Establishment method of programmed necrosis inducing system

A technology of programmed necrosis and establishment method, applied in the field of establishment of programmed necrosis induction system, can solve problems such as poor effect, and achieve the effect of high efficiency, rapid induction of cell necrosis and strong specificity

Inactive Publication Date: 2017-09-15
LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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  • Claims
  • Application Information

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Problems solved by technology

[0006] The purpose of the present invention is to provide a method for establishing a programmed necrosis in

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  • Establishment method of programmed necrosis inducing system
  • Establishment method of programmed necrosis inducing system
  • Establishment method of programmed necrosis inducing system

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Embodiment Construction

[0021] The present invention will be described in detail below in combination with specific embodiments.

[0022] In the present invention, through the puromycin screening of the lentiviral packaging system, we have constructed stable cell lines of A549 and Hela cells that can induce the expression of RIPK3-2×FKBP, and added different concentrations of Doxycycline (hereinafter referred to as Doxycycline) to the cell culture medium. "Dox" for short) induced the expression of RIPK3-2×FKBP protein, and then performed Western blot experiments, figure 1 It is a schematic diagram showing that the expression of RIPK3-2×FKBP depends on Dox; the results show that in A549 and Hela cells, the expression of RIPK3-2×FKBP is very high when the concentration of Dox is 1 μg / mL, while in the control without adding DOX The expression of RIPK3-2×FKBP was not detected in the cells. Meanwhile, the induction effect of 1μg / mL DOX in Hela was even better than that of 4μg / mL and 10μg / mL Dox. These r...

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Abstract

The invention discloses an establishment method of a programmed necrosis inducing system. The establishment method comprises the following steps: linking a target gene segment RIPK3-2*FKBP to lentiviral vector containing a Tet-On system, so that a system which can induce expression is constructed; co-transfecting a vector which is correctly sequenced and a lentiviral element vector into 293T cells, collecting virus liquid infected Hela cells and A549 cells, and implementing puromycin screening, so that a stable cell line is obtained; inducing stable expression of RIPK3-2*FKBP protein of the cells by adding doxycycline to a culture medium, and then adding a dimer so as to induce dimerization of RIPK3; and on the basis of Western blotting and related experimental technological means, analyzing an experimental result. The inducing system provided by the invention has the beneficial effects that programmed necrosis of cells is induced through the Dox (the doxycycline) and Idimer; the system is higher in sensitivity and efficiency and stronger in specification; and necrosis of the cells can be rapidly induced.

Description

technical field [0001] The invention belongs to the field of gene technology and relates to a method for establishing a programmed necrosis induction system. Background technique [0002] Tumor necrosis factor-α, a pleiotropic cytokine, is involved in multiple cellular responses, including cell survival, cell proliferation, and cell death. Cell death induced by TNF-α requires the combination of TNF-α and TNFR1 located on the cell membrane. After activation, TNFR1 recruits a series of proteins in the cell to form different complexes. When TNF-α binds to TNFR1, it can induce the aggregation of itself and its downstream protein death domain and other molecules to form the TNF receptor-associated death domain (TRADD), RIP1, TNFR-associated factor 2 (TRAF2), and intracellular apoptosis. Inhibitor of death protein 1 (cIAP1), cIAP2 complex. These protein molecules will respond to different stimuli or changes in the microenvironment, trigger the activation of different signaling p...

Claims

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Application Information

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IPC IPC(8): C12N15/867C12N5/10C12N9/12
CPCC12N9/1205C12N15/86C12N2740/15043C12N2830/003
Inventor 张志东秦晓东甘晓丽李彦敏
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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