介绍一种信号传导研究简单易行的方法

虽然把这个帖子放在信号传导前沿区里，但是这种方法已经不是什么前沿方法了，实际上就是stratagene的path detect 系统，利用荧光素酶报道基因检测外源基因或是外源刺激因子对目的基因的影响。其中主要是用于研究MAPK信号途径。这是网页及介绍：
http://www.stratagene.com/products/displayProduct.aspx?pid=466

The activation of cis-acting elements often reflects the activation of more than one transcriptional factor. It is known that many signal transduction pathways converge at a protein kinase, which phosphorylates and activates a specific transcriptional factor. Therefore, the activation of a transcriptional factor, as used in the PathDetect trans-reporting systems, will give a more precise assessment of the involvement of a protein or compound of interest in a specific signal transduction pathway.

Sensitive and Easy Method to Study Protein Function
PathDetect trans-reporting systems allow you to determine if your protein or compound of interest directly or indirectly activates one of six transcription factors: c-Jun, Elk1, CREB, c-Fos, ATF2 or CHOP. Each PathDetect trans-system consists of a reporter plasmid, a trans-activator plasmid and both positive and negative control plasmids. Simply cotransfect the reporter plasmid, trans-activator plasmid and an expression vector containing your gene of interest into your cell line of choice. Expression of the luciferase reporter gene indicates that your gene of interest is directly or indirectly involved in the phosphorylation and activation of the transcriptional factor in the trans-activator plasmid, and hence involved in the respective signal transduction pathway.

Choice of Reporter Plasmids
We have constructed a series of reporter plasmids that enable the use of the PathDetect system with reporter proteins in addition to luciferase. These reporter proteins include humanized green fluorescent protein from a novel marine organism (Vitalty® hrGFP), chloramphenicol acetyltransferase (CAT), b-galactosidase (b-Gal), and secreted alkaline phosphatase (SEAP).

Each reporter protein offers certain advantages depending on your experiment. hrGFP can be easily detected in vivo using a fluorescent microscope, fluorescent activated cell sorting (FACS), or fluorometer. ß-Gal activity can be analyzed using a spectrophotometer or a microplate reader which makes it a good choice when a 96-well plate format is desired. The CAT reporter protein has an in vivo half-life of about 50 hours. This is advantageous when an accumulative vs. a dynamic change type of result is desired. The SEAP reporter can be continuously monitored without lysing the cells. This facilitates the automation of the sampling and assay procedures. Of the four types of reporters listed here, we have found luciferase to be the most sensitive reporter, therefore, the luciferase reporter should be used when very low levels of reporter gene expression are expected.

These new reporter plasmids have the same backbone as the original pFR-Luc plasmid and have been validated to function with the PathDetect System.

Pathway-Specific Activation
The pathway-specific trans-activator vectors express a chimeric trans-activator protein that will be phosphorylated if the upstream signal transduction pathway is activated. Phosphorylation can be either direct or indirect through other signaling intermediaries. Each PathDetect trans-activator vector allows a different pathway to be studied—if a trans-activator plasmid is not available, simply make your own with the pFA-CMV vector. This vector contains the GAL4 DNA binding domain upstream of a multiple cloning site controlled by the CMV promoter### for easy construction of fusion trans-activator plasmids.

Low Background and High Activation
The GAL4 DNA binding domain is a yeast transcriptional activator and no mammalian transcriptional factors can efficiently bind to it. This feature of the fusion activators creates very low assay backgrounds when using the PathDetect trans-reporting systems. Activation as high as 200-fold has been observed using these systems.

Stable Cell Lines
The HLR cell lines are double-stable fusion trans-activator cell lines with reporter gene activity. These cell lines were constructed in a series of transfection and selection experiments. For this process, the pFR-Luc plasmid with a hygromcyin expression cassette was transfected into HeLa cells. Single colonies were isolated and tested with the PathDetect CREB, ELk1, c-Jun, CHOP trans-reporting systems to select the cell line that demonstrated the highest levels of luciferase activity when cotransfected with fusion trans-activator and control plasmids as well as the lowest background of luciferase activity. This clonal line was designated the HeLa luciferase reporter (HLR) cell line. In order to construct the double-stable cell lines, the HLR cell line was transfected with the fusion trans-activator pFA2-CREB, pFA2-Elk1, pFA2-cJun, or pFA2-CHOP plasmids. After antibiotic selection, the clones that yielded the highest responses with the positive control vectors from the PathDetect kits were identified and maintained.

Eliminate Transfections
The HLR-CREB, HLR-Elk1, HLR-cJun, and HLR-CHOP cell lines are used for studying signal transduction pathways that converge at the transcription factors CREB, Elk1, c-Jun, and CHOP, respectively. These stable HeLa cell lines feature constitutive expression of the GAL4-CREB, GAL4-Elk1, GAL4-cJun, or GAL4-CHOP fusion proteins and encode the firefly luciferase gene controlled by a promoter that responds to GAL4 fusions. Upon activation by phosphorylation, these fusion proteins induce luciferase expression when bound to the promoter. Luciferase activity in the stable cell lines reflects the activation status of the fusion trans-activator protein and the activation status of the corresponding signaling pathways. The effects of extracellular stimuli on signaling pathways that converge at these activators can be studied directly, without transfection.
用这种办法只要做个瞬时转染加上荧光素酶活性分析就行了，三天搞定。我们实验室用这个方法已经发了5篇3.0 的SCI论文了，省时省力的好办法。