Two reporter genes are better than one (2/2)

Very generally speaking, in a cell the protein pool detain the 90% of the executive power: proteins execute the algorithms encoded in the DNA genes (don't report this statement to a scientist or he will freak out in spontaneous combustion). The point is that protein functions rely on their ability to engage into specific protein-protein interactions. Several approaches (two-hybrid system, interaction trap) have therefore been developed by functional genomics to study these interactions (here a review in protein interactions). One of these approaches is Resonance Energy Transfer (RET), a proximity based assay that rely on the non-radiative transfer of energy between donor and acceptor molecules according to a so-called Forster mechanism. In my knowledge two RETs are actually developed: FRET (Fluorescence) and BRET (Bioluminescence), both with different advantages and disadvantages belonging to Fluorescence vs Bioluminescence (I'm writing a post about that).
Until now, BRET imaging development at the subcellular level was hampered by the low level of light intrinsic to the bioluminescent luciferase reaction compared to fluorescent ones, so Xiaodong Xu (Vanderbilt University) and Vincent Coulon (French INSERM), started to establish the appropriate experimental conditions to visualize and quantify protein-protein interactions with BRET. Recently they demonstrated respectively on the Proceeding of the National Academy of Science and on the Biophysical Journal, that BRET imaging offers enough resolution to detect signals that originate selectively from sub-cellular compartements. This is true also for plant and animal tissues, so now it is possible to track these interactions and have a knowledge if they occur in the nucleus or plasma membrane or endocytic vesicles directly by microscopy in alive cells, while in the past BRET was preferentially used in microplate readers of cell lysates.

Because BRET is made by simultaneously using of two reporter genes (Renilla luciferase and Yellow Fluorescent Protein) I can tell you once again that two is better than one.

Two reporter genes are better than one (1/2)

Sometimes choosing the right reporter gene is a hard matter. Let choose fluorescent proteins: they are very bright, but they need also an external source of light for excitation and because of that, some specimen suffer from ligth toxicity or results in autofluorescence; do you choose luciferase enzymes? they are less bright - so you need more sophisticated instruments - and if you want to record endpoint (RLU) proportional to gene expression, your have to give saturating amounts of ATP and luciferin to your sample. Although with cell cultures it is quite simple to fulfill the requirements of reporter gene, or eventually move to another reporter system to integrate intrisinc limitations of a single reporter measurement, this is not true for reporter animals. Once you knock your GFP mouse, your EGFP zebrafish, or your GUS Arabidobsis, you have to deal forever with specific limitation typical of the selected reporter.

Multimodality technologies effort to fill that gap by coupling one promoter with two or three different reporter genes. Up to date, there are three strategies in plasmid construction that claim for multimodality: bicistronic vectors, fusion proteins and bidirectional promoters. Recently, the bicistronic vector was the strategy choosed by Luisa Ottobrini and colleagues from Milan University to develop multimodality imaging of estrogen receptor transcriptional activity, as reporterd in the latest print issue of the European Journal of Nuclear Medicine and Molecular Imaging (a forum for the exchange of clinical and scientific information for the community involved in molecular investigation of diseases).

In the developed construct, a promoter activated by the estrogen receptor, drive the expression of two reporter genes, the firefly luciferase - for in vivo bioluminescent imaging (BLI) -, and a mutated form of the domaminergic D2 receptor (D2R80A) for positron emission tomography (PET). Thanks to the internal ribosome entry site (IRES) of the encephalomyocarditis virus, the two reporter genes can be translated from a single RNA transcript. Finally, insulator sequences (MAR) flanks the construct and prevents enhancer-mediated activation, or repression of transcription by chromatin, assuring the reporter to be transcriptionally accessible in every cell in which estrogen receptors are expressed.

According to the authors,

The coupling of a nuclear with an optical reporter gene yields highly informative data.

So... two is better than one, and now it remains to wait for the first reporter animal generated with such kind of construct.

