CN108588121B - A method for inducing homozygous hemoglobin phenotype in Epoa knockout zebrafish embryos - Google Patents

Abstract

The invention provides a method for inducing Epoa gene knockout of a hemoglobin phenotype of a zebra fish embryo homozygote, which induces the Epoa gene knockout of the hemoglobin phenotype of the zebra fish embryo homozygote by carrying out gene silencing on an Epob gene of the Epoa gene knockout of the zebra fish embryo homozygote. The method of the invention induces Epoa gene knockout zebra fish embryo homozygote hemoglobin phenotype by gene silencing of Epob gene, and has simple operation and stable and obvious induction effect. In the process of the method, gene sequencing and RT-PCR determination can be simultaneously realized on a single zebra fish embryo.

Description

A method for inducing homozygous hemoglobin phenotype in Epoa knockout zebrafish embryos

technical field

The invention belongs to the field of biomedicine and relates to zebrafish, in particular to a method for inducing the homozygous hemoglobin phenotype of Epoa gene knockout zebrafish embryos.

Background technique

As an emerging biological model, zebrafish is widely used in scientific research and the preparation of gene knockout models because of its transparent embryos, easy to observe, low cost of breeding, and easy gene editing operations. Previous studies on gene down-regulation in zebrafish mostly performed gene down-regulation RNA intervention through Morpholino injection of the corresponding gene. However, since Morpholino only works for 5-7 days in zebrafish, more and more studies have questioned the inevitable off-target effect of Morpholino. Current research suggests using emerging gene editing technologies (such as CRISPR/Cas9) to construct genes Knockout zebrafish model to achieve stable and long-term gene function inspection and intervention.

At the same time, with the extensive development of gene editing biological models and experimental observations, many studies suggest that Morpholino and gene knockout may lead to different zebrafish phenotypes. This phenotypic difference is not only due to the side effects and off-target effects of Morpholino technology, but more from the compensatory effects of related genes after gene knockout. However, due to the importance of some target genes in biological development, none of their gene knockout homozygous models can survive, and only stable reproductive heterozygous adult zebrafish can be obtained. Therefore, the gene compensation study of homozygous zebrafish for these target genes can only be observed when there are still surviving homozygous individuals in the embryonic stage. In addition, it is necessary to first cross the heterozygotes to obtain zebrafish embryos with a mixture of homozygotes, wild-type and heterozygotes, and further sequence the embryonic zebrafish to screen out the homozygous zebrafish, and then perform the homozygous zebrafish embryos. Phenotype observation and analysis or the use of RT-PCR and other techniques for gene compensation research.

Erythropoietin (EPO) is a hormone-like substance secreted by the kidney and liver, which plays an important role in inhibiting the apoptosis of primitive erythrocytes and promoting erythropoiesis. However, due to the importance and complexity of the role of EPO, previous studies targeting EPO knockout animal models failed to survive effectively due to severe anemia, limiting further understanding and research on EPO. At present, studies on gene compensation in homozygous embryos of Epoa knockout zebrafish are very scarce, and there is no related method for inducing hemoglobin phenotype.

SUMMARY OF THE INVENTION

In view of the deficiencies in the prior art, the purpose of the present invention is to provide a method for inducing hemoglobin phenotype for Epoa gene knockout zebrafish embryo homozygotes, and to solve the technical gap in this field in the prior art.

In order to solve the above-mentioned technical problems, the present invention adopts the following technical solutions to realize:

A method for inducing homozygous hemoglobin phenotype in Epoa knockout zebrafish embryos by gene silencing of Epoa knockout zebrafish embryos homozygous for hemoglobin expression type.

The present invention also has the following distinguishing technical features:

The method specifically includes the following steps:

Step 1, construction of Epoa gene knockout zebrafish animal model:

1) Establish an E. coli plasmid containing the target sequence of the EPO gene fragment:

1-0) Obtain zebrafish EPO gene sequence from Ensemble database

ENSDART00000020288.9, the database URL is http://www.ensembl.org;

1-1) Use Zifit Target Version4.2 for target sequence selection and primer design for the EPO gene exon 2 region, and then synthesize the designed primers;

Among them, the target sequence of the selected EPO exon 2 region is:

CATCTGTGACCTGCGCGT;

Among them, the designed and synthesized primers are:

forward primer TAGGACGCGCAGGTCACAGATG;

reverse primer AAACCATCTGTGACCTGCGCGT;

1-2) Synthesize the E. coli plasmid containing the target sequence of the EPO gene fragment;

1-2A) Primer annealing: mix 2 μL forward primer, 2 μL reverse primer, 2 μL NEB buffer, 14 μL distilled water, incubate the mixture at 95°C for 5 minutes, then cool down to 50°C at a rate of 0.1°C/sec, incubate at 50°C for 10 minutes, Then cool down to 4°C at 1°C/sec to obtain annealed oligomers;

1-2B) Restriction: 1 μL of annealed oligos obtained from 1-2A), 400 ng pT7-gRNA plasmid, 1 μL NEB buffer, 1 μL T4 DNA ligase, 0.5 μL BsmBI enzyme, 0.3 μL BglII enzyme, 0.3 μl SalI enzyme , 0.5 μL T4 ligase and DNA-free water were mixed, and the total volume after mixing was 10 μL; the mixture was first incubated at 37 °C for 60 min, then at 16 °C for 45 min, and so on for three cycles; then the mixture was heated to 37 °C and incubated for 30 min, Then heat up to 55°C and incubate for 30min, then heat up to 80°C and incubate for 15min, and cool down to 4°C to limit the endocut product;

