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Directional modification method of specific gene of animal genome and application thereof

A specific gene and genome technology, applied in plant genetic improvement, botanical equipment and methods, biochemical equipment and methods, etc., can solve the problems of long operation cycle, time-consuming and labor-intensive zinc finger domain, and incomplete matching of binding ability, etc. To achieve the effect of effective targeted mutagenesis

Active Publication Date: 2012-09-05
NANJING UNIV
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Problems solved by technology

However, its disadvantage is that it is time-consuming and laborious to screen zinc finger domains by these methods, and it is not always possible to screen effective zinc finger nucleases for a given gene, and the binding ability of the zinc finger domains screened out is comparable to that of the pair in vivo. Whether zinc finger nucleases can effectively cut target genes is not completely matched
At the same time, the existing zinc finger nuclease construction and screening systems do not take into account the mutation patterns that the designed zinc finger nuclease can obtain in vivo, and how to efficiently realize the screening of progeny
Therefore, targeted mutagenesis of the genome with zinc finger nucleases designed and screened based on existing methods has defects such as low cutting mutagenesis efficiency and long operating cycle

Method used

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  • Directional modification method of specific gene of animal genome and application thereof
  • Directional modification method of specific gene of animal genome and application thereof
  • Directional modification method of specific gene of animal genome and application thereof

Examples

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Embodiment 1

[0066] Embodiment 1. Targeted knockout of the myostatin gene in the genome of the yellow catfish

[0067] In fishery production, obtaining large-scale finished fish and increasing the muscle content in finished fish is one of the main goals of fish genetic engineering breeding. Myostatin ("myostatin" for short) is a negative regulator of muscle growth, and mutations in the myostatin gene cause animals to grow more muscle and thus make animals bigger. For example, cattle with natural mutations in the myostatin gene have increased muscle and become double muscle cattle; mice with myostatin gene knockout have increased muscle and become larger and become super mice; zebrafish myostatin After gene knockout, the individual body weight of the fish increased by more than 40%. Correspondingly, it is the purpose of this embodiment to knock out the myostatin gene of other fish such as yellow catfish, remove the gene's inhibitory function on myogenesis, and accelerate the growth rate of...

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Abstract

The invention relates to a method for modifying an animal genome by directed mutagenesis, in particular relates to a novel directional transformation method of a specific gene of an animal genome by utilizing zinc finger nuclease. The method comprises the following steps: designing the zinc finger nuclease which specifically identifies and cuts a targeting gene according to a sequence in a target animal target gene region; validating the directional cutting capability and efficiency of the zinc finger nuclease to the target gene by using an independent expression system; and carrying out directional genetic modification on the target gene of the embryo of a target animal to obtain a stable genetic character by utilizing the selected zinc finger nuclease. Furthermore, the invention also relates to a main gene mutation mode which is obtained by using the independent expression system, selecting and utilizing the zinc finger nuclease, and a filial generation obtained through efficient screening and oriented genetic modification by utilizing the selected gene mutation mode. On the other hand, the invention relates to the zinc finger nuclease which is obtained by utilizing the method and is capable of specifically identifying the myostatin gene of pelteobagrus fulvidraco and directionally knocking out the myostatin gene of the pelteobagrus fulvidraco.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a method for directional transformation of a specific gene in an animal genome and an application thereof. Background of the invention [0002] It is a basic research strategy used in contemporary life science research to study the mechanism of action of genes in the occurrence and inheritance of animals, to create disease animal models, or to create new animal strains with excellent production traits by mutagenizing the genome. Animal genome mutagenesis technology is divided into non-directed mutation technology and directed mutagenesis technology for specific genes. Because researchers can select mutation sites and make full use of existing animal genome sequence information, directed mutagenesis technology has become the "gold standard" for researchers to conduct related research. [0003] Gene targeting in directed mutagenesis is a genetic technique that uses the principle of homo...

Claims

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Application Information

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IPC IPC(8): C12N15/09C12N9/22C12N15/55C12N15/63C12Q1/68C12Q1/02A01K67/033
Inventor 董张及赵庆顺
Owner NANJING UNIV
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