Construction method of dpy19l1l gene-deleted zebra fish
阅读说明：本技术 一种dpy19l1l基因缺失型斑马鱼的构建方法 (Construction method of dpy19l1l gene-deleted zebra fish ) 是由 陆辉强 徐朝鹏 黄勇 廖信军 曹子岗 于 2020-07-09 设计创作，主要内容包括：本发明涉及基因敲除及斑马鱼模型技术领域,具体涉及一种dpy19l1l基因缺失型斑马鱼的构建方法。在斑马鱼的dpy19l1l基因上设计合适的打靶位点,在体外合成的特异性gRNA和Cas9蛋白,显微共注射进入斑马鱼一细胞内,将胚胎培养48h后,通过选取胚胎进行基因型分析,从而证实了所选位点的有效性,经培养获得稳定遗传dpy19l1l纯合突变斑马鱼品系。本发明能更高效且更精确地在生物体基因组中沉默特定基因,且制作简单、成本低,且可同时对靶基因上多个位点进行切,沉默任意数目的单个基因,且敲除dpy19l1l基因斑马鱼胚胎出现未出现的发育畸形,构建成功dpy19l1l突变体先天性脊柱弯曲,为脊柱弯曲模型提供了一个很好的斑马鱼模型,有助于研究骨骼相关疾病。(The invention relates to the technical field of gene knockout and zebra fish models, in particular to a construction method of a dpy19l1l gene-deleted zebra fish. Designing a proper targeting site on a ppy 19l1l gene of zebra fish, injecting a specific gRNA and a Cas9 protein synthesized in vitro into a zebra fish cell by microinjection, culturing an embryo for 48h, and then selecting the embryo to perform genotype analysis, thereby confirming the effectiveness of the selected site, and obtaining a stable genetic ppy 19l1l homozygous mutant zebra fish strain by culture. The invention can silence specific gene in organism genome more efficiently and more accurately, has simple manufacture and low cost, can simultaneously cut a plurality of sites on the target gene to silence any number of single genes, knocks out the development deformity of the dpy19l1l gene zebra fish embryo, successfully constructs the congenital spinal curvature of the dpy19l1l mutant, provides a good zebra fish model for the spinal curvature model, and is beneficial to researching bone related diseases.)
1. A construction method of a ppy 19l1l gene-deleted zebra fish is characterized by comprising the following steps:
s1, querying a genome DNA sequence of a zebra fish dpy19l1l gene, analyzing a functional structural domain of the zebra fish dpy19l1l gene, finding a target site of a dpy19l1l gene at the No.1 exon of the dpy19l1l gene according to a CRISPR/Cas9 knockout principle, wherein the sequence is 5'-GGAATAACAGTGCTGATTCT-3', and 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, primer star PCR to obtain gRNA in vitro transcription template;
the gDNA Vector Template is obtained by cloning Cas9cDNAs with double NLS into a pXT7 Vector and performing linearization by using XbaI endonuclease;
the sequence of the gDNA adaptor Primer is 5'-AGCACCGACTCGGTGCCACTT-3';
s3, carrying out in vitro transcription according to the gRNA in vitro transcription template obtained in S2, and extracting and purifying to obtain a purified gRNA;
s4, injecting the purified gRNA and the Cas9 protein into fertilized eggs of the zebra fish at a one-cell stage in a micro-injection manner, and culturing to obtain a stable genetic dpy-19l1l homozygous mutant zebra fish strain, namely a dpy19l1l gene-deleted zebra fish.
2. The method for constructing zebrafish with deletion of ppy 19l1l gene according to claim 1, wherein the S2 reaction system is: gDNA Vector Template 1. mu.l, target site length Primer 1. mu.l, gDNA linker Primer, dNTP Mix 1. mu.l, 5 XPrimer stage buffer 10. mu.l, Primer stage DNA polymerase 1. mu.l, ddH2O32. mu.l; the gDNA Vector Template was obtained by cloning Cas9cDNAs with double NLS into pXT7 Vector and linearizing with XbaI endonuclease.