1-2C) Transfer the restriction endonuclease product into Escherichia coli: store Escherichia coli in a -80°C refrigerator, place it on ice for 20 to 30 min after taking it out, and combine the restriction endonuclease product synthesized in step 1-2B) with 50μL of E. coli was mixed, the mixture was placed on ice for 20min, then placed in a water bath at 42°C for 90sec, and then placed on ice for at least 90sec; 1mL of LB culture medium was added to the above mixture and incubated at 37°C, 220rpm incubator for 45min. Then, the above mixture was centrifuged at 6000 rpm at 4°C for 5 minutes, and 900 μL of the supernatant after centrifugation was taken, spread in LB medium, and cultured in a constant temperature incubator at 37°C overnight;

1-2D) Select a single colony from the overnight cultured colony in 3 mL of culture solution, cultivate overnight at 37°C and 220 rpm; use the GETM Healthcare Illustra kit to extract plasmids for gene sequencing; obtain the plasmid containing the target gene sequence as a positive plasmid, and EPO expression Sub-2 region target sequence: CATCTGTGACCTGCGCGT;

2) Establish and cultivate an EPO knockout zebrafish model based on CRISPR gene knockout technology:

2-1) Mix 1～3ug of positive plasmid containing the target sequence, 5μL of NEB 3:1 buffer, 5μL of 10% BSA, and 1μL of BamHI-HF, adjust the volume to 50μL with DNA-free water, and water bath at 37°C overnight; add 100ug /mL proteinase K 0.5μL and 0.5% SDS 2.5μL, heated at 50°C for 20min, using QiagenTM PCR purification kit to extract DNA, agar gel electrophoresis to verify, the target DNA was obtained;

T7Transcription Kit，

T7Transcription Kit的商标品牌为Invitrogen，将目标DNA逆转录为EPO gRNA；使用不含目的Epo靶序列的空白质粒提取对照RNA，不含目的Epo靶序列的空白质粒即不含有EPO外显子2区域靶序列的质粒；对照RNA即无法造成基因敲除的对照序列；2-2) Use

T7Transcription Kit,

The brand name of T7Transcription Kit is Invitrogen, which reverse-transcribes the target DNA into EPO gRNA; uses a blank plasmid without the target Epo target sequence to extract the control RNA, and the blank plasmid without the target Epo target sequence does not contain the target of EPO exon 2 region The plasmid of the sequence; the control RNA is the control sequence that cannot cause gene knockout;

2-3) The Cas9 plasmid comes from the No. #63154 plasmid with the trademark brand of Addgene, and uses the T7/T3 Transcription Kit to synthesize Cas9 RNA, and the trademark brand of the T7/T3 Transcription Kit is Invitrogen;

2-4) Mix EPO gRNA and Cas9RNA in 0.1mol/L KCl solute, and inject into zebrafish zygote single cell; the concentration of EPO gRNA after mixing is 200pg/μL, and the concentration of Cas9RNA after mixing is 200pg/μL, using Mixed injection of control RNA and Cas9 was used as the control observation group;

72 hours after fertilization, the genomic DNA of the embryos was extracted for PCR amplification, gene sequencing was performed, and sequence alignment was performed using ClustalW2 to further clarify the effectiveness of gene knockout. The website of ClustalW2 is http://www.ebi.ac.uk/ Tools/msa/clustalw2/;

2-5) The chimeric fish obtained after EPO gRNA injection was cultured to the size of an adult fish in about 3 months. After EPO gRNA injection, a chimeric fish, that is, a chimeric zebrafish containing multiple mixed gene mutant cells, was obtained, and the fin tissue was taken for gene sequencing. , EPO+/- heterozygous zebrafish were obtained by crossing the gene sequencing positive adult fish with wild background zebrafish, and EPO-/- homozygous zebrafish embryos were obtained by crossing the adult heterozygous background zebrafish, and the homozygous gene variation was judged by the gene sequencing results. sequence;

Step 2, to obtain a mixed embryo population:

A mixed population of homozygous, heterozygous and wild-type embryos containing the Epoa gene was obtained by crossing Epoa heterozygous adult zebrafish;

Step three, gene silencing induction:

Perform gene silencing on the Epob gene of the mixed embryo group obtained in step 2 to induce the hemoglobin phenotype of Epoa gene knockout zebrafish embryos;

Step 4: Obtain the embryo homozygote of Epoa knockout zebrafish after induction:

Cut a part of 1/5 of the total length of the tail of each embryo in the induced mixed embryo group obtained in step 3, and perform genomic DNA extraction and gene sequencing for inspection;

The remaining part of each embryo was subjected to RNA extraction and cDNA reverse transcription, and the obtained cDNA was subjected to Epob and Epoa gene expression RT-PCR detection respectively. According to the detection results, the induced Epoa gene knockout zebrafish embryo homozygotes were obtained .

Specifically, in step 3, the specific process of gene silencing induction is as follows:

In step 3.1, the mixed embryo population of Epoa knockout zebrafish was injected with Epob morpholine reagent for gene silencing at the embryo single-cell or double-cell stage, where: the injection concentration was 6 μg/μl, and the solute was 0.1 M KCL solution; The morpholino sequence is 5'-GCTCCAATGTAATGGCTTACCTGAA-3 (Gene ToolTM);

Step 3.2, incubate in a 28.5°C incubator, collect the successfully hatched embryos 8 hours after embryo fertilization, and continue to incubate in a 28.5°C incubator;

In step 3.3, microtweezers were used to decapsulate the embryos 24 hours after the embryos were fertilized, and the embryos were collected 48 hours after the embryos were fertilized.

