective mutation detection on the adult fish and wild zebra fish outcrossing, and screening heritability effective mutation F 1 Feeding to adult fish; the hoxb1a F is obtained by genotyping 1 Mutant zebrafish;
a3, hoxb1a F of the same mutation 1 Carrying out internal crossing on the mutant zebra fish to obtain hoxb1a F 2 Mutant zebrafish;
a4, identified as F 2 The homozygote of the hoxb1a gene knockout in the generation is the zebra fish hoxb1a gene mutant with stable inheritance.
4. The method for preparing the zebrafish hoxb1a gene deletion mutant according to claim 3, wherein in the step A1, primers used for detecting hoxb1a gene knockout comprise an upstream primer hoxb1a-F with the sequence shown in SEQ ID NO. 4 and a downstream primer hoxb1a-R with the sequence shown in SEQ ID NO. 5.
5. The method for preparing the zebrafish hoxb1a gene deletion mutant according to claim 1, wherein the step S6 comprises the following steps:
(1) designing primers around the target point, so that the distance between the primers and the two sides of the target point is greater than 100bp, and the absolute value of the difference of the distances between the primers and the target point is greater than 100 bp; selecting a pair of healthy WT zebra fishes as parents, cutting tails to perform PCR, directly sending PCR products to sequence, detecting whether the gene to be knocked out of parent fishes is homozygote, requiring that target sequences of adult fishes to be injected are homozygote, and if the sequencing result shows that the target sequences are heterozygote, reselecting the adult fishes to be injected;
(2) microinjection of gRNA and Cas9 protein into zebrafish, mixing the final concentration of gRNA in the injection system: 100 ng/. mu.L; cas9 protein: 800 ng/. mu.L; total microinjection volume V ═ 1 nL;
(3) picking out dead eggs in the evening on the injection day, changing half of fresh water, changing water once in the morning and at the evening, 48h after fertilization, detecting double-outer-side primer PCR, and knocking out successfully F 0 Feeding zebra fish;
(4) after 3-4 months of sexual maturity of zebra fish, mutating F 0 Hybridizing zebra fish with wild zebra fish to obtain heterozygote with certain probability, collecting embryo, extracting genome, performing PCR with detection primer, TA cloning, sequencing to determine genotype, and determining F capable of being inherited and having frame shift mutation 1 Feeding zebra fish;
(5) after sexual maturity of 3-4 months, F 1 And (3) cutting tails of adult male fishes and female fishes of the mutant zebra fishes again, carrying out genotype identification and screening, and mating the mutant zebra fishes again to obtain the zebra fish hoxb1a gene deletion homozygous mutant.
6. The method for preparing the zebrafish hoxb1a gene deletion mutant according to any one of claims 1 to 5, wherein the zebrafish hoxb1a gene mutant is characterized in that pericardial edema and abnormal cardiac cyclization heart malformation are observed at 4.5dpf, and the chambers of atria and ventricles of the in situ hybridization mutant are not abnormally developed.
7. Use of zebrafish hoxb1a gene deletion mutant as animal model for researching biological function of hoxb1a gene and diseases related to hoxb1a gene deletion, wherein the zebrafish hoxb1a gene deletion mutant is obtained by the preparation method of the zebrafish hoxb1a gene deletion mutant as described in any one of claims 1-6.
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| WO2007138691A1 (en) * | 2006-05-31 | 2007-12-06 | National University Corporation Nagoya University | Polycystic kidney disease-related gene and use thereof |
| CN114540355A (en) * | 2022-02-23 | 2022-05-27 | 中国农业大学 | HHEX cartilage tissue specificity knockout mouse animal model and construction method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2007138691A1 (en) * | 2006-05-31 | 2007-12-06 | National University Corporation Nagoya University | Polycystic kidney disease-related gene and use thereof |
| CN114540355A (en) * | 2022-02-23 | 2022-05-27 | 中国农业大学 | HHEX cartilage tissue specificity knockout mouse animal model and construction method thereof |
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