3. The method for constructing zebrafish with deletion of dpy19l1l gene according to claim 2, wherein the amplification process of S2 comprises: 2min at 98 ℃; the following steps were repeated for 35 cycles: 15s at 98 ℃, 15s at 58 ℃ and 20s at 72 ℃; 5min at 72 ℃; 30min at 72 ℃.
4. The method for constructing zebrafish with deletion of ppy 19l1l gene according to claim 3, wherein the transcription system of S3 is as follows: template DNA 100 + 200ng, 10 × RNA polymerase Reaction buffer 1ul, 25mM rNTP Mix 1ul, RNase Inhibitor 1ul, T7 RNA pol ymease 0.5ul, RNase free H2And O is metered to 10 ul.
5. The method for constructing zebrafish with deletion of ppy 19l1l gene according to claim 4, wherein the transcription system is: 37 ℃ for 3 h.
6. The method for constructing zebrafish with deletion of ppy 19l1l gene according to claim 5, wherein the culture of S4 comprises the following steps:
s1, culturing and incubating the fertilized eggs subjected to microinjection, carrying out Sanger sequencing to detect the effectiveness of a target site, screening out the fertilized eggs with gene mutation, recording the fertilized eggs as F0 generations, and feeding the fertilized eggs to adult fishes;
s2, crossing the F0 adult fish with WT to obtain an F1 generation;
s3, determining F1 generation of the gene knockout of ppy 19l1l by using a genotype identification method;
s4, selecting F1 generations with the same mutation types from the F1 generation mutants to mate to obtain F2 generations;
s5 and genotype identification F2 generations, wherein the homozygous knockout of the gene dpy19l1l is homozygous mutant zebra fish strain capable of stably inheriting the gene dpy-19l1l, namely the gene dpy19l1l deletion zebra fish.
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-deleted zebra fish.
Background
The DPY19L1L gene is located on zebrafish chromosome 16 and is one member of the DPY19 gene family, which encodes the DPY19(dumpy-19, DPY-19) protein family comprising DPY19L1 (DPY-19-like 1), DPY19L2, DPY19L3 and DPY19L4, and the protein family has the classical function of mannose transferase activity. The DPY19L1 protein is a multi-transmembrane protein, mainly participates in the development of a nervous system, regulates the radial migration of glutamatergic neurons in the development process of cerebral cortex, and is required for the growth of axons. The DPY19L2 gene plays an important role in the spermatogenesis process, and the gene deletion is an important pathogenic factor of the sperm round head disease. It has been shown that round-nose sperm sterility is associated with a balance between migration and gene deletion of the DPY19L2 gene at the chromosome break. In conclusion, the DPY19 gene mainly plays a role in the processes of neurodevelopment and spermatogenesis, is not reported in the fields related to skeletal development and diseases, and is deficient in the research of the DPY19L1L gene.
Based on the above, the method provides the zebra fish with the deletion of the DPY19L1L gene, and the discovery of the new function and the genetic mechanism of the DPY19L1L gene in the development of the zebra fish is particularly important.
Disclosure of Invention
The invention aims to provide a construction method of a dpy19l1l gene-deleted zebra fish, and the dpy19l1l gene is related to the skeletal development of the zebra fish and can be used for researching a deep molecular mechanism of the skeletal development of the zebra fish.
In order to achieve the purpose, the invention adopts the following technical scheme:
a construction method of a ppy 19l1l gene-deleted zebra fish comprises the following steps:
s1, searching a genome DNA sequence of a zebra fish dpy19l1l gene on NCBI, analyzing a functional structural domain of the zebra fish dpy19l1l gene, finding a target site of the dpy19l1l gene at the No.1 exon of the dpy19l1l gene according to a CRISPR/Cas9 knockout principle, wherein the sequence is 5'-GGAATAACAGTGCTGATTCT-3', designing a target site length primer and a gDNA joint primer, and the sequence of the dpy19l1l target site length primer is 5'-Taatacgactcactatag GGAATAACAGTGCTGATTCTgttttagagctagaaatagc-3', gDNA joint primer and is 5'-AGCACCGACTCGGTGCCACTT-3';
s2, primer star PCR to obtain gRNA in vitro transcription template;
s3, carrying out in vitro transcription according to the gRNA in vitro transcription template obtained in S2, and extracting and purifying to obtain a purified gRNA;
s4, injecting the purified gRNA and the Cas9 protein into fertilized eggs of the zebra fish at a one-cell stage in a micro-injection manner, and culturing to obtain a stable genetic dpy-19l1l homozygous mutant zebra fish strain, namely a dpy19l1l gene-deleted zebra fish.
