Description:
NRF2/ARE Responsive Luciferase Reporter NIH3T3 Stable Cell Line is derived from Mouse fibroblast, and stably express firefly luciferase reporter gene under the control of the NRF2/ARE response element. This cell line is an ideal cellular model for monitoring the activation of Antioxidant Response Pathway triggered by stimuli treatment, enforced gene expression and gene knockdown.Principle:
NRF2 plays a crucial role in cellular anti-oxidant defense, making it a therapeutic target for neurodegenerative diseases and cancer. Under normal conditions, NRF2 localizes in the cytosol and is rapidly degraded by the proteasome. Under oxidative stress, NRF2 is stabilized and translocates to the nucleus where it binds to a DNA promoter and initiates gene expression. In the nucleus, NRF2 forms a heterodimer with a small Maf protein and binds to the Antioxidant Response Element in the upstream promoter region of many antioxidative genes, and initiates their transcription.
This NRF2 luciferase reporter stable cell line has been stably transfected with pTA-ARE-luciferase reporter vector, which contains 4 repeats of antioxidant response binding sites, a minimal promoter upstream of the firefly luciferase coding region, along with a hygromycin expression vector. Following selection, the hygromycin resistant clones were subsequently screened for TBHQ-induced luciferase activity. The clone with the highest fold induction was selected and expanded to produce this stable cell line.
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Principle behind TF luciferase reporter. TF luciferase reporter stable cell line utilizes artificial promoter constructs to drive luciferase expression. The promoter region can consists of multiple repeats of a cis-element TF binding site, a DNA fragment from the promoter region of a known TF downstream gene, or a DNA fragment containing putative/known TF binding sites. There are several ways that a TF can be activated, such as through extracellular stimuli or through intracellular signaling pathways. Once activated, the TF translocates to the nucleus and often interacts with relevant co-factors to drive gene expression. Once luciferase is expressed, it can generate light in an enzymatic assay and the amount of light measured is positively correlated with the level of TF activation. |
Data:
Analysis of the NRF2 Pathway Reporter NIH3T3 Stable Cell Line in response to stimuli. The cells were seeded on a 96-well plate for 8 hours or overnight with DMEM including 10% FBS. The cells then were treated with or without 10μM TBHQ or 10μM 4HNE respectively in DMEM and 0.1% FBS for 16 hours.
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推荐了解“主细胞库”“工作细胞库”这两个名词。
1.将外源基因插入慢病毒载体
2.将构建完成的载体与慢病毒包装质粒混合，共转染靶细胞
3.收集病毒液
4.用病毒液感染靶细胞
5.用载体上带的抗生素进行筛选，如果没有，可以用无限稀释法
6.获得稳转株
如果经费充足的话可以找公司包装病毒，如武汉的普健可以提供各种载体的构建及细胞株构建的技术服务。