Specifically, in step 4, an experimental device is used in the cutting process, and the experimental device is an experimental device for simultaneously performing gene sequencing and phenotype observation of zebrafish embryos, including a main body of the orifice plate, and the main body of the orifice plate is provided with There are reagent holes distributed in an array along the horizontal and vertical directions, and the main body of the orifice plate is equipped with a groove-making top cover and a cutting top cover;

The bottom surface of the groove-making top cover is provided with first top plugs that are matched with the reagent holes in one-to-one correspondence, and each first top plug is provided with the same groove-making plate, which is located in each first top of the same longitudinal row. The groove-making plate on the plug is arranged in the same longitudinal vertical plane;

The bottom surface of the cutting top cover is provided with second top plugs matched with the reagent holes in one-to-one correspondence, each second top plug is provided with the same sample cutting knife, and each second top plug located in the same horizontal row The cutting knives on the top are arranged in the same horizontal and vertical plane;

The horizontal vertical plane where the sample cutting knife is located is perpendicular to the longitudinal vertical plane where the groove-making plate is located;

The vertical length of the sample cutting knife is greater than the vertical length of the groove-making plate.

The grooved plate is located in the longitudinal vertical plane where the longitudinal diameter of the end face of the first top plug is located.

The sample cutting knife is located on a transverse cutting chord parallel to the transverse diameter on the end face of the second plug, and the distance between the transverse cutting chord and the center of the end face of the second plug is 30% of the diameter of the second plug.

The four corners of the top surface of the orifice plate body are provided with guide pins, the groove-making top cover is provided with first guide through holes that cooperate with the guide pins, and the cutting top cover is provided with guide pins that cooperate with the guide pins. The second guide through hole.

The main body of the orifice plate is a 96-well plate.

Compared with the prior art, the present invention has the following technical effects:

(I) The method of the present invention proposes to induce the homozygous hemoglobin phenotype of Epoa gene knockout zebrafish embryos by silencing the Epob gene, the method is simple to operate, and the induction effect is stable and obvious. In the process of the method of the present invention, gene sequencing and RT-PCR assay can be simultaneously performed on a single zebrafish embryo.

(II) The device of the present invention can simply and accurately realize gene sequencing and phenotype observation of zebrafish embryos, and provide a simple and feasible experimental device for zebrafish research.

(III) The device of the present invention can cut a plurality of zebrafish embryo samples at one time, and each sample can be accurately cut into one-fifth and four-fifth parts, which is standard for sample preparation and increases the accuracy of the experiment.

Description of drawings

Figure 1 shows the hemoglobin staining of zebrafish embryos under different conditions of control, Epoa gene down-regulation and Epoa gene knockout at 48 hours, suggesting that there may be gene compensation in Epoa gene knockout zebrafish.

Figure 2 is a schematic diagram of the separation of zebrafish body and tail under the microscope.

Figure 3 shows the results of gene sequencing of Epoa gene knockout homozygous, heterozygous, and wild-type zebrafish embryos.

Figure 4 is a comparison chart of the expression levels of Epoa and Epob in Epoa gene knockout homozygotes, heterozygotes, and wild-type zebrafish embryos.

FIG. 5 is a schematic diagram of the external overall structure of the present invention.

Fig. 6 is a schematic view of the structure of the main body of the orifice plate.

FIG. 7 is a schematic view of the front structure of the groove-making plate.

Fig. 8 is a left side view of the structure of the groove-making plate.

Figure 9 is a schematic view of the front structure of the sample cutting knife.

Figure 10 is a schematic view of the structure of the left side of the cutting knife.

Fig. 11 is a schematic diagram of the use state of the trough-making plate.

Figure 12 is a schematic diagram of the use state of the sample cutter.

Figure 13 is a schematic diagram of the effect of hemoglobin staining in zebrafish embryos.

Figure 14 is the Epob real-time quantitative PCR results.

The meaning of each label in the figure is: 1- well plate body, 2- reagent well, 3- groove top cover, 4- cutting top cover, 5- guide pin, 6- agar medium, 7- groove; 31- The first top plug, 32 - the groove plate, 33 - the first guide through hole; 41 - the second top plug, 42 - the sample cutter, 43 - the second guide through hole.

The specific content of the present invention will be further explained in detail below in conjunction with the embodiments.

Detailed ways

As shown in Figure 1, our research group found in the construction of Epoa knockout zebrafish using CRISPR technology, Epoa knockout zebrafish larvae can survive through the oxygen in the external environment within 10 days of embryonic development, so it is possible The Epoa gene knockout zebrafish was constructed to realize the phenotype observation of Epoa gene knockout in the embryonic stage. However, further research results suggest that there are different hemoglobin staining phenotypes in Epoa knockout zebrafish embryos constructed by CRISPR technology and Epoa gene knockdown embryos under Morpholino technology. Except that the hemoglobin staining in zebrafish embryos was basically the same as the control normal zebrafish hemoglobin staining. This difference in phenotype suggests that there may be gene compensation in Epoa knockout zebrafish. Reviewing the NCBI gene bank, we found that there is a previously unstudied Epob gene in zebrafish. Then, whether Epob gene silencing can further induce hemoglobin phenotype changes in Epoa knockout zebrafish embryos is the key question of this research method.