Ottobrini, L., Ciana, P., Moresco, R., Lecchi, M., Belloli, S., Martelli, C., Todde, S., Fazio, F., Gambhir, S.S., Maggi, A., Lucignani, G. (2008). Development of a bicistronic vector for multimodality imaging of estrogen receptor activity in a breast cancer model: preliminary application. European Journal of Nuclear Medicine and Molecular Imaging, 35(2), 365-378. DOI: 10.1007/s00259-007-0578-z

When barriers stem new roads

In vivo molecular BLI is rapidly becoming a standard method for measuring gene expression changes in normal tissues, cancer, and specific cell populations, such as stem cells. The principal gene reporter involved in BLI is firefly luciferase that, by catalyzing the oxidation of D-luciferin, emits yellow light. Although in reporter systems luciferase is directly expressed by the cell, its substrate luciferin must be added by external sources. For instance, in transgenic luciferase mice, luciferin is injected intraperitoneally, where it is thought to enter in the bloodstream reaching almost every cell of the organism. But some barriers need to be accounted. When Yimao Zhang from Johns Hopkins Medical Institution, was studying inhibitors of tumor promoting hedgehog signaling pathway, he uncovered that luciferin uptake by the cells is influenced by cell expression of the ABC transporter family member ABCG2/BCRP. In the Vol 67 No 19 of Cancer Research (the most frequently cited cancer journal in the world) Zhang and colleagues show that D-luciferin is a substrate for ABCG2/BCRP. Because any event that changes the activity of this transporter can substantially alter cell-based or in vivo bioluminescent imaging endpoints independent of other physiologic processes under investigation, this work confirm once again that drug-resistence gene modulation must be considered in BLI studies. To be optimist, better remember that side-effects in developing technologies could give new hints: "why don't establish the feseability of novel high-throughput methods for identifying new ABCG2 inhibitors just by bioluminescence imaging?" Said Zhang...

Shine enantio-tools for preparative chemist

In the beginning it was only to help beetle to mate. Then it became a reporter gene in transfection assays. Finally it was the main character of bioluminescence imaging (BLI), as a way to detect bacterial pathogens in living hosts first, then as a way to monitor tumor growth, measuring protein-protein interactions, observing the trafficking of immune cells, and to study gene expression in vivo. Not a surprise that its bioluminescent properties represent a routine in drug development in pharmaceutical industry. Obviously, we are talking about firefly luciferase (fLuc). Now, at University of Hyogo, the similarity of the the sequences between firefly luciferase and some acyl-CoA synthetase (so-called LACS1), lead Dai-Ichiro Kato and colleagues to hypothize and demonstrate that this bright enzyme has also thioesterification activity versus some nonsteroidal anti-inflammatory drugs like ketoprofen. These results, published in Vol 274 of FEBS Journal, a collector of papers that advance new concepts in the area of molecular life sciences, suggest that this old reporter gene would be also a new option for the preparative chemist.

---/ citation /--- --- ---
Kato, D., Teruya, K., Yoshida, H., Takeo, M., Negoro, S., & Ohta, H. (2007). New application of firefly luciferase - it can catalyze the enantioselective thioester formation of 2-arylpropanoic acid FEBS Journal, 274 (15), 3877-3885 DOI: 10.1111/j.1742-4658.2007.05921.x

Imaging splicing in vivo using reporter mice

Imaging technologies are influencing the way we study regulatory processes in vivo. Several groups have just taken advantage of imaging technologies to develop reporters capable of reflecting alternative splicing events in living organisms such as rodents and worms. Now a detailed protocol in which these advances are explained step by step appeared in the Vol 2 No 9 (2007) of Nature Protocols, a online resource for authoritative and peer-reviewed protocols: Vivian I Bonano and colleagues from Duke University Medical Center, developed a fluorescence reporter named "Gint" that use enhanced GFP (EGFP) expression as an indication of silencing in vivo. The transgenic model can be analyzed both by macro-imaging and epifluorescence microscopy, and the strategy described can be adapted also to examine other types of alternative splicing and other RNA processing events. In vivo imaging is the brightest application of reporter genes.

Fiat lux

After the breaking of the Abbe limit, biophotonics became once again a new and exciting research field that is already delivering substantial dividends in a wide range of applications from fundamental medical research to diagnosis, therapy and surgery. This analyzes Philip Hunter, in Vol 8 | No 10 of EMBO reports, the journal of the European Molecular Biology Organization that sharply focuses all the areas of molecular biology. Technical advances like quantitative phase contrasting imaging; optical coherence tomography; raman spectroscopy and two-photon laser scanning microscopy, bestowed optical imaging the ability to observe biochemical processes in real time. In the latter technique, the use of fluorescence underlight how reporter genes can generate innovations, allowing scientists to shine light on the manifold mysteries off the cell at a level of detail only previously possible with electron microscopy.