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Method for building transgenic mouse animal model

A technology of transgenic mice and animal models, applied in the direction of introducing foreign genetic material using vectors, recombinant DNA technology, animal husbandry, etc., can solve the problems of complicated detection of CD69 expression, and achieve the effect of specificity in function and physiological significance

Inactive Publication Date: 2014-12-10
ZHEJIANG UNIV
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AI Technical Summary

Problems solved by technology

Two kinds of transgenic mice found that CD69 can be expressed in thymus T cells, but not in peripheral lymphoid organs, and the detection of CD69 expression is complicated

Method used

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  • Method for building transgenic mouse animal model
  • Method for building transgenic mouse animal model
  • Method for building transgenic mouse animal model

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Experimental program
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Embodiment 1

[0028] Construction of 1pInsulater-LCK-CD69-IRES-EGFP recombinant plasmid

[0029] 1) Extract RNA from mouse lung tissue

[0030] Material: B6 mice

[0031] Reagents: RNA extraction reagent RNAiso Plus (Takara company), 0.1% volume concentration of diethyl pyrocarbonate (DEPC solution), RNase-free water (DEPC-treated water), 75% volume concentration of ethanol solution (treated with DEPC water), chloroform.

[0032] One B6 mouse (B6 is the strain of the mouse) was sacrificed, the lung tissue of the mouse was removed, weighed, quickly frozen with liquid nitrogen, then moved to a mortar pre-cooled with liquid nitrogen and ground into powder, during which the Add liquid nitrogen to keep the temperature low. Add 1ml RNAiso Plus to every 50-100mg tissue of the ground sample to completely cover the tissue powder, let it stand at room temperature until the tissue completely dissolves and becomes a transparent liquid, centrifuge at 12000g×4min at 4°C, draw the supernatant and trans...

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Abstract

The invention discloses a method for building a transgenic mouse animal model. The method comprises the following steps: building a recombinant plasmid pInsulater-LCK-CD69-IRES-EGFP by utilizing the cDNA of the mouse lung tissue CD69 as a target gene, the LCK as a controlling element and the IRES-EGEP (Internal Ribosome Entry Site-Enhanced Green Fluorescent Protein) as a vector; linearizing the pInsulater-LCK-CD69-IRES-EGFP by virtue of restriction enzyme digestion, recovering and purifying one segment of LCK-CD69-IRES-EGFP of 8081bp; performing micro-injection of mouse male pronucleus to obtain a positive transgenic mouse. The built transgenic mouse animal model has higher specificity for studying the functions and physiological significance of the CD69. Meanwhile, for study of inflammatory diseases, the continuous expression of the CD69 in the peripheral blood lymphocytes is more conductive to observing the influence of the CD69 on inflammations.

Description

(1) Technical field [0001] The invention relates to the construction of an animal model, in particular to a method for the construction of a transgenic mouse animal model. (2) Background technology [0002] CD69 is a cell surface activation marker antigen, discovered in 1981, earlier known as AIM, EA-1, LEU-23, MLR-3, etc., is a member of the C-type lectin receptor family, and is also a NK cell signal A member of the conduction gene complex family. CD69 is expressed on almost all myeloid cells. In addition to the continuous structural expression in monocytes, platelets and other cells, its expression in most cells only appears in activated cells. Resting T or NK cells do not express CD69, and can express CD69 on the cell surface 1 hour after receiving the cell stimulation signal, and reach the peak rapidly and last until 72 hours (see Nakamura S, Sung SS, Bjorndahl JM, et al.Human T cell activation. IV. T cell activation and proliferation via the early activation antigen ...

Claims

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Application Information

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IPC IPC(8): C12N15/63A01K67/027
Inventor 汪慧英
Owner ZHEJIANG UNIV
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