However, as mentioned above, since Epoa homozygous adult fish cannot survive, the zebrafish embryos we obtained are the offspring of Epoa heterozygous hybridization. It is necessary to first screen Epoa homozygotes, heterozygotes, and wild-type by gene sequencing, and then cross hybridization. The homozygotes, heterozygotes and wild-type obtained from Epoa were silenced by Epob gene, and then the hemoglobin phenotypes of Epoa homozygotes, heterozygotes and wild-type were observed respectively.

The purpose of the present invention is to provide a method for simultaneously performing gene sequencing verification of Epoa heterozygous hybrid embryos and compensatory expression of Epob for the gene compensation of Epoa gene knockout zebrafish embryos. The purpose of the present invention is to propose a method for verifying the gene compensation of the embryos after hybridization of the gene knockout heterozygous zebrafish adult fish. The purpose of the present invention is also to provide a primer design and RT-PCR verification method for Epob gene expression after constructing Epoa gene knockout zebrafish animal model based on CRISPR technology.

The Epoa gene in the present invention refers to the zebrafish Erythropoietin a gene, and the NCBIGene ID of the Epoa gene is: 100004455; Ensembl: ENSDARG00000055163; FIN: ZDB-GENE-061218-3.

The Epob gene described in the present invention refers to the zebrafish Erythropoietin b gene, and the NCBIGene ID of the Epob gene is: 100002479; Ensembl: ENSDARG00000100737; ZFIN: ZDB-GENE-141216-428.

The gene sequences of Epoa gene and Epob gene can be obtained by querying according to the index ID of the above-mentioned National Center for Biotechnology Information.

Specific embodiments of the present invention are given below. It should be noted that the present invention is not limited to the following specific embodiments, and all equivalent transformations made on the basis of the technical solutions of the present application fall into the protection scope of the present invention.

Example 1:

This example provides a method for inducing the hemoglobin phenotype of homozygous Epoa knockout zebrafish embryos. The method induces Epoa knockout zebrafish by silencing the Epob gene of Epoa knockout zebrafish embryos homozygous Embryos homozygous hemoglobin phenotype.

The method specifically includes the following steps:

Step 1, construction of Epoa gene knockout zebrafish animal model:

1) Establish an E. coli plasmid containing the target sequence of the EPO gene fragment:

1-0) Ensemble database (http://www.ensembl.org) to obtain the zebrafish EPO gene sequence (ENSDART00000020288.9);

1-1) Use Zifit Target Version4.2 for target sequence selection and primer design for the EPO gene exon 2 region, and then synthesize the designed primers;

Among them, the target sequence of the selected EPO exon 2 region is:

CATCTGTGACCTGCGCGT;

Among them, the designed and synthesized primers are:

forward primer TAGGACGCGCAGGTCACAGATG;

reverse primer AAACCATCTGTGACCTGCGCGT;

1-2) Synthesize the E. coli plasmid containing the target sequence of the EPO gene fragment;

1-2A) Primer annealing: mix 2 μL forward primer, 2 μL reverse primer, 2 μL NEB buffer, 14 μL distilled water, incubate the mixture at 95°C for 5 minutes, then cool down to 50°C at a rate of 0.1°C/sec, incubate at 50°C for 10 minutes, Then cool down to 4°C at 1°C/sec to obtain annealed oligomers;

1-2B) Restriction: 1 μL of annealed oligos obtained from 1-2A), 400 ng pT7-gRNA plasmid, 1 μL NEB buffer, 1 μL T4 DNA ligase, 0.5 μL BsmBI enzyme, 0.3 μL BglII enzyme, 0.3 μl SalI enzyme , 0.5 μL T4 ligase and DNA-free water were mixed, and the total volume after mixing was 10 μL; the mixture was first incubated at 37 °C for 60 min, then at 16 °C for 45 min, and so on for three cycles; then the mixture was heated to 37 °C and incubated for 30 min, Then heat up to 55°C and incubate for 30min, then heat up to 80°C and incubate for 15min, and cool down to 4°C to limit the endocut product;

1-2C) Transfer the restriction endonuclease product into Escherichia coli: store Escherichia coli in a -80°C refrigerator, place it on ice for 20 to 30 min after taking it out, and combine the restriction endonuclease product synthesized in step 1-2B) with 50μL of E. coli was mixed, the mixture was placed on ice for 20min, then placed in a water bath at 42°C for 90sec, and then placed on ice for at least 90sec; 1mL of LB culture medium was added to the above mixture and incubated at 37°C, 220rpm incubator for 45min. Then, the above mixture was centrifuged at 6000 rpm at 4°C for 5 minutes, and 900 μL of the supernatant after centrifugation was taken, spread in LB medium, and cultured in a constant temperature incubator at 37°C overnight;

1-2D) Select a single colony from the overnight cultured colony in 3 mL of culture solution, cultivate overnight at 37°C and 220 rpm; use the GETM Healthcare Illustra kit to extract the plasmid for gene sequencing; obtain the target sequence containing the target gene sequence (EPO exon 2 region target sequence). : the plasmid of CATCTGTGACCTGCGCGT) is a positive plasmid;

2) Establish and cultivate an EPO knockout zebrafish model based on CRISPR gene knockout technology:

2-1) Mix 1～3ug of positive plasmid containing the target sequence, 5μL of NEB 3:1 buffer, 5μL of 10% BSA, and 1μL of BamHI-HF, adjust the volume to 50μL with DNA-free water, and water bath at 37°C overnight; add 100ug /mL proteinase K 0.5μL and 0.5% SDS 2.5μL, heated at 50°C for 20min, using QiagenTM PCR purification kit to extract DNA, agar gel electrophoresis to verify, the target DNA was obtained;

