CN111793653B - Construction method of dpy19l1l gene deletion type zebra fish - Google Patents
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Abstract
The invention relates to the technical field of gene knockout and zebra fish models, in particular to a construction method of a dpy19l1l gene deletion type zebra fish. Designing a proper targeting site on a dpy19l1l gene of the zebra fish, microinjecting the specific gRNA and Cas9 protein synthesized in vitro into a zebra fish cell, culturing the embryo for 48 hours, and then carrying out genotype analysis by selecting the embryo, thereby confirming the effectiveness of the selected site, and obtaining the stable genetic dpy19l1l homozygous mutant zebra fish strain through culture. The invention can silence specific genes in the genome of an organism more efficiently and accurately, has simple manufacture and low cost, can simultaneously cut a plurality of sites on a target gene, silence any number of single genes, knock out the development deformity of dpy19l1l gene zebra fish embryos, construct successful dpy19l1l mutant congenital spinal curvature, provide a good zebra fish model for a spinal curvature model, and is helpful for researching bone related diseases.
Description
Technical Field
The invention relates to the technical field of gene knockout and zebra fish models, in particular to a construction method of a dpy19l1l gene deletion type zebra fish.
Background
The DPY19L1L gene is located on chromosome 16 of zebra fish, and is a member of the DPY19 gene family, which encodes the DPY19 (dumpy-19, DPY-19) protein family, including DPY19L1 (DPY-19-like 1), DPY19L2, DPY19L3 and DPY19L4, which has mannose transferase activity as a classical function. DPY19L1 protein is multi-transmembrane protein, mainly participates in nervous system development, regulates radial migration of glutamatergic neurons in the process of cerebral cortex development, and is simultaneously required for axon growth. The DPY19L2 gene plays an important role in the spermatogenesis process, and the gene deletion is an important causative factor of the oligospermia. Studies have indicated that the sterility of a round-headed sperm is related to a balance between migration of the DPY19L2 gene and gene deletion at chromosomal breakpoints. In conclusion, the DPY19 gene mainly plays a role in the processes of neural development and spermatogenesis, and is not reported in the fields related to skeletal development and diseases, so that the research on the DPY19L1L gene is deficient.
Based on the above, it is important to provide a DPY19L1L gene deletion type zebra fish and to excavate the new function and genetic mechanism of the DPY19L1L gene in the development of the zebra fish.
Disclosure of Invention
The invention aims to provide a construction method of a dpy19l1l gene deletion type zebra fish, wherein in the invention, the dpy19l1l gene is related to zebra fish bone development, and can be used for researching a deep molecular mechanism of zebra fish bone development.
In order to achieve the above purpose, the invention adopts the following technical scheme:
a construction method of dpy19l1l gene deletion type zebra fish comprises the following steps:
s1, inquiring a genomic DNA sequence of a dpy19l1l gene of the zebra fish on NCBI, analyzing a functional domain of the genomic DNA sequence, finding a target site of the dpy19l1l gene from an exon 1 of the dpy19l1l gene according to a CRISPR/Cas9 knockout principle, designing a target site long primer and a gDNA joint primer, wherein the sequence of the target site long primer of the dpy19l1l is 5'-Taatacgactcactatag GGAATAACAGTGCTGATTCT gttttagagctagaaatagc-3', gDNA joint primer sequence 5'-AGCACCGACTCGGTGCCACTT-3';
s2, obtaining an in vitro transcription template of the gRNA by primer star PCR;
s3, in-vitro transcription is carried out according to the gRNA in-vitro transcription template obtained in the S2, and the gRNA is extracted and purified to obtain purified gRNA;
s4, microinjecting the purified gRNA and Cas9 protein to fertilized eggs of the zebra fish in a cell stage, and culturing to obtain a stable inheritance dpy-19l1l homozygous mutant zebra fish strain, namely dpy19l1l gene deletion type zebra fish.
Further, the S2 reaction system is as follows: gDNA Vector Template 1. Mu.l, 1. Mu.l of target site long primer, gDNA adapter primer, 1. Mu.l of dNTP Mix, 5X Primer star buffer. Mu.l, primer star DNApolymerase. Mu.l, ddH 2 O32 μl; the gDNA Vector Template is obtained by cloning Cas9cDNAs with double NLS into a pXT7 vector and linearizing with XbaI endonuclease.
