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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然后你得确定，这个细胞株对于你得研究来说不可或缺，具有明确的代表性。
上面两个都没有问题的话，祝你顺利。
① 在构建载体时，目的基因直接整合到细胞染色体组上，最好不要通过先瞬转在筛选稳定细胞株的这种方法，因为转染效率没有保证
② 高表达载体的构建，哺乳动物表达量一直是它自身的缺点，最好根据高表达载体定向的驯化细胞，提高蛋白表达量
③ 细胞的选择，筛选稳定细胞株我们常用的细胞是CHO，中国仓鼠卵巢细胞，由于CHO具有诸多的优点因此适合用于筛选稳定细胞株，而HEK293细胞则常用于瞬时转染
④ 后期的筛选，双抗预防污染，筛选细胞的时候抗生素浓度一定要做预实验，而且转染的时候不能有抗生素，关于细胞转染 稳定细胞系构建的相关理论
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。