T7Transcription Kit(InvitrogenTM)将目标DNA逆转录为EPO gRNA；使用不含目的Epo靶序列的空白质粒(即不含有EPO外显子2区域靶序列的质粒)提取对照RNA(即无法造成基因敲除的对照序列)；2-2) Use

T7Transcription Kit (InvitrogenTM) reverse-transcribes the target DNA into EPO gRNA; uses a blank plasmid without the target Epo target sequence (that is, a plasmid that does not contain the target sequence of the EPO exon 2 region) to extract the control RNA (that is, the one that cannot cause gene knockout) control sequence);

2-3) The Cas9 plasmid was from AddgeneTM (Plasmid#63154), and Cas9 RNA was synthesized using T7/T3 Transcription Kit (InvitrogenTM);

2-4) Mix EPO gRNA and Cas9RNA in 0.1mol/L KCl solute, and inject into zebrafish zygote single cell; the concentration of EPO gRNA after mixing is 200pg/μL, and the concentration of Cas9RNA after mixing is 200pg/μL. Mixed injection of control RNA and Cas9 was used as a control observation group;

Embryos genomic DNA was extracted for PCR amplification 72 hours after fertilization. Perform gene sequencing and use ClustalW2 (http://www.ebi.ac.uk/Tools/msa/clustalw2/) for sequence alignment to further clarify the effectiveness of gene knockout;

2-5) After injecting EPO gRNA to obtain a chimeric fish (ie, a chimeric zebrafish containing a variety of mixed gene mutant cells), culture it to the size of an adult fish in about 3 months, take the fin tissue for gene sequencing, and sequence the gene sequencing-positive adult fish. EPO+/- heterozygous zebrafish were obtained by crossing with wild background zebrafish, and EPO-/- homozygous zebrafish embryos were obtained by crossing adult heterozygous background zebrafish. Homozygous gene variant sequences were determined by gene sequencing results.

Step 2, to obtain a mixed embryo population:

A mixed population of homozygous, heterozygous and wild-type embryos containing the Epoa gene was obtained by crossing Epoa heterozygous adult zebrafish;

The specific process of step 2 is as follows:

Step 2.1, one day before mating, put one male and one male Epoa heterozygous adult zebrafish into a special incubator for zebrafish, and isolate them with a partition;

Step 2.2, take out the separator the next day, and zebrafish freely mate for 2-4 hours;

Step 2.3, put the mated zebrafish back into the fish tank, and use a mesh screen to collect the zebrafish eggs;

Step 2.4, add zebrafish special E3 culture medium, select successfully hatched embryos and transfer them to a clean sterile petri dish, add zebrafish special E3 culture medium again, and incubate in a 28.5 ℃ incubator for 24 hours;

In step 2.5, the embryos were taken out after 24 hours, and the embryos were ruptured using the special sharp forceps for the microscope. After the ruptured membranes were screened, the intact embryos were added to the special E3 medium for zebrafish, and transferred to a 28.5°C incubator for further incubation for 24 hours, that is, 48 hours after the embryos were fertilized. A mixed population of embryos was obtained.

Step three, gene silencing induction:

Perform gene silencing on the Epob gene of the mixed embryo group obtained in step 2 to induce the hemoglobin phenotype of Epoa gene knockout zebrafish embryos;

The specific process of gene silencing induction is as follows:

In step 3.1, the mixed embryo population of Epoa knockout zebrafish was injected with Epob morpholine reagent for gene silencing at the embryo single-cell or double-cell stage, where: the injection concentration was 6 μg/μl, and the solute was 0.1 M KCL solution; The morpholino sequence is 5'-GCTCCAATGTAATGGCTTACCTGAA-3 (Gene ToolTM);

Step 4.2, incubate in a 28.5°C incubator, collect successfully hatched embryos 8 hours after embryo fertilization, and continue to incubate in a 28.5°C incubator;

In step 4.3, microtweezers were used to decapsulate the embryos 24 hours after the embryos were fertilized, and the embryos were collected 48 hours after the embryos were fertilized.

As shown in Figure 13, under Epob gene silencing and control conditions, zebrafish embryos were stained for hemoglobin at 48 h under the control, ie, Epoa gene knockout condition, and the hemoglobin decreased after Epob silencing compared with the control.

As shown in Figure 14, A: the expression of Epob in zebrafish embryos was significantly reduced after Epob silencing compared with the control; B: the morpholine binding site of zebrafish Epob.

Step 4: Obtain the embryo homozygote of Epoa knockout zebrafish after induction:

Cut a part of 1/5 of the total length of the tail of each embryo in the induced mixed embryo group obtained in step 3, and perform genomic DNA extraction and gene sequencing for inspection;

An experimental device is used in the cutting process, and the experimental device is an experimental device for simultaneously performing gene sequencing and phenotype observation of zebrafish embryos, as specifically described in Example 2.

The remaining part of each embryo was subjected to RNA extraction and cDNA reverse transcription, and the obtained cDNA was subjected to Epob and Epoa gene expression RT-PCR detection respectively. According to the detection results, the induced Epoa gene knockout zebrafish embryo homozygotes were obtained .

This step yields Figures 3 and 4.

It can be seen from Figure 3: (a) Epoa gene knockout zebrafish wild-type, heterozygous, homozygous gene sequences and gene sequencing results. (b) CRISPR gene knockout target sequence: CATCTGTGACCTGCGCGT. (c) The sequencing result of the wild-type zebrafish target is consistent with the gene sequence, and the sequencing result obtained using reverse PCR primers is:

AGGACGCGCAGGTCACAGATGG, gene sequencing waveform did not see other waveforms. (d) The homozygous target sequencing results were inconsistent with the gene sequence, the terminator ATT appeared after AGGACGCGCAGGT, and no other waveforms were found in the gene sequencing waveform. (e) Heterozygote sequencing results are determined according to the dominant base, and homozygous and wild-type gene sequencing waveforms can be seen in the gene sequencing waveforms.