Further, the amplification procedure of S2 is: 98 ℃ for 2min; the following steps were repeated for 35 cycles: 98℃15s,58℃15s,72℃20s;72 ℃ for 5min; 30min at 72 ℃.
Further, the transcription system of S3 is: template DNA10ul, 10X RNA polymerase Reaction buffer2ul,25mM rNTP Mix0.8ul,RNase Inhibitor0.5ul,T7 RNA polymerase2ul,RNase free H 2 O4.7ul。
Further, the transcription system is: 37℃for 3h.
Further, the culturing of S4 specifically comprises the following steps:
s1, culturing and hatching microinjected fertilized eggs, detecting the effectiveness of target sites by Sanger sequencing, screening out gene mutation, marking as F0 generation, and breeding to adult fish;
s2, laterally crossing the F0 adult fish with the WT to obtain an F1 generation;
s3, determining F1 generation of dpy19l1l gene knockout by using a genotype identification method;
s4, selecting F1 generations with the same mutation type from the F1 generation mutants to mate to obtain F2 generation;
s5, genotype identification is carried out on the homozygous of the dpy19l1l gene knockout in the F2 generation, namely the stable inheritance dpy-19l1l homozygous mutant zebra fish strain, namely the dpy19l1l gene deletion type zebra fish.
Compared with the prior art, the invention has the following beneficial effects:
(1) The invention can silence specific genes in the genome of an organism more efficiently and accurately, has simple manufacture and low cost, can cut a plurality of sites on a target gene at the same time, silence any number of single genes, and knock out the development malformation of dpy19l1l gene zebra fish embryos.
(2) The DPY19l1 gene deletion type zebra fish congenital spinal curvature successfully constructed by the invention provides a good zebra fish model for a spinal curvature model, and is beneficial to researching bone related diseases and screening medicines for treating or relieving bone diseases. .
Drawings
FIG. 1 is a peak plot of wild type and Dpy19l1l heterozygotes py19l1l gene sequences of example 1, wherein A is wild type, B is one heterozygote and C is another heterozygote.
FIG. 2 is an alignment of wild type and Dpy19l1l heterozygotes py19l1l gene sequences and base mutation of example 1, wherein A is wild type, B is one heterozygote and C is another heterozygote.
FIG. 3 is a comparison of wild type and dpy19l1 gene deleted zebra fish phenotypes of example 1, wherein A is wild type and B is dpy19l1 gene deleted zebra fish.
Detailed Description
The present invention will now be described in detail with reference to the drawings and specific examples, which should not be construed as limiting the invention. Unless otherwise indicated, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples are commercially available unless otherwise indicated.
Example 1
Construction method of dpy19l1l gene deletion type zebra fish
S1, inquiring a genomic DNA sequence of a dpy19l1l gene of the zebra fish on NCBI, analyzing a functional domain of the genomic DNA sequence, finding a target site of the dpy19l1l gene from an exon 1 of the dpy19l1l gene according to a CRISPR/Cas9 knockout principle, and designing a target site long primer, wherein the sequence is 5'-GGAATAACAGTGCTGATTCT-3', as shown in SEQ ID NO.1, and the sequence of the dpy19l1l target site long primer is 5'-Taatacgactcactatag GGAATAACAGTGCTGATTCT gttttagagctagaaatagc-3', as shown in SEQ ID NO. 2;
s2, obtaining an in vitro transcription template of gRNA by primer star PCR, wherein the total reaction volume is 50 μl, and the system is shown in Table 1:
TABLE 1 primer star PCR System
The gDNA Vector Template is obtained by cloning Cas9cDNAs with double NLS into a pXT7 vector and linearizing with XbaI endonuclease;
the sequence of gDNA adaptor Primer is 5'-AGCACCGACTCGGTGCCACTT-3'; as shown in SEQ ID NO. 3.
The T7 promoter and target site carried on the long primer are connected to gDNA Vector through PCR mode in vitro amplification, then DNA fragment is recovered through DNA purification technology mode, and the concentration of the DNA fragment is determined, and the fragment is the in vitro transcription template.
