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Breeding method for knocking out fish MC4R gene by using CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 system

A system knockout, fish technology, applied in biochemical equipment and methods, using vectors to introduce foreign genetic material, nucleic acid vectors, etc., can solve problems such as slow growth of fish and aquatic animals, and achieve low cost and high accuracy. , The effect of pure line time is short

Active Publication Date: 2016-12-07
CHONGQING INST OF GREEN & INTELLIGENT TECH CHINESE ACADEMY OF SCI
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  • Abstract
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  • Claims
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Problems solved by technology

[0003] In view of the shortcomings of the prior art described above, the purpose of the present invention is to provide a breeding method for knocking out the fish MC4R gene using the CRISPR-Cas9 system, which is used to solve the problem of slow growth of fish and aquatic animals in the prior art, etc. question

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  • Breeding method for knocking out fish MC4R gene by using CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 system
  • Breeding method for knocking out fish MC4R gene by using CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 system
  • Breeding method for knocking out fish MC4R gene by using CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 system

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[0027] Embodiments of the present invention are described below through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the content set forth in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various modifications or changes can be made to the details in this specification based on different viewpoints and applications without departing from the spirit of the present invention.

[0028] In this embodiment, zebrafish is taken as an example. Of course, the present invention is also applicable to other economic fishes, specifically including carp, crucian carp, tilapia, grass carp, silver carp, bighead carp, snakehead, snakehead and other economic fish.

[0029] 1. Determination of the target site of zebrafish MC4R gene

[0030] First, look up the gene sequence of the zebrafish MC4R gene in the gene database, compare its ...

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Abstract

The invention provides a breeding method for knocking out fish MC4R gene by using a CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 system. The method comprises the following steps: determination of MC4R gene targeted site, preparation of gRNA, in-vitro microinjection and knock-out verification, screening of MC4R-gene-knock-out fish, culture of hybrid fish, and the like. The breeding method is suitable for all commercial fishes, and can implement quick growth and breeding of fishes by knocking out the fish MC4R gene. Compared with the traditional breeding process, the method provided by the invention has the characteristics of high accuracy, low cost and short pure line acquisition time. Compared with the transgenic breeding, the gene knock-out can cause gene malfunction of the fish, and can not bring in any external gene, thereby avoiding the problem of transgenic safety.

Description

technical field [0001] The invention relates to the field of molecular breeding of fish, in particular to a breeding method for knocking out the MC4R gene of fish by using a CRISPR-Cas9 system. Background technique [0002] Melanocortin receptor 4 (Melanocortin 4 receptor, MC4R) belongs to the family of G-protein coupled receptors. It is a kind of peptide substance secreted by the ventromedial nucleus of the hypothalamus. effect. As early as 1997, Huszar et al. conducted research on mice knocking out the MC4R gene and found that the mice knocking out the MC4R gene were genetically obese, showing symptoms such as polyphagia, excessive insulin secretion, and obesity (Huszar D, Lynch C A, Fairchild-Huntress V, et al. Cell, 1997, 88(1):131-141.). A series of research results in recent years have shown that MC4R gene mutation or polymorphism has a significant impact on animal growth performance. In 2009, overexpression of AGRP, which inhibits the activity of MC4R, showed the c...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85A01K67/027
CPCA01K67/0276A01K2217/075A01K2227/40C12N15/85C12N2800/106C12N2800/80
Inventor 裴得胜王超
Owner CHONGQING INST OF GREEN & INTELLIGENT TECH CHINESE ACADEMY OF SCI
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