It can be seen from Figure 4 that: (a) the relative expression levels of Epoa gene knockout zebrafish wild-type, heterozygous, and homozygous; the expression level is 1 with wild-type as a reference. (b) Epoa is not expressed in homozygotes and is intermediate between the expression in heterozygotes. (c) The expression of Epob in homozygotes is about twice that of wild type, and the expression in heterozygotes is not significantly different from wild type.

Specifically, as shown in Figure 2, the zebrafish embryos were separated into B head and C tail with microscope-specific sharp tweezers at part A, which were used for RT-PCR expression sequencing and genome sequencing respectively. 48h after embryo fertilization, transfer the to-be-observed plate to a 96-well plate, and put 1 embryo in each well; use the microscope-specific sharp forceps to clip off 1/5 of the tail of the zebrafish, and remove 4/5 of the body of the zebrafish. Transfer to EP tubes for subsequent RT-PCR validation, and transfer 1/5 of the zebrafish tail to PCR tubes for genomic DNA extraction.

Specifically, the process of RT-PCR verification of the 4/5 part of the zebrafish body is as follows:

D30，EppendorfTM)测定mRNA浓度。(a) Single embryo mRNA extraction was performed using RNeasy Mini Kit (Cat No./ID: 74104, QiagenTM), and a spectrophotometer (Eppendorf

D30, EppendorfTM) to measure mRNA concentration.

First Strand cDNA Synthesis Kit(E6300S，BioLabsTM)对步骤(a)中获得mRNA进行cDNA提取。(b) use

First Strand cDNA Synthesis Kit (E6300S, BioLabsTM) performs cDNA extraction on the mRNA obtained in step (a).

(c) The extracted cDNA was verified by RT-PCR against Epoa, Epob and the housekeeping gene β-actin, and the primer sequences for verification are shown in Table 1.

Table 1 | RT-PCR primer sequences

Specifically, the genomic DNA extraction and sequencing process for the 1/5 part of the zebrafish tail is as follows:

(a) Put 1/5 part of zebrafish tail into RT-PCR tube, numbered to mark;

(b) use a syringe to remove excess liquid from the tube;

(c) Add 20 μL of lysis buffer (10 mM Tris pH=8, 1 mM EDTA, 0.3% Tween, 0.3% Glycerol) to each tube;

(d) put it into a PCR machine at 98°C for 10min;

(e) Add 10uL proteinase K (10mg/mL) to each tube;

(f) Continue to put it into the PCR apparatus at 55°C for more than 5 hours, and then at 98°C for 10 minutes, and mix the obtained materials to obtain genomic DNA;

(g) Take 4.25 μL of genomic DNA, 6.25 μL of Taq Mix Green, and 1 μL of pre-sequence and reverse-sequence primers for PCR amplification of the Epoa gene fragment; the sequences of Epoa PCR amplification primers are shown in Table 2.

PCR扩增引物序列Table 2|

PCR amplification primer sequences

(h) PCR process: 95℃-3min; (95℃-30s, 57℃-30s, 72℃-45s)*37 cycles; 72℃-10min; 4℃-∞;

(i) DNA purification using QIAquick PCR Purification Kit (Cat No./ID: 28104, QiagenTM)

(j) Send the purified DNA to a gene sequencing company for testing and verification.

Example 2:

This embodiment provides an experimental device for simultaneous gene sequencing and phenotype observation of zebrafish embryos. As shown in Figures 5 to 12, it includes a well plate body 1, and the well plate body 1 is provided with horizontal and vertical directions. The reagent wells 2 are distributed in an array, and the well plate main body 1 is equipped with a groove-making top cover 3 and a cutting top cover 4;

The bottom surface of the groove-making top cover 3 is provided with first top plugs 31 that are matched with the reagent holes 2 in one-to-one correspondence, and each first top plug 31 is provided with the same groove-making plate 32, which is located in the same longitudinal row. The groove-making plates 32 on each first top plug 31 are arranged in the same longitudinal vertical plane;

The bottom surface of the cutting top cover 4 is provided with second top plugs 41 that are matched with the reagent holes 2 in one-to-one correspondence, and each second top plug 41 is provided with the same sample cutting knife 42, which is located in each of the same horizontal row. The sample cutting knives 42 on the second top plugs 41 are arranged in the same horizontal vertical plane;

The transverse vertical plane where the sample cutting knife 42 is located is perpendicular to the longitudinal vertical plane where the groove-making plate 32 is located;

The vertical length of the sample cutting knife 42 is greater than the vertical length of the groove-making plate 32 .

As a preferred solution of this embodiment, the groove-making plate 32 is located in the longitudinal vertical plane where the longitudinal diameter of the end face of the first top plug 31 is located.

As a preferred solution of this embodiment, the sample cutting knife 42 is located on a transverse cutting chord on the end face of the second top plug 41 that is parallel to the transverse diameter, and the distance between the transverse cutting chord and the center of the end face of the second top plug 41 is 30% of the diameter of the second top plug 41 . This enables each sample to be accurately cut into fifths and fourths.

As a preferred solution of this embodiment, four corners of the top surface of the orifice plate main body 1 are vertically provided with guide pins 5 , and the groove-making top cover 3 is provided with first guide through holes 33 that cooperate with the guide pins 5 , The cutting top cover 4 is provided with a second guide through hole 43 which is matched with the guide pin 5 . Easy to align.

