Construction method of AURKA-CKO1-N condition gene knockout mouse model

A technology for gene knockout mice and construction methods, which can be used in genetic engineering, plant genetic improvement, botany equipment and methods, etc., and can solve the problems of mouse embryo lethality, etc.

Active Publication Date: 2019-01-15
XUZHOU MEDICAL UNIV
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  • Claims
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Problems solved by technology

However, the mechanism of Aurora-A involved in tumorigenesis and development needs to be further elucidated
However, since Aurora-A systemic knockout mice are prone to mouse embryonic lethality

Method used

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  • Construction method of AURKA-CKO1-N condition gene knockout mouse model
  • Construction method of AURKA-CKO1-N condition gene knockout mouse model
  • Construction method of AURKA-CKO1-N condition gene knockout mouse model

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Embodiment 1

[0046] This embodiment provides a method for constructing an AURKA-CKO1-N conditional gene knockout mouse model, comprising the following steps:

[0047] (1) Construction of a recombinant targeting vector for AURKA conditional gene knockout

[0048]Use the ES cell (JM8A3ES cell, derived from C57 / BL6N strain) vector to target the AURKA gene, and use the bacterial artificial chromosome (BAC) for targeting, so that two loxP sites are inserted into intron 2 and intron 3 respectively. When Cre is expressed When exon 3 of the mouse AURKA gene can be knocked out, and replaced with the Neo gene, resulting in a frameshift mutation, the protein translation is terminated in exon 3 in advance;

[0049] The upstream arm is 4.2kb (DNA kilobase pairs), the downstream arm is 4.0kb, and the downstream arm is flanked by the phosphoglycerate kinase promoter-mediated negative selection marker gene expression cassette of herpes simplex virus thymidine kinase, and finally the AURKA conditional Gen...

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Abstract

The invention relates to a construction method of an AURKA-CKO1-N condition gene knockout mouse model. A recombination targeting carrier is constructed, embryonic stem cell are transfected after linearization, embryonic stem cell with homologous recombination are screened for clone, a donor mice blastula is injected after amplification to produce a chimera mouse; the chimera mouse copulates with an flp mouse to obtain a male F1 generation of mouse; and the male F1 generation of mouse copulates with a mouse whose tissues include specific Cre recombinase, and the AURKA-CKO1-N condition gene knockout mouse model is obtained in the next generation. According to the AURKA-CKO1-N condition gene knockout mouse model, an AURKA gene loses functions in specific tissues and cells, and an ideal modelis provided for research on an action mechanism of Aurora-A in tumorigenesis development.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a method for constructing an AURKA-CKO1-N conditional gene knockout mouse model. Background technique [0002] Gene knockout is a new technology developed in the second half of the 1980s by applying the principle of DNA homologous recombination. The definition of gene knockout refers to a gene whose structure is known but whose function is unknown, an experiment is designed at the molecular level to remove the gene, and then the experimental animal is observed as a whole to speculate on the function of the corresponding gene. [0003] Aurora-A is an important class of serine / threonine protein kinase involved in the regulation of cell mitosis, and its coding gene is located in the 20q13.2 chromosome region where translocation, deletion and amplification are active. Means that its expression pattern is inherently unstable. Abnormal expression of Aurora-A can le...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01K67/027C12N15/54C12N15/85
CPCA01K67/0276A01K2217/075A01K2227/105C12N9/12C12Y207/11001
Inventor 杨晶郑葵阳汤仁仙
Owner XUZHOU MEDICAL UNIV
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