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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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
由于质粒的不兼容性,拥有同种复制子的质粒不能在同一细胞内稳定共存,经过几代的复制,会质粒丢失,所以并不是任何两个或两个以上质粒都可以在同一细胞内稳定存在,但是可以同时进入.
稳定转染的细胞株，就是转染后质粒可以稳定整合到基因组上不会因细胞分裂而丢失。区别于质粒瞬时转染不能长时间保留质粒在细胞里。
对于瞬时转染的检测：如果有报告基因，就通过报告基因的表达看转染成功与否；没有报告基因的话，就在24～96h内收获细胞通过RT-PCR和WB来鉴定。
至于到底好不好转就跟你的细胞有关了，和细胞是否是稳转株关系不大。
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
推荐了解“主细胞库”“工作细胞库”这两个名词。
建立稳定细胞株,一般是根据不同基因载体中所含有的抗性标志选用相应的药物对靶...专注整体实验·服务生命科学已开展了数千个实验外包项目
