Transgenic zebrafish expressing gene Cas9 and construction method and application of transgenic zebrafish

A construction method and zebrafish technology, applied in the field of transgenic research, can solve the problems of difficult, time-consuming and labor-intensive knockout fish

Inactive Publication Date: 2018-07-06
YANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it is difficult and time-consuming to obtain homozygous knockout fish using traditional methods

Method used

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  • Transgenic zebrafish expressing gene Cas9 and construction method and application of transgenic zebrafish
  • Transgenic zebrafish expressing gene Cas9 and construction method and application of transgenic zebrafish
  • Transgenic zebrafish expressing gene Cas9 and construction method and application of transgenic zebrafish

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0030] (1) Preparation of sgRNA template

[0031] System: 5×Buffer5μL,

[0032] dNTP (10mM) 0.5 μL,

[0033] sgRNA-F (μM) 1 μL,

[0034] sgRNA-R (μM) 1 μL,

[0035] DNA Polymerase 0.5 μL,

[0036] RNase-free water 17μL,

[0037] Total25μL.

[0038] Conditions: 98°C for 2 minutes, 50°C for 10 minutes, and 72°C for 10 minutes.

[0039] (2) Product recovery

[0040] Take 2 μL of the product and use 2.5% agarose gel for electrophoresis detection.

[0041] (3) sgRNA in vitro transcription

[0042]

[0043] Conditions: overnight at 37°C.

[0044] (4) In vitro purification (removal of free bases, T7 enzyme, template DNA)

[0045] 1) Add RNase-free water to the sgRNA in vitro transcription product to make up to 100 μL. Add an equal volume (100 μL each) of Tris-saturated phenol-chloroform, centrifuge at 15,000 rpm at 4°C for 10 min, and slowly absorb the supernatant;

[0046] 2) Add 1 mL of 100% ethanol, shake for 10 seconds, centrifuge at 15,000 rpm at 4°C for 10 minutes,...

Embodiment 1

[0058] In Example 1, the zebrafish Mitfα gene was used as the Cas9 gene insertion site, and a mixture of Cas9 mRNA, MitfasgRNA, and Cas9 gene donor fragment was injected into fertilized eggs by microinjection. The integration of the Cas9 gene in the F0 generation of zebrafish was identified, and its expression in the offspring (F1) was detected, and finally the homozygous line was selected in the F1 generation by using pigment extinction.

[0059] Specifically include the following steps:

[0060] (1) Scientifically raise zebrafish, select healthy male fish and female fish, produce fertilized eggs through natural mating (light cycle: 14h light, 10h dark, male to female ratio 2:1), and collect fertilized eggs.

[0061] (2) The zebrafish Mitfα gene was used as the Cas9 gene integration site to synthesize Mitfα sgRNA and Cas9 mRNA, respectively. The Ef1α promoter of zebrafish itself was combined with the front end of the Cas9 gene in the MLM3613 vector, and the recombinant plasm...

Embodiment 2

[0079] In Example 2, by editing the Tyr gene, it was proved that the constructed zebrafish expressing the Cas9 gene can be used as a gene editing tool. Embryos of transgenic Cas9 zebrafish and wild-type TU zebrafish were used for microinjection, and a control group was set up. The samples of injection group at 48hpf development were collected, and genomic DNA was extracted for identification.

[0080] Specifically include the following steps:

[0081] (1) Scientifically raise zebrafish, select healthy wild zebrafish and Cas9 transgenic zebrafish males and females, respectively, and naturally mate and reproduce to produce fertilized eggs (light cycle: 14h light, 10h dark, male to female ratio 2:1 ), and fertilized eggs were collected separately. A control group (no injection), a traditional method embryo injection group, and a Cas9 transgenic zebrafish injection group were set up. In addition, some transgenic zebrafish embryos were left without any treatment.

[0082] (2) F...

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Abstract

The invention relates to a transgenic zebrafish expressing a gene Cas9 and a construction method and the application of the transgenic zebrafish. According to the transgenic zebrafish expressing the gene Cas9, the gene Cas9 is inserted into a gene Mitfa of the zebrafish. The transgenic zebrafish capable of expressing the gene Cas9 is constructed in a targeting way at the fixed point of the pigmentgene Mitfa through the CRISPR/Cas9 transgenic technology, and pigment fading is taken as a mark for screening the homozygous knockout line. Using the transgenic zebrafish to edit a gene Tyr and a gene ZFERV proves that the transgenic zebrafish is more effective in the gene knockout, and a homozygous knockout individual is obtained on the F0 generation. Compared with the application of the traditional CRISPR/Cas9 technology to the zebrafish, the gene editing efficiency of the Cas9 transgenic zebrafish is higher; and a simple effective tool is provided for the large-scale gene screening and theestablishment of a disease model.

Description

technical field [0001] The invention relates to the field of transgenic research, in particular to a method for constructing a transgenic zebrafish expressing a Cas9 gene by genome editing technology. Background technique [0002] As early as the 1930s, zebrafish became a classic model of embryonic development in biomedical research. Since then, zebrafish have been used for the first time to study vertebrate embryonic development and related issues. In the past ten years, gene editing technologies such as ZFNs, TALENs and CRISPR / Cas9 have been applied to the study of fish gene function, opening up new ways for the study of gene function. These nucleic acid-based genome editing tools target specific sites of interest by using different recognition modules, precisely induce double-strand breaks at specific genetic loci, and repair DNA through non-homologous recombination. [0003] The use of these gene editing tools in zebrafish research is of epoch-making significance, prov...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85A01K67/027
CPCC12N15/8509A01K67/0276A01K2217/075A01K2227/40A01K2267/03C12N2800/80C12N2810/10
Inventor 崔恒宓杨钰杨哲戴振清
Owner YANGZHOU UNIV
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