As a preferred solution of this embodiment, the orifice plate body 1 is a 96-well plate.

When the device of this embodiment is used, firstly, the agar is heated and dissolved and introduced into the reagent well 2 of the orifice plate body 1 to form the agar medium 6, and the tank top cover 3 is covered so that the first top plug 31 is closed. On the reagent well 2, the trough-making plate 32 extends into the reagent well 2 and is submerged in the agar, rests for a few minutes and waits for the agar to cool, then removes the trough-making top cover 3, and a groove 7 is formed on the agar medium 6.

Then, place the zebrafish embryo on the groove 7 according to the observation position required for the experiment, add zebrafish culture medium and anesthetic, and observe under the corresponding microscope.

Finally, cover and cut the top cover 4, and cut the fish tail of the zebra embryo. After the cutting is completed, remove the cutting top cover 4, and use a sampler to suck up the tail cut tissue from the top blank, and put it into the corresponding container for genomic DNA extraction. ; The remaining part of the zebrafish embryo can continue to be observed in subsequent cultivation.

Claims (8)

1. A method for inducing Epoa gene knockout zebrafish embryo homozygote hemoglobin phenotype is characterized in that the Epoa gene knockout zebrafish embryo homozygote hemoglobin phenotype is induced by carrying out gene silencing on Epob genes of Epoa gene knockout zebrafish embryo homozygotes.

2. The method according to claim 1, characterized in that it comprises in particular the steps of:

step one, constructing an Epoa gene knockout zebra fish animal model:

1) establishing an Escherichia coli plasmid containing an EPO gene fragment target sequence:

1-0) acquiring a zebra fish EPO gene sequence ENSDART00000020288.9 by an Ensemble database, wherein the website address of the database is http:// www.ensembl.org;

1-1) performing Target sequence selection and primer design using Zifit Target version4.2 for an EPO gene exon 2 region, and then synthesizing the designed primer;

wherein the selected EPO exon 2 region has the target sequence:

CATCTGTGACCTGCGCGT；

wherein the designed and synthesized primers are as follows:

a forward primer TAGGACGCGCAGGTCACAGATG;

reverse primer AAACCATCTGTGACCTGCGCGT;

1-2) synthesizing an Escherichia coli plasmid containing an EPO gene fragment target sequence;

1-2A) primer annealing: mixing 2 mu L of forward primer, 2 mu L of reverse primer, 2 mu L of EB buffer solution and 14 mu L of distilled water, incubating the mixture for 5min at 95 ℃, then cooling to 50 ℃ at the speed of 0.1 ℃/sec, incubating for 10min at 50 ℃, and then cooling to 4 ℃ at the speed of 1 ℃/sec to obtain annealing oligomer;

1-2B) restriction endonuclease: 1. mu.L of the annealed oligomer obtained in 1-2A), 400ng of pT7-gRNA plasmid, 1. mu.L of NEB buffer, 1. mu. L T4DNA ligase, 0.5. mu.L of BsmBI enzyme, 0.3. mu.L of BglII enzyme, 0.3. mu.L of SalI enzyme, 0.5. mu. L T4 ligase and DNA-free water were mixed together, and the total volume after mixing was 10. mu.L; the mixture is firstly incubated at 37 ℃ for 60min and then at 16 ℃ for 45min, and the process is circulated for three times; then heating the mixture to 37 ℃ and incubating for 30min, then heating to 55 ℃ and incubating for 30min, heating to 80 ℃ and incubating for 15min, and cooling to 4 ℃ to obtain a restriction endonuclease product;

1-2C) transfer of restriction products into E.coli: storing Escherichia coli in a refrigerator at-80 deg.C, taking out, standing on ice for 20-30 min, mixing the restriction endonuclease product synthesized in step 1-2B) with 50 μ L of Escherichia coli, standing the mixture on ice for 20min, standing in water bath at 42 deg.C for 90sec, and standing on ice for at least 90 sec; adding 1mLLB culture solution into the mixture, culturing for 45min in a incubator culture plate at 37 ℃ and 220rpm, centrifuging the mixture for 5min in a 4 ℃ centrifuge at 6000rpm, taking 900 mu L of centrifuged supernatant, spreading in LB culture medium, and culturing at 37 ℃ in the incubator overnight;

1-2D) selecting a single colony from overnight culture flora, placing the single colony in 3mL culture solution, and carrying out overnight culture at 37 ℃ and 220 rpm; extracting plasmids by using GETMEALThcare Illustra kit for gene sequencing; obtaining a plasmid containing a target gene sequence as a positive plasmid, wherein the EPO exon 2 region target sequence: CATCTGTGACCTGCGCGT, respectively;

2) an EPO gene knockout zebra fish model is established and cultivated based on CRISPR gene knockout technology:

2-1) mixing 1-3 ug of positive plasmid containing target sequence, 5 muL of NEB 3:1 buffer solution and 5 muL of 10% BSA 5 mu L, BamHI-HF1 muL, adjusting the volume to 50 muL without DNA water, and carrying out 37 ℃ overnight water bath; adding 100ug/mL proteinase K0.5 μ L and 0.5% SDS 2.5 μ L, heating at 50 deg.C for 20min, extracting DNA with Qiagen (TM) PCR purification kit, and performing agarose gel electrophoresis to obtain target DNA;

