Description:
STAT3 Responsive Luciferase Reporter Hela Stable Cell Line is derived from human cervical cancer, and stably express firefly luciferase reporter gene under the control of the STAT3 response element. This cell line is an ideal cellular model for monitoring the activation of JAK-STAT Receptor Signaling Pathway triggered by stimuli treatment, enforced gene expression and gene knockdown.Principle:
STAT3 is activated in response to various cytokines and growth factors including IFNγ, IL-6 and OSM. The activation of STAT3 results in formation of STAT3 homodimers and STAT3/STAT1 heterodimers that translocate to the cell nucleus and induce transcription of genes by binding to the consensus element on the promoter region of target genes associated with cell growth and apoptosis. Signosis has established Stat3 luciferase reporter HeLa stable cell line, which can be used as a reporter system for monitoring the activation of Stat3 triggered by stimuli treatment, enforced gene expression and gene knockdown.
The cell line was established by transfection using a pTA-Stat3-luciferase reporter vector, which contains 4 repeats of Stat3 binding sites, a minimal promoter upstream of the firefly luciferase coding region, along with hygromycin expression vector followed by hygromycin selection. The hygromycin resistant clones were subsequently screened for oncostatin induced luciferase activity. The clone with the highest fold induction (10 fold) was selected and expanded to produce this stable cell line.
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 HeLa/STAT3-luciferase activity of control and treatment with Oncostatin. The cells were seeded on a 96-well plate for overnight with DMEM including 10% FBS. The cells then were treated with or without 10ng/ml Oncostatin respectively in DMEM and 0.1% FBS for 8 hours. Stat3 luciferase reporter cell line showed more than 25-fold increase in luciferase activity in response to Oncostatin M treatment.
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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
一种是脂质体转染后,单克隆筛选稳定细胞株.另外一种是应用逆转录病毒,慢病毒转染,筛选稳定细胞株.
脂质体转染:在转染24小时后,消化细胞并计数.将细胞种到96孔板,保证每个孔2-3个细胞,这样才能得到单克隆.待细胞贴壁后,加入抗生素筛选.筛选时间和浓度视细胞而定.一般G418一个星期作用,嘌呤霉素2-3天.
脂质体法筛单克隆时间较长,且效率低,大概只有1%.
病毒转染:先要用包装细胞,一般为293细胞,包装出病毒,再用病毒转染目的细胞.包装病毒视不同类型的病毒而定,一般要3-5天的时间.包装好的病毒要测滴度,根据滴度决定转染目的细胞的病毒量.转染目的细胞1-2天后加抗生素筛选得到稳定细胞株.
病毒转染得到稳定细胞株的效率高,只是步骤繁琐.
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