After the reaction system was mixed uniformly, primer star PCR was performed on a PCR apparatus for amplification reaction, and the reaction procedure was as shown in Table 2:
TABLE 2 primer star PCR amplification procedure
The final PCR product concentration was 31.8ng/ul and OD was 1.85.
S3, carrying out in-vitro transcription according to the in-vitro transcription template obtained in the step S2, wherein the total reaction volume is 10ul, and the system is as follows:
TABLE 3 in vitro transcription reaction System
After the system is added, the electrophoresis running gel is detected immediately after the synthesis in an oven at 37 ℃ for 3 hours, and the result is seen. After 1ul DNase I was added, gRNA extraction was performed for 1 h.
Extracting and purifying the successfully transcribed gRNA of S3 by a trizol method, operating according to a kit protocol, and finally adding 10ul of RNase free H 2 O。
The protocols were as follows:
a. adding 500ul of trizol and 100ul of chloroform, shaking vigorously, mixing, standing and layering for 5min.
b. 15000xg maximum rotation speed at 4 ℃ for 15min.
c. Taking the supernatant into a new EP tube, adding an equal volume of pre-cooled isopropanol at-20 ℃, vibrating for 15s, and precipitating at-20 ℃ for half an hour.
d. 15000xg maximum rotation speed at 4 ℃ for 10min.
e. Removing the supernatant, taking 1ml of 75% ethanol, and washing for 2 times to precipitate; shaking for 15s, and carrying out maximum rotation speed of 15000xg at 4 ℃ for 5min.
f. Air-dried on ice, and dissolved by adding 10ul RNase free H2O.
The purified gRNA had a concentration of 583.1ng/ul and an OD of 2.08, and was stored at-80 ℃.
S5, sucking embryos by using a suction tube within 30min after fertilization of the zebra fish, transferring the embryos to a special microinjection culture dish made of agarose, preparing a mixed solution of Cas9 protein and gRNA (ribonucleic acid) before microinjection, enabling the final concentration of the Cas9 protein to be 5 mu g/ul, enabling the final concentration of the gRNA to be 100ng/ul, and injecting 3 mu L of the mixed solution of the Cas9 protein and the gRNA into fertilized eggs in a cell stage; placing the injected fertilized eggs in fish liquid water, and incubating at 28.5 ℃;
the microinjection system is as follows:
table 4 microinjection system
The culture of the step specifically comprises the following steps:
(1) Culturing and incubating microinjected fertilized eggs, detecting the effectiveness of target sites by Sanger sequencing, screening out the gene mutation, marking the gene mutation as F0 generation, and breeding the gene mutation to adult fish;
the Sanger sequencing assay specifically comprises the following steps:
a, extracting genome of zebra fish
After 48 hours of fertilization of zebra fish embryos (48 hpf), wild-type embryos (as control) and post-injection embryos were collected separately in 1.5ml Ep tubes, 10 embryos per tube, and genomic DNA was extracted as follows: 20 μl of 50mm NaOH solution was added to the embryo-filled Ep tube, and the mixture was placed in a metal bath for cleavage at 95deg.C for 15min; after cooling at 4℃2ul Tris-HCl (ph 8.0) was added for neutralization to obtain a crude genomic DNA.
b, PCR amplifying the target sequence
After extracting the genome DNA, designing a primer sequence to amplify a target DNA fragment;
the PCR reaction system is as follows: dpy19l1l sequencing primer: an upper primer (forward primer 5'-GTCTGCGAGAGCCAAACTAC-3' shown as SEQ ID NO. 4) and a lower primer (reverse primer 5'-TTGTTTACAATCCGAGCTCC-3' shown as SEQ ID NO. 5).
c, the PCR product is sent to a Yingjun biological company for sequencing to analyze the insertion and deletion of mutation site bases, the peak diagram and the sequence given after sequencing are compared with a standard target sequence on NCBI, the mutation type of each monoclonal is analyzed according to the comparison result, the result is shown in fig. 1 and 2, the dpy19l1l heterozygote mutant sequence is two, and nonsense mutation is generated by frame shifting.
