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Preparation method of transgenic mouse of specificity expression Cre recombinase of hematopoietic system

A technology of transgenic mice and recombinant enzymes, which is applied in recombinant DNA technology, the use of vectors to introduce foreign genetic material, animal husbandry, etc., can solve the problems of sensitive response to inducers, blocked proliferation of K562 cells, and decreased colony formation ability

Inactive Publication Date: 2013-01-09
INST OF RADIATION MEDICINE ACAD OF MILITARY MEDICAL SCI OF THE PLA
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Problems solved by technology

However, EDAG is highly expressed in peripheral blood cells of leukemia patients and leukemia cells K562. After Hemin, Pokeweed PMA, etc. induce K562 cells to differentiate into erythroid or megakaryotic lineage, the expression of EDAG is rapidly down-regulated; after antisense nucleic acid is used to inhibit the expression of EDAG, The proliferation of K562 cells is blocked, the ability of colony formation is reduced, and the cells are more sensitive to the inducer response

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  • Preparation method of transgenic mouse of specificity expression Cre recombinase of hematopoietic system
  • Preparation method of transgenic mouse of specificity expression Cre recombinase of hematopoietic system
  • Preparation method of transgenic mouse of specificity expression Cre recombinase of hematopoietic system

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[0019] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these examples are only used to illustrate the present invention but not to limit the scope of the present invention.

[0020] Materials and methods

[0021] 1. Materials

[0022] The recombinase Cre expression plasmid A1.0-Cre was donated by Professor Yang Xiao from the Institute of Bioengineering, Academy of Military Medical Sciences. The plasmid pEDAG-GFP containing EDAG promoter was constructed and preserved in our laboratory. Bacterial strains DH5a and JM109 were prepared and preserved in our laboratory. Restriction enzymes and T4 ligase were purchased from NEB Company. C57B / 6 mice were provided by the Experimental Animal Center of our hospital. ROSA26 reporter gene mice were provided by the Institute of Model Animals, Nanjing University. EDAG-Cre transgenic mice were successfully constructed and kept in the Experimental Animal Center o...

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Abstract

The invention relates to a preparation method of a transgenic mouse of specificity expression Cre recombinase of a hematopoietic system. The invention discloses a transgenic mouse animal model, a deoxyribonucleic acid (DNA) fragment pEDAG-Cre is integrated in a genome of the transgenic mouse animal model, the DNA fragment comprises a promoter at an end of a gene (EDAG) 5' which is related to human embryonic development, a Cre recombinase gene and a human growth hormone gene polyA fragment. The invention also comprises a preparation method of the transgenic mouse animal model. The method comprises the steps of cloning 2.3 kb of a control region of the promoter at the end of the EDAG gene 5' by using a polymerase chain reaction (PCR) method, constructing a transgenic expression vector pEDAG-Cre, leading 5.6 kb of transgenic fragments into a male pronucleus of the mouse by using a micro-injection method, and identifying a positive transgenic mouse by using the PCR method. The RT-PCR method and a mouse-identifying result report ROSA26LacZ show that the specificity expression Cre recombinase of the transgenic mouse can be achieved only in the hematopoietic system.

Description

technical field [0001] The present invention relates to a preparation method of a transgenic mouse model, in particular to cloning the 2.3kb 5' end promoter regulatory region of the human embryonic development-related gene EDAG gene by PCR method, using the regulatory sequence to construct an expression vector and preparing A method for preparing transgenic mice specifically expressing Cre recombinase in the hematopoietic system. Background technique [0002] Mammalian hematopoiesis is a complex and dynamic process. At embryonic day 7.5 in mice, blood islands are composed of endothelium and hematopoietic cells derived from extraembryonic mesoderm-derived cells. At this time, hematopoietic cells are mainly primitive red blood cells with round nuclei, large cell bodies, and expression of embryonic hemoglobin. This process lasts for a short time. Known as primitive hematopoiesis. At embryonic day 10.5, the intraembryonic site aorta-gonad-mesonephros (AGM) begins to generate a...

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IPC IPC(8): A01K67/02C12N15/85
Inventor 葛常辉许望翔李长燕杨晓明
Owner INST OF RADIATION MEDICINE ACAD OF MILITARY MEDICAL SCI OF THE PLA
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