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Dual-luciferase report cell capable of detecting activation of NF-kappaB and construction method of dual-luciferase report cell

A dual-luciferase, cell technology, applied in genetically modified cells, biochemical equipment and methods, cells modified by introducing foreign genetic material, etc., can solve the problem of inability to correct cell number and cytotoxicity, low sensitivity and low price. And other issues

Inactive Publication Date: 2018-03-27
YANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

(4) Secreted alkaline phosphatase (SEAP): SEAP is a mutant of human placental alkaline phosphatase. Its advantage is that it does not need to lyse the cells, and the enzyme activity can be detected only with the culture medium. When PNPP) is the substrate, the standard colorimetric method can be used to measure the enzyme activity, which is simple to operate, short in reaction time, cheap in price, but low in sensitivity
[0005] Most of the existing NF-κB activation reporter cells use secreted alkaline phosphatase (SEAP) as the reporter system to detect through chromogenic reaction (www.invivoGen.com), and the detection sensitivity of SEAP needs to be further improved
Secondly, almost all reporter cells lack an internal reference reporter system, which cannot correct for differences caused by cell number and cytotoxicity

Method used

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  • Dual-luciferase report cell capable of detecting activation of NF-kappaB and construction method of dual-luciferase report cell
  • Dual-luciferase report cell capable of detecting activation of NF-kappaB and construction method of dual-luciferase report cell
  • Dual-luciferase report cell capable of detecting activation of NF-kappaB and construction method of dual-luciferase report cell

Examples

Experimental program
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Effect test

Embodiment 1

[0027] Example 1: Construction of dual luciferase reporter cells that can detect NF-κB activation

[0028] (1) Monoclonal cells stably expressing NF-κB FLuc:

[0029] After human embryonic kidney cells (HEK293, ATCC product number: CRL-1573) were digested with 0.25% trypsin, single cells were harvested, counted and analyzed in 0.5X10 6 Each well was divided into 6-well cell culture plates and placed in a cell culture incubator for overnight culture. The next day, the cells were infected with lentivirus (G&P biosciences, product number LTV-NFkB-Luc) expressing NF-κB FLuc at 1.0 MOI, and 48 hours after infection, the infected cells and non-infected control cells were simultaneously killed with 10 μg / ml blast fungus (Blasticidin, InvivoGen, Cat. No. ant-bl-05) screening. After 1-2 weeks of selection, all the control cells died, and a small amount of blasticidin-resistant cells grew in the infected cells, so that HEK293 cells stably expressing NF-κB FLuc were obtained. The abov...

Embodiment 2

[0032] Example 2: Specific response of NF-κB dual luciferase reporter cells to stimulators

[0033] The NF-κB dual-luciferase reporter cells obtained in Example 1 were further subjected to experimental application and verification. Except TNF-α used in Example 1 can activate NF-κB in cells, other substances such as interleukin 1 (IL-1) and phorbol ester (PMA) can also effectively activate NF-κB. NF-κB dual luciferase reporter cells at 0.4X10 5 Each well was divided into 96-well cell culture plates and placed in a cell culture incubator for overnight culture. On the second day, use different concentrations of human IL-1β (12.5, 25, 50, 100ng / ml, source: Peprotech, item number: 200-01B-2) and PMA (50, 100, 200ng / ml, source: InvivoGen, item number: tlrl-pma ) stimulation for 8 hours. At the same time, 10 and 20 μg / ml high molecular weight artificial double-stranded RNA similar synthetic Poly(I:C) (HMW) (InvivoGen, product number: tlrl-pic) were stimulated for 8 hours as a cont...

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Abstract

The invention belongs to the technical field of biology and particularly relates to a dual-luciferase report cell capable of detecting activation of NF-kappaB and a construction method of the dual-luciferase report cell. The report cell is an HEK293 cell in which NF-kappaB firefly lueiferase (Fluc) and internal reference sea pansy luciferase (Rluc) are introduced. The construction method comprisesthe steps of stably expressing NF-kappaB Fluc / TK Rluc monoclonal cells. The highly-sensitive NF-kappaB firefly lueiferase and internal reference sea pansy luciferase are sequentially introduced intothe HEK293 cell, and the NF-kappaB dual-luciferase HEK293 stable report cell is constructed and invented by virtue of a specific stable cell screening technique, so that an NF-kappaB report cell system is improved and replaced.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a dual-luciferase reporter cell capable of detecting NF-κB activation and a construction method thereof. Background technique [0002] Transcription factor NF-κB is a very conserved signaling molecule present in all cells of low to high mammals, and plays an important role in various cellular activities such as apoptosis, virus replication, tumorigenesis, inflammation and autoimmune diseases [1,2] . The NF-κB family consists of P50 (a processing product of p105, both known as NF-κB1), P52 (a processing product of p100, both known as NF-κB2), REL (also known as c-REL ), REL-A (also known as P65) and REL-B ( figure 1 ). These proteins dimerize to form functional NF-κB. Except that REL-B can only effectively combine with P50 or P52, there are all combinations of homologous or heterologous dimers, and these different dimers all have NF-κB activity. Each member of the NF-...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/867
CPCC12N5/0686C12N9/0069C12N15/85C12N2510/00C12N2800/107C12Y113/12005C12Y113/12007
Inventor 朱建中李双洁何姗周荣云朱美芹夏芃芃敖大
Owner YANGZHOU UNIV
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