(2) Laterally crossing the F0 adult fish with the WT to obtain an F1 generation;
(3) Determining F1 generation of dpy19l1l gene knockout by using a genotyping method;
(4) F1 generation of the same mutation type is selected from the F1 generation mutants to mate to obtain F2 generation;
(5) The homozygous of the dpy19l1l gene knockout in the F2 generation is the stable inheritance dpy-19l1l homozygous mutant zebra fish strain, namely the dpy19l1l gene deletion type zebra fish, and the dpy19l1l deletion type zebra fish has a spinal curvature phenotype after screening as shown in figure 3.
Example 2
Application of dpy19l1l gene deletion type zebra fish constructed in example 1
In this example, a study of bone-related diseases was conducted on dpy19l1l gene-deleted zebra fish constructed in example 1, and a drug for treating or alleviating bone diseases was selected to alleviate the bending degree of zebra fish ridges, which was the objective drug.
While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. It is therefore intended that the following claims be interpreted as including the preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.
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Claims (6)
- The application of the dpy19l1l gene in constructing a congenital spinal curvature zebra fish model is characterized in that the construction method of the congenital spinal curvature zebra fish model comprises the following steps:s1, inquiring a genomic DNA sequence of a dpy19l1l gene of the zebra fish, analyzing a functional domain of the genomic DNA sequence, finding a target site of the dpy19l1l gene from an exon 1 of the dpy19l1l gene according to a CRISPR/Cas9 knockout principle, designing a target site long primer and a gDNA joint primer, wherein the sequence of the dpy19l1l target site long primer is 5'-Taatacgactcactatag GGAATAACAGTGCTGATTCT gttttagagctagaaatagc-3', gDNA joint primer sequence 5'-AGCACCGACTCGGTGCCACTT-3';s2, obtaining an in vitro transcription template of the gRNA by primer star PCR;the gDNAVector Template is obtained by cloning Cas9cDNAs with double NLS into a pXT7 vector and linearizing with XbaI endonuclease;the sequence of gDNAadaptor Primer is 5'-AGCACCGACTCGGTGCCACTT-3';s3, in-vitro transcription is carried out according to the gRNA in-vitro transcription template obtained in the S2, and the gRNA is extracted and purified to obtain purified gRNA;s4, microinjection of purified gRNA and Cas9 proteins into fertilized eggs of the zebra fish in a cell stage, and culturing to obtain a stable genetic dpy-19l1l homozygous mutant zebra fish strain, namely a congenital spinal curvature zebra fish model.
- 2. The use according to claim 1, wherein the S2 reaction system is: gDNA Vector Template 1. Mu.l, 1. Mu.l of target site long primer, gDNA adapter primer, 1. Mu.l of dNTP Mix, 5X Primer star buffer. Mu.l, primer star DNA polymerase. Mu.l, ddH 2 O32 μl; the gDNA Vector Template is obtained by cloning Cas9cDNAs with double NLS into a pXT7 vector and linearizing with XbaI endonuclease.
- 3. The use according to claim 2, wherein the amplification procedure of S2 is: 98 ℃ for 2min; the following steps were repeated for 35 cycles: 98℃15s,58℃15s,72℃20s; 5min at 72 ℃; 30min at 72 ℃.
- 4. The use according to claim 3, wherein the transcription system of S3 is: template DNA 100-200ng, 10X RNA polymerase Reaction buffer 1ul,25mM rNTP Mi x 1ul,RNase Inhibitor 1ul,T7 RNApolymerase 0.5ul,RNase free H 2 O was fixed to a volume of 10 ul.
- 5. The use according to claim 4, wherein the transcription conditions are: 37℃for 3h.
- 6. The use according to claim 5, wherein the cultivation of S4 comprises the steps of:s1, culturing and hatching microinjected fertilized eggs, detecting the effectiveness of target sites by Sanger sequencing, screening out gene mutation, marking as F0 generation, and breeding to adult fish;s2, laterally crossing the F0 adult fish with the WT to obtain an F1 generation;s3, determining F1 generation of dpy19l1l gene knockout by using a genotype identification method;s4, selecting F1 generations with the same mutation type from the F1 generation mutants to mate to obtain F2 generation;s5, genotype identification is carried out on the homozygote of the dpy19l1l gene knockout in the F2 generation, namely the stable inheritance dpy-19l1l homozygote mutant zebra fish strain, namely the congenital spinal curvature zebra fish model.
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| CN111793653A (en) | 2020-10-20 |
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