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gRNA targeting mouse Atp7b gene and method for constructing Wilson disease mouse model

A mouse and targeted technology, applied in the field of molecular biology, can solve the problem of lack of Wilson disease mouse model, and achieve the effect of short cycle and simple method.

Pending Publication Date: 2021-12-03
CYAGEN BIOSCI INC
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Problems solved by technology

In order to study the pathogenesis of the disease and develop effective treatment methods, the construction of an experimental model of the disease is indispensable for the study of the disease. The mouse model is currently recognized as the most effective disease model for preparing human disease models and studying genome functions. However, there is still a lack of efficient methods for constructing Wilson disease mouse models

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  • gRNA targeting mouse Atp7b gene and method for constructing Wilson disease mouse model
  • gRNA targeting mouse Atp7b gene and method for constructing Wilson disease mouse model
  • gRNA targeting mouse Atp7b gene and method for constructing Wilson disease mouse model

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Embodiment

[0037] 1. Knockout protocol design

[0038] According to the requirements, look up the detailed information of the Atp7b gene, select the gene knockout region as much as possible to include the functional domain of the gene, and select the knockout region according to the actual situation, we choose the Atp7b gene knockout region Exon2-Exon20 with a total of 33183bp sequence, including the entire Atp7b gene 91.91% of the coding region, as shown in Figure 1.

[0039] 2. gRNA sequence design

[0040] According to the sequence of the mouse Atp7b gene, three gRNA sequences targeting the gene were designed and synthesized, and the sequence information is as follows:

[0041] gRNA1=SEQ ID NO 1=5'-GGCACCCAGGCCAAATGTAGGGG-3';

[0042] gRNA2=SEQ ID NO 2=5'-GACTCTTACCACTGCTAAGTGGG-3';

[0043] gRNA3 = SEQ ID NO 3 = 5'-TCTTTGCTAAGGAGTAAAACTGG-3'.

[0044] 3. Microinjection of Cas9 / sgRNA

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Abstract

The invention belongs to the field of molecular biology, and particularly discloses a group of gRNA targeting a mouse Atp7b gene and a method for constructing a Wilson disease mouse model. Three gRNAs specifically targeted to the Atp7b gene are designed, Exon2-Exon20 of the Atp7b gene is knocked out by utilizing cas9 protein, the sequence of a knocked region is not 3 times, frameshift mutation is caused, and the knocked-out region contains 91.91% of a coding region of the gene, so that the aim of knocking out the gene is fulfilled; and the method for constructing the Atp7b gene knockout mouse model by using the CRISPR / Cas9 system is simple and easy to implement, short in period and high in probability of obtaining positive mice, the pathogenesis of the disease can be fully researched by utilizing the mouse model, and service is provided for further developing a treatment mode aiming at the disease.

Description

technical field [0001] The invention belongs to the field of molecular biology, and specifically discloses a group of gRNA targeting mouse Atp7b gene and a method for constructing a Wilson disease mouse model. Background technique [0002] Wilson's disease (WD), also known as hepatolenticular degeneration, is a rare autosomal recessive genetic copper metabolism disease. This disease is a mixed connective tissue disease among rheumatic diseases. Clinically, liver disease and neurological symptoms are the main symptoms. feature. Increased accumulation of copper in tissues is a major cause of liver disease, neurological lesions, brown-green rings around the cornea (Kay-Ferring rings), kidney and other organ morbidity, and tissue damage can cause bilateral softening of the basal ganglia in cirrhosis, transsexual. According to the National Institute of Diabetes and Digestive and Kidney Diseases in the United States, about 1 / 10,000 to 1 / 30,000 people in the world suffer from thi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/873C12N15/89A01K67/027
CPCC12N15/1137C12N15/873C12N15/89A01K67/0276C12N9/14C12Y306/03004C12N2310/20A01K2227/105A01K2267/0306
Inventor 刘玉姜伟孙敏
Owner CYAGEN BIOSCI INC
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