Although it is true that some tumors or diseases are caused by alterations in the genetic information of the genome itself, epigenetic regulation can sometimes better elucidate the mechanism of tumor or disease development. In the progress of the project, especially after the phenotype is determined, we often study the mechanism of phenotyping from the direction of epiregulation, transcriptional regulation, or protein modification. The relationship between differential genes and phenotypes is often easy to determine, and the mechanism is relatively complicated. Genome-wide screening is a strategy for epiregulation, as is the discovery of key genes through bioinformatics analysis.
A complete gene structure includes many elements, such as coding regions, including exons and introns; The leading region, located upstream of the coding region, is equivalent to RNA5'terminal non-coding regions, containing regulatory regions, including promoters and enhancers; Caudal region, located at RNA3'Downstream of the coding region, which corresponds to the terminal non-coding region. The two sides of the genetically coding region are also called flanking sequences. The leader region regulates gene expression and is defined as a cis-acting element, including promoters, enhancers, regulatory sequences, and inducible elements. The cis-acting element does not encode any protein on its own, but only provides a site of interaction with the trans-acting factor. The essence is the nucleotide sequence, i.e., the dn** segment.
Trans acting factors bind to specific cis-acting elements, which are involved in the regulation of gene expression. The gene encoding the trans action factor is not on the same chromosome as the gene that is regulated by the trans action factor. Trans-acting factors have two important functional domains: the DNA binding domain and the transcriptional activation domain, which are essential structures for trans-acting factors to exert transcriptional regulatory functions. Trans-acting factors can be induced to be synthesized, and their activity is also regulated by a variety of factors. The trans action factor here is protein in nature. Transcription factors are important components of trans acting factors and can be induced for expression.
The DNA binding domain of the transcription factor and the cis-acting element are covalently bound to inhibit or enhance the expression of genes. The luciferase reporter system is a method to detect the interaction of transcription factors with the DNA of the promoter region of the gene of interest. The principle is to clone the promoter sequence before the firefly luciferase, and use the promoter of interest to initiate the expression of the firefly luciferase. As shown in the figure below, if you want to study the expression regulation of IFNA or IFN, you can clone the promoter of IFNA or IFN into Firefly Luciferase, and the internal control is to use the promoter of the TK gene to initiate the expression of Renilla Luciferase.
Luciferase reporter principle (Rachael Kenworthy et al.). 2009. nucleic acids res）
If we want to investigate whether a transcription factor can interact with a target promoter fragment. First, the target gene promoter or other regulatory elements of the gene to be studied are inserted into front of the luciferase reporter gene to construct a reporter gene plasmid. Then, cells are co-transfected with a reporter plasmid that can express the transcription factor to be detected; If this transcription factor activates the target promoter, the luciferase gene is expressed, and the amount of luciferase expressed is proportional to the intensity of the transcription factor; A specific luciferase substrate is then added, and luciferase reacts with the substrate to produce fluorescence. The activity of luciferase can be measured by measuring the intensity of fluorescence to determine whether the transcription factor can interact with the target promoter fragment.
At the same time, in order to reduce the influence of intrinsic factors such as cell number, cell transfection and lysis efficiency on the accuracy of the experiment, the plasmid prl-TK with Renilla luciferase gene was co-transfected with the reporter plasmid as the control plasmid to provide an internal control control for transcriptional viability, so that the test results were not interfered with by the change of experimental conditions. During the measurement, a firefly fluorescence signal is generated when luciferase detection reagent is added, so that the firefly luciferase reporter gene is measured first. After quantifying the fluorescence intensity of the fireflies, the reaction reagent was added to the same sample, the reaction was quenched, and the renilla luciferase reaction was initiated at the same time, and a second measurement was taken at the same time. So, how do you use a luciferase reporter system to study the promoter and transcription factor regulation of genes?
In August 2023, the internationally renowned academic journal J Hematol Oncol published a study titled Synergistic Efficacy of Simultaneous Anti-TGF-VEGF Bispecific Antibody and PD-1 Blockade in Cancer Therapy**, in which Luciferase was used The Reporter system was used to detect the blocking effect of TGF-VEGF bispecific antibody on TGF- signal.
Experimental procedure: 30,000 A549 or MDA-MB-231 cells were seeded in a 96-well plate and cultured overnight. The next day, transfect 02 g SBE4 Luciferase Reporter plasmid. After 24 h, they are treated with 10 ng ml TGF-1 and 106 pM of the specific antibody Y332D for 24 h, respectively; Fluorescence reporter detection is then performed.
Results and Conclusions:Research revealed that TGF- mediates the transcription of smad-binding element-containing luciferase reporter construct, SBE4-luc therefore, sbe4 luciferase reporter assay was performed to test the blocking capability of y332d on tgf-β/smad pathway. the results showed that y332d remarkedly blocked tgf-β1 signaling in a549 and mda-mb-231 cells. also, y332d significantly antagonized tgf-β1-regulated emt in a549 and mda-mb-231 cells.4fxngdjnlty4w7g
Luciferase reporter genes are routinely executed.
In response to the different needs of reporter gene testing, three detection systems have been launched one by one.
Characterization of luciferase reporter genes:
1.The detection sensitivity is high and the signal stability is good.
HEK293-NFK B-Luc cells were stimulated with serial dilution of TNF at 37C and 5% CO for 6 hours, and then the signal was detected with enhanced, hypersensitive, and stable detection reagents, respectively.
2.Easy to operate.
Simply mix the substrate with luciferase assay buffer and add it directly to the cells.
3.Good stability.
At the same time, in 10 repeated freeze-thaw experiments, the full range of reporter gene detection kits showed strong stability.