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Construction method of Adgrv1 gene Y6236fsX1 mutant animal model

A technology of animal model and construction method, which is applied in the field of construction of Adgrv1 gene Y6236fsX1 mutant animal model, which can solve the problems of mouse models without simulated deafness mutation

Active Publication Date: 2018-11-23
山东大学深圳研究院 +1
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  • Application Information

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Problems solved by technology

However, the search found that although there are Adgrv1 gene knockout mice, there is no mouse model that simulates human-related deafness mutations. Therefore, it is of great significance to establish a method for constructing an animal model of the Adgrv1 gene Y6236fsX1 mutation

Method used

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  • Construction method of Adgrv1 gene Y6236fsX1 mutant animal model
  • Construction method of Adgrv1 gene Y6236fsX1 mutant animal model
  • Construction method of Adgrv1 gene Y6236fsX1 mutant animal model

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Embodiment 1

[0018] Example 1: The construction method of Adgrv1 gene Y6236fsX1 mutant animal model based on CRISPR / Cas9 technology is realized by the following steps:

[0019] (1) Selection and design of Guide RNA targeting mouse Adgrv1 gene and repair template DNA

[0020] Design the Adgrv1 gene Y6236fsX1 mutant mouse construction strategy, such as figure 1 shown.

[0021] According to the construction strategy, design a suitable Guide RNA at the corresponding position of the Adgrv1 gene, and order the corresponding repair template DNA, wherein the nucleotide sequence of Guide RNA target site1 is shown in SEQ ID NO.1, and the nucleotide sequence of Guide RNA target site2 The sequence is shown in SEQ ID NO.2, the nucleotide sequence of the repair template DNA is shown in SEQ ID NO.3, and the specific sequence is as follows:

[0022]

[0023] (2) In vitro synthesis of Guide RNA, Cas9 mRNA and repair template DNA

[0024] Guide RNA, Cas9 mRNA and repair template DNA were synthesized b...

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Abstract

The invention discloses a construction method of an Adgrv1 gene Y6236fsX1 mutant animal model based on CRISPR / Cas 9 technology. The method comprises the following steps: (1) designing and choosing theGuide RNA (ribonucleic acid) and repair template DNA (deoxyribonucleic acid) of proper target mouse Adgrv1 genes; (2) synthesizing Cas9 mRNA, the Guide RNA and the repair template DNA in vitro; (3) enabling the Guide RNA, the Cas9 mRNA and the repair template DNA to be jointly prepared into an injection sample in a mole ratio so as to be injected into a fertilized ovum; (4) identifying obtained F0-generation mice; and (5) enabling positive F0-generation mice to perform passage and obtaining and identifying F1-generation mice. The method can be used for successfully obtaining an Adgrv1 gene Y6236fsX1 mutant mouse model corresponding to Y6244fsX1 pathogenic mutations in human, thereby laying a foundation for researching relevant factor and diagnostic treatment of the Y6244fsX1 pathogenic mutations in human.

Description

technical field [0001] The invention belongs to the technical field of making gene mutation animal models by using gene modification technology, in particular to a method for constructing Adgrv1 gene Y6236fsX1 mutant animal models based on CRISPR / Cas9 technology. Background technique [0002] CRISPR / Cas (Clustered Regularly Interspaced Shot Palindromic repeats / CRISPR-associated) system is an acquired immune system in prokaryotes that resists foreign gene invasion. In bacteria and archaea, the CRISPR system integrates phage or plasmid DNA that invades the host fragments to the CRISPR site, and then guide the Cas endonuclease to cut the exogenous DNA sequence through the corresponding CRISPR RNAs (crRNAs), thereby resisting the invasion of viruses or phages. The CRISPR / Cas system is divided into three types (TypeⅠ, TypeⅡ, and TypeⅢ), among which the TypeⅡ system has been widely used since it successfully realized gene editing in mammalian cells in 2013 due to its simplicity, h...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/90C12N9/22A01K67/027
CPCA01K67/0275A01K2227/105A01K2267/0306C12N9/22C12N15/907
Inventor 徐志刚杜海波王燕飞
Owner 山东大学深圳研究院
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