2-2) use of

T7Transcription Kit，

The trademark brand of T7Transcription Kit is Invitrogen, and target DNA is reversely transcribed into EPO gRNA; extracting control RNA by using a blank plasmid without a target Epo target sequence, wherein the blank plasmid without the target Epo target sequence does not contain an EPO exon 2 region target sequence; the control RNA is a control sequence which cannot cause gene knockout;

2-3) Cas9 plasmid from the #63154 plasmid under the trademark Addgene, Cas9RNA was synthesized using the T7/T3Transcription Kit, the trademark of T7/T3Transcription Kit is Invitrogen;

2-4) mixing EPO gRNA and Cas9RNA in 0.1mol/LKCl solute, and injecting in zebra fish fertilized egg single cells; the concentration of EPO gRNA after mixing is 200 pg/mu L, the concentration of Cas9RNA after mixing is 200 pg/mu L, and control RNA and Cas9 are mixed and injected to be used as a control observation group;

extracting embryo genome DNA for PCR amplification after 72 hours of fertilization, carrying out gene sequencing, and carrying out sequence comparison by using ClustalW2 to further confirm the gene knockout effectiveness, wherein the website of a database of ClustalW2 is http:// www.ebi.ac.uk/Tools/msa/ClustalW 2/;

2-5) obtaining chimeric fish after EPO gRNA injection and culturing the chimeric fish to the size of adult fish about 3 months, obtaining the chimeric fish after EPO gRNA injection, namely chimeric zebra fish containing multiple mixed gene mutant cells, taking fin tissue gene sequencing, hybridizing the gene sequencing positive adult fish with wild background zebra fish to obtain EPO +/-heterozygote zebra fish, hybridizing the adult heterozygote background zebra fish to obtain EPO-/-homozygote zebra fish embryos, and judging a homozygote gene variant sequence through a gene sequencing result;

step two, obtaining a mixed embryo group:

obtaining a mixed population of embryos comprising homozygotes, heterozygotes and wild types of the Epoa gene by crossing adult zebrafish of the Epoa heterozygotes;

step three, gene silence induction:

carrying out gene silencing on the Epob gene of the mixed embryo group obtained in the step two to induce the Epoa gene to knock out the hemoglobin phenotype of the zebra fish embryo;

step four, obtaining an induced Epoa gene knockout zebra fish embryo homozygote:

performing genome DNA extraction and censored gene sequencing on the part of the total length of each embryo with the cut tail 1/5 in the induced mixed embryo group obtained in the third step;

and (3) carrying out RNA extraction and cDNA reverse transcription on the remaining embryo part of each embryo, respectively carrying out gene expression RT-PCR detection on Epob and Epoa on the obtained cDNA, and obtaining the embryo homozygote of the induced Epoa gene knockout zebra fish according to the detection result.

3. The method of claim 2, wherein in step three, the specific process of gene silence induction is:

step 3.1, injecting Epob morpholine reagent to perform gene silencing in a single-cell or double-cell stage of an embryo of a mixed embryo group of Epoa gene knockout zebra fish, wherein: KCL solution with injection concentration of 6 μ g/μ l and solute of 0.1M; morpholine sequence 5'-GCTCCAATGTAATGGCTTACCTGAA-3';

step 3.2, culturing in a constant temperature box at 28.5 ℃, collecting successfully hatched embryos 8 hours after embryo fertilization, and continuously culturing in the constant temperature box at 28.5 ℃;

and 3.3, performing embryo demoulding by using micro forceps 24h after embryo fertilization, and collecting embryos 48h after embryo fertilization.

4. The method as claimed in claim 2, wherein in the fourth step, an experimental device is adopted in the cutting process, the experimental device is an experimental device for simultaneously performing gene sequencing and phenotype observation on zebra fish embryos, and comprises a pore plate main body (1), reagent holes (2) are arranged on the pore plate main body (1) in an array manner along the transverse direction and the longitudinal direction, and the pore plate main body (1) is matched with a groove-making top cover (3) and a cutting top cover (4);

the bottom surface of the groove-making top cover (3) is provided with first top plugs (31) which are correspondingly matched with the reagent holes (2) one by one, each first top plug (31) is provided with the same groove-making plate (32), and the groove-making plates (32) on each first top plug (31) positioned in the same longitudinal column are distributed in the same longitudinal vertical plane;

the bottom surface of the cutting top cover (4) is provided with second top plugs (41) which are correspondingly matched with the reagent holes (2) one by one, each second top plug (41) is provided with the same sample cutting knife (42), and the sample cutting knives (42) on each second top plug (41) in the same transverse row are distributed in the same transverse vertical plane;

the transverse vertical plane of the sample cutting knife (42) is mutually vertical to the longitudinal vertical plane of the groove making plate (32);

the vertical length of the sample cutting knife (42) is greater than that of the groove making plate (32).

5. A method according to claim 4, characterized in that the grooving plate (32) is located in a longitudinal vertical plane in which the longitudinal diameter of the end face of the first plug (31) is located.

6. The method according to claim 4, characterized in that the sample cutting knife (42) is located on a transverse cutting chord parallel to the transverse diameter of the end face of the second plug (41), and the distance of the transverse cutting chord from the center of the end face of the second plug (41) is 30% of the diameter of the second plug (41).

7. The method according to claim 4, characterized in that the top surface of the orifice plate body (1) is vertically provided with guide pins (5) at four corners, the grooving top cover (3) is provided with first guide through holes (33) matched with the guide pins (5), and the cutting top cover (4) is provided with second guide through holes (43) matched with the guide pins (5).

8. The method according to claim 4, wherein the well plate body (1) is a 96 well plate.

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