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A mouse rtn4-a/b gene knockout method

A gene knockout, rtn4-a technology, applied in recombinant DNA technology, introduction of foreign genetic material using vectors, etc., can solve problems such as unclear mechanism of action

Inactive Publication Date: 2016-05-04
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Rtn4-B is widely expressed in peripheral organs and tissues, including lungs, cardiovascular tissues, etc. It is mainly involved in the regulation of the body's inflammatory response and the repair of tissue damage. In addition, it is also involved in pathology such as tumor cell apoptosis and neuron degeneration Physiological process, but the specific mechanism of action is still unclear

Method used

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  • A mouse rtn4-a/b gene knockout method
  • A mouse rtn4-a/b gene knockout method
  • A mouse rtn4-a/b gene knockout method

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Embodiment 1

[0026] (1) Recombinant plasmid preparation: use BAC vector (BAC: pBR322) to target rtn4-a / b gene (EnsemblGeneID: ENSMUSG00000020458). The 5' and 3' arms homologous to the Rtn4-a / b gene and the expression cassette marked with the NEO resistance gene were knocked into the BAC targeting vector to replace the rtn4-a / b gene region to be knocked out. Wherein, the Frt sequences at both ends of neo can be recognized by Flp recombinase to delete the neo gene. The 5'arm contains 2888bp bases, and the 3'arm contains 4398bp bases ( figure 1 ). 100 μg of Rtn4-A / B-KO plasmid DNA was linearized with NotI (enzyme dosage: 150 U), the enzyme digestion system was 1500 μl, digested overnight at 37°C, treated with equal volumes of phenol chloroform and chloroform, precipitated with absolute ethanol, and washed with 1000 μl sterile PBS Resuspend the linearized plasmid DNA for later use. The constructed homologous targeting plasmid was digested with BamHI and electrophoresed as follows: figure ...

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Abstract

The invention relates to a mouse Rtn4-A / B gene knockout method which comprises the following steps: knocking 5' and 3' arms homologous with a mouse Rtn4-A / B gene and an expression cassette marked with a NEO resistance gene into a BAC targeting vector; and linearizing the obtained Rtn4-A / B-KO plasmid DNA (deoxyribonucleic acid) with Not I, digesting over night, treating with isometric phenol-chloroform and chloroform, adding anhydrous ethanol to precipitate, and suspending the linearized plasmid DNA with sterile PBS for later use; and (2) performing cell transfection on an ES cell with the linearized Rtn4-A / B-KO DNA; and extracting the resistance-cloned genome DNA, and performing long-chain PCR (polymerase chain reaction) identification across the 5' or 3' arm and an insertion element. The invention effectively knocks out the gene expression of Rtn4-A, B1 and B2, and has no obvious influence on the expression of other Rtn4 subtypes, thus laying a foundation for further researching the biological function of Rtn4-A / B.

Description

technical field [0001] The invention belongs to the field of Rtn4 gene, in particular to a mouse Rtn4-A / B gene knockout method. Background technique [0002] Rtn4 (also known as Nogo) protein is a member of reticulon protein family 4 (reticulon protein family 4, Rtn4), located on chromosome 11, with a length of nearly 50kb, consisting of 11 exons and 8 introns, this family includes RTN4 -A, B, C three proteins. Among them, Rtn4-A and B are obtained by alternative splicing of the RTN4 gene through the same promoter in different ways, and both have the same carboxy-terminal sequence (containing 188 amino acid residues). Rtn4-A is mainly expressed in the central nervous system, and there is only a small amount of expression in the testis and heart tissue in the peripheral tissues. Rtn4-B is widely expressed in peripheral organs and tissues, including lungs, cardiovascular tissues, etc. It is mainly involved in the regulation of the body's inflammatory response and the repair ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/09C12N15/63
Inventor 李强胥武剑朱莹
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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