Method for constructing mouse model capable of specifically knocking out IKKalpha gene in hippocampus region, targeting vector and kit

A technology for targeting vectors and mouse models, applied in pathology and physiology, which can solve problems such as sequence inversion and lack of research

Active Publication Date: 2017-02-15
CENT SOUTH UNIV
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Problems solved by technology

[0006] 2. If the two LoxP sites are located on one DNA strand, but in opposite directions, Cre recombinase can cause sequence inversion between the two LoxP sites;
iRFP720 is a near-infrared fluorescent protein gene, which is isolated ...

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  • Method for constructing mouse model capable of specifically knocking out IKKalpha gene in hippocampus region, targeting vector and kit
  • Method for constructing mouse model capable of specifically knocking out IKKalpha gene in hippocampus region, targeting vector and kit
  • Method for constructing mouse model capable of specifically knocking out IKKalpha gene in hippocampus region, targeting vector and kit

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Embodiment Construction

[0052] (1) PCR identification of IKKα and CamK2α in transgenic mice

[0053] Three weeks after the birth of the mouse, put it in a 1.5ml EP tube, add 500μl lysate and 20μl proteinase K, put it into a 1.5ml EP tube, incubate overnight in a constant temperature metal bath at 60°C, and centrifuge at 500g the next day. For the supernatant, measure the concentration of the obtained crude gDNA, and dilute the gDNA concentration to 30ng / μl for subsequent PCR reaction verification. Perform PCR verification according to the above primers. The PCR product is 643bp, indicating that IKKαknock in has been integrated into the chromosomal genome of the mouse. The homozygous type has only a simple 643bp band, and the heterozygous type has two bands of 299bp and 643bp. Wild-type mice Then there is only 299bp, which is a negative mouse. If a image 3 with Figure 4 .

[0054] (2) Construction of Cre-LoxP system

[0055] After the stable transgenic mice IKKα and CamK2α were obtained, the tra...

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Abstract

The invention discloses a method for constructing a mouse model capable of specifically knocking out an IKKalpha gene in a hippocampus region, a targeting vector and a kit. The IKKalpha gene is knocked out from specific mouse tissues through utilizing a Cre-LoxP recombination system, so that a transgenic mouse with the knocked-out IKKalpha gene is established, and an NF-omicronB signal channel and an acting mechanism on diseases, particularly tumors, are researched on the whole level of animals. Meanwhile, in order to determine and monitor whether specific knockout of the IKKalpha gene in the hippocampus region causes influences on the behavioral science of the mouse, red luciferase RFP720 is connected into a transgenic carrier. A gene targeting technology is adopted, and expression of the endogenous IKKalpha gene in the hippocampus region of the mouse is reduced based on iRFP720 near-infrared fluorescent protein and a Cre-LoxP gene knockout system, so that living body imaging of the transgenic mouse is monitored through an animal imaging system.

Description

technical field [0001] The invention belongs to the technical fields of imaging, pathology, physiology, genetics, molecular biology and behavior. It specifically relates to a method for constructing a mouse model for specifically knocking out the IKKα gene in the hippocampus region, as well as a targeting vector and a kit. Background technique [0002] IKKα is a regulatory subunit that constitutes the IKK complex, which can affect the activation of the NF-κB signaling pathway. Studies on glioma (a tumor that occurs in the brain) have shown that NF-κB and other signaling pathways related to inflammation are abnormally activated, but the mechanism of IKKα in brain tumors is currently unclear. Whether it will affect the occurrence and development of brain tumors is not yet clear. At the same time, as an inflammation-related molecule, whether it will affect its behavioral function after complete knockout in the normal mammalian hippocampus is not clear. Therefore, using animal...

Claims

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Application Information

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IPC IPC(8): C12N15/85A01K67/027
CPCA01K67/0276A01K2217/075A01K2217/15A01K2227/105A01K2267/03C12N9/12C12N15/8509C12N2800/107C12N2800/30
Inventor 陶永光赖巍巍刘双
Owner CENT SOUTH UNIV
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