Further, the reaction system of S2 is: gDNA Vector Template 1. mu.l, target site length Primer 1. mu.l, gDNA linker Primer, dNTP Mix 1. mu.l, 5 XPrimer stage buffer 10. mu.l, Primer stage DNApolymerase 1. mu.l, ddH2O32. mu.l; the gDNA Vector Template was obtained by cloning Cas9cDNAs with double NLS into pXT7 Vector and linearizing with XbaI endonuclease.
Further, the amplification procedure of S2 is: 2min at 98 ℃; the following steps were repeated for 35 cycles: 15s at 98 ℃, 15s at 58 ℃ and 20s at 72 ℃; 5min at 72 ℃; 30min at 72 ℃.
Further, the transcription system of S3 is: template DNA10ul, 10 XRNA polymerase reaction buffer2ul, 25mM rNTP Mix0.8ul, RNase Inhibitor0.5ul, T7 RNA polymerase2ul, RNaseFERE H2O4.7ul。
Further, the transcription system is: 37 ℃ for 3 h.
Further, the cultivation of S4 specifically includes the following steps:
s1, culturing and incubating the fertilized eggs subjected to microinjection, carrying out Sanger sequencing to detect the effectiveness of a target site, screening out the fertilized eggs with gene mutation, recording the fertilized eggs as F0 generations, and feeding the fertilized eggs to adult fishes;
s2, crossing the F0 adult fish with WT to obtain an F1 generation;
s3, determining F1 generation of the gene knockout of ppy 19l1l by using a genotype identification method;
s4, selecting F1 generations with the same mutation types from the F1 generation mutants to mate to obtain F2 generations;
s5 and genotype identification F2 generations, wherein the homozygous knockout of the gene dpy19l1l is homozygous mutant zebra fish strain capable of stably inheriting the gene dpy-19l1l, namely the gene dpy19l1l deletion zebra fish.
Compared with the prior art, the invention has the following beneficial effects:
(1) the invention can silence specific gene in organism genome more efficiently and more accurately, has simple manufacture and low cost, can simultaneously cut a plurality of sites on the target gene to silence any number of single genes, and knocks out the development deformity of the dpy19l1l gene zebra fish embryo.
(2) The invention successfully constructs the congenital spinal curvature of the zebra fish with the gene deletion of dpy19l1, provides a good zebra fish model for the spinal curvature model, and is favorable for researching bone-related diseases and screening medicines for treating or relieving the bone diseases. .
Drawings
FIG. 1 shows the peak sequence of the gene sequence of py19l1l in example 1, which is a hybrid of wild type and Dpy19l1l, wherein A is wild type, B is a hybrid, and C is another hybrid.
FIG. 2 shows the sequence alignment and base mutation of example 1, wherein the wild type and Dpy19l1l are heterozygote py19l1l gene, A is wild type, B is a heterozygote, and C is another heterozygote.
FIG. 3 is a comparison of the phenotype of wild-type zebrafish deleted from the gene dpy19l1 of example 1, wherein A is wild-type and B is zebrafish deleted from the gene dpy19l 1.
Detailed Description
The invention is described in detail below with reference to the figures and the specific embodiments, but the invention should not be construed as being limited thereto. The technical means used in the following examples are conventional means well known to those skilled in the art, and materials, reagents and the like used in the following examples can be commercially available unless otherwise specified.