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A method for targeted knockdown of multiple copies of genes in the zebrafish genome

A genome and zebrafish technology, applied in recombinant DNA technology, cells modified by the introduction of foreign genetic material, and introduction of foreign genetic material using vectors, etc., can solve the problem of not finding homologous genes, and achieve a wide range of applications.

Active Publication Date: 2019-09-03
NANJING MATERNITY & CHILD HEALTH CARE HOSPITAL +2
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Problems solved by technology

However, the KRAB domain has no homologous genes in zebrafish, and a dCas9 fusion system is urgently needed. Its transcription-inhibiting domain exists in zebrafish. nearby altered gene expression

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  • A method for targeted knockdown of multiple copies of genes in the zebrafish genome

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Embodiment Construction

[0021] The present invention will be further explained below in conjunction with specific embodiments.

[0022] refer to figure 1 , a method for directional knocking down multi-copy genes in the zebrafish genome proposed by the present invention, comprising the following steps:

[0023] Step 1: Preparation of zebrafish embryos. Zebrafish are kept in a circulating water system at a room temperature of 28.5°C, with 14 hours of light and 10 hours of darkness every day. They are fed once in the morning and in the afternoon. The female fish and the male fish are placed at both ends of the mating box, separated by a partition in the middle. When the light is on the next morning, the partition is pulled out, and the male and female mate. The embryos produced will sink to the bottom of the mating box due to gravity and collected. embryos, and place the embryos in a constant temperature incubator at 28.5°C for future use;

[0024] Step 2: Construction of the expression vector of the ...

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Abstract

The invention discloses a method for directional knockdown of a multi-copy gene in zebrafish genome. The method comprises the following steps: I, preparing a zebrafish embryo; II, constructing an expression vector of dCas9-Eve fusion protein; III, conducting in-vitro transcription of sgRNA, dCas9-Eve, Cas9 and gene, and purifying the sgRNA, dCas9-Eve, Cas9mRNA and gene mRNA, which are synthesized through the in-vitro transcription, by virtue of RNeasy Mini Kit; IV, identifying activity of the sgRNA; and V, knocking out expression of the multi-copy gene znfl1s by virtue of a dCas9-Eve method, and judging the situation that the gene expression is knocked out by detecting the expression of the gene by virtue of an embryo whole-mount in situ hybridization method. The method provided by the invention, by virtue of the dCas9-Eve, can specifically knock down the transcription of the gene under the circumstance of implementing the effective targeted identification of sgRNA existence of a gene promoter, so that the researched gene is prevented from being affected by MO toxicity or non-specific phenotype caused by target missing; and moreover, since an Eve inhibiting transcription structural domain is derived from zebrafish, the dCas9-Eve is applicable to knockdown of any zebrafish genes at a transcription level.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for directional knocking down multi-copy genes in the zebrafish genome. Background technique [0002] When studying the function of genes in early embryonic development, we usually design morpholino (MO) in a certain region of the gene to knock down the expression of the gene. The MO of the splice site is used to verify the function of the gene, and MO toxicity or off-target often leads to non-specific phenotypes. CRISPR / Cas9 is an adaptive immune defense formed during the long-term evolution of bacteria and archaea, which can be used to fight against invading viruses and foreign DNA. Changing genome editing tools. In recent years, researchers have built some new gene regulation tools based on the traditional CRISPR / Cas9 gene editing system: CRISPR interference (CRISPRi) technology for down-regulating genes. [0003] CRISPRi refers to the co-expression of Cas9 (dCas9) lac...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/85C12N5/10
Inventor 李俊赵庆顺李景云顾淳董晓华
Owner NANJING MATERNITY & CHILD HEALTH CARE HOSPITAL
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