Construction method of Ano5 gene knockout mouse model

A gene knockout, gene technology, applied in the fields of genetics, biology, and pathology, to achieve stable and stable effects

Active Publication Date: 2019-04-30
BEIJING STOMATOLOGY HOSPITAL CAPITAL MEDICAL UNIV
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Problems solved by technology

[0005] At present, the animal models for Ano5 gene knockout mainly include two mouse models established by Griffin by knocking out the 8th and 9th exons and by Xu by replacing the 1st exon and its upstream 1.6kb sequence. However, these models only introduce Ano5-related phenotypic changes in muscle-related diseases were detected, but no changes in bone tissue were found

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  • Construction method of Ano5 gene knockout mouse model
  • Construction method of Ano5 gene knockout mouse model
  • Construction method of Ano5 gene knockout mouse model

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Embodiment 1

[0041] Example 1 Construction of Ano5 knockout mouse model

[0042] 1. According to the sequence of exons 11 and 12 of the mouse Ano5 gene (GenBank: NC_000073.6), sgRNA was designed based on the CRISPR-Cas9 system; the sgRNA sequence of Ano5 is as follows ( figure 1 -A):

[0043] Recognition site on exon 11: 5′-CCTGGGATGTTATCTGGCTTGC-3′

[0044] Recognition site on exon 12: 5′-CCAGGGATGTGTGCACACTAGGC-3′

[0045] 2. PMSG-treated C57 / BL6 female mice (3-week-old, average body weight 15g), injected with hCG 46 hours later, and mated with male mice. The fertilized eggs were collected the next day for microinjection. 100ng / ml) and the mRNA of Cas9 nuclease (50ng / ml) were transcribed in vitro and injected into fertilized eggs together, and the fertilized eggs that survived the injection were taken and transplanted into pseudopregnant female mice to produce mice, that is, the F0 generation mice.

[0046] 3. Extract the tail DNA of the F0 generation mice, and sequence the PCR ampli...

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Abstract

The invention provides a construction method of an Ano5 gene knockout mouse model. A CRISPR-Cas9 gene knockout technology is adopted to remove 11th and 12th exons of the Ano5 gene. After Ano5 gene knockout, mice all have clinical symptoms of human GDD patients such as lateral bending of shin bones, hyperplasia of cortical bone, hypertrophy of lower jawbone, increasing of alkaline phosphatase in blood, and the like. The established mouse model is used to simulate the human GDD disease, is stable, has stable heredity and similar symptoms of the human GDD disease, can be applied to research on GDD pathogenesis and GDD gene therapy, and is economic, simple and reliable.

Description

technical field [0001] The invention relates to the fields of pathology, genetics and biotechnology, in particular to a method for constructing an Ano5 gene knockout mouse model. Background technique [0002] GDD (Gnathodiaphyseal dysplasia) is a dominant hereditary disease involving the oral cavity and long bones. Cortical thickening. GDD patients often have fractures or repeated fractures in adolescence, and the level of alkaline phosphatase in the blood increases. Anoctamin 5 (ANO5) gene is the causative gene of GDD. In recent years, related reports on GDD pedigrees and sporadic cases have emerged. Because the disease causes great pain to patients and seriously affects their quality of life, it has become the focus of scholars to explore the etiology of GDD, and then guide the diagnosis, prevention and treatment of the disease. [0003] GDD is an autosomal dominant genetic disorder. In 2003, Tsutsumi first mapped the causative gene to the 11p14.3-15.1 region of human...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/90C12N5/10A01K67/027C12R1/91
CPCA01K67/0276A01K2217/075A01K2227/105A01K2267/0306C07K14/47C12N15/85C12N15/8509C12N15/907C12N2800/107C12N2810/10
Inventor 胡颖王小予刘秀董蕊
Owner BEIJING STOMATOLOGY HOSPITAL CAPITAL MEDICAL UNIV
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