Gaussia luciferase (GLuc) holds the promise to became a great reporter. In the native form, GLuc is secreted. This feature gives additional advantages, but markedly attenuates its application for in vivo imaging. At MSKCC.org, Elmer Santos and colleagues recently described on Nature Medicine a membrane anchored external GLuc (termed extGluc) genetically engineered through the addition of a CD8 transmembrane domain to the carboxy terminus of the enzyme. The strategy to put the reporter outside the cell should be advantageous: the substrate luciferin do not need to enter the cell and variability previously inferred to drug-resistant genes should be avoided. In effect, the new reporter was enough sensitive to monitor in vivo T cells by means of classical bioluminescence imaging on a IVIS workstation.


Santos, E., Yeh, R., Lee, J., Nikhamin, Y., Punzalan, B., Punzalan, B., Perle, K., Larson, S., Sadelain, M., & Brentjens, R. (2009). Sensitive in vivo imaging of T cells using a membrane-bound Gaussia princeps luciferase Nature Medicine, 15 (3), 338-344 DOI: 10.1038/nm.1930

Posted in labels: in vivo, luciferase, molecular imaging 0 referee

A Science letter by Robert Stevenson focused my attention on the eventual patentability of new "automated" discoveries. This is of course a letter in response to the "automation of science" previously reviewed. Apparently, it should be legally difficult to patent any invention made by a robot: the American patent law strictly refer to the inventor as "a person", while the European law seems more broad. Thus, supposing a brilliant robot scientist is ever build, no man might protect/deserve those inventions for its proprietary benefit. Legacy is not considered: A invents robot/algorythm B, B invents drug C, but C is not patentable. Honestly, once developed such a robot, it should take no longer to develop a second robot mimicking human creativity (i.e., writing good and bad dates and results on a paper lab-book). Are we so close to Singularity? Interestingly, in a previous Science paper, Debra Meloso from the italian Bocconi University has modeled the patent system and proposed a better way to promote intellectual discovery (maybe including generation of robot-scientists) that should be based on a sort of 2.0 trading of discoveries. Might a machine sell a product? Ask to lawyer Crawford.

Posted in labels: outliers 0 referee

In vivo optical imaging of deep tissues in animals is most feasible between 650 and 900 nm because such wavelengths minimize the absorbance by hemoglobin, water, and lipids, as well as light-scattering. Roger Tsien, last year's Nobel Prize in chemistry for his research on fluorescent proteins, introduced in a Science report, a modified version of the Deinococcus radiodurans phytochrome turned to be a infrared fluorescent protein (IFP). Carrying IFP into the mouse liver through an adenovirus-vector, the infrared fluorescence performed better than mKate a red fluorescent protein as imaged by a Maestro spectral imager. More background inf available at Brainwindows

Noninvasive fluorescence molecular tomography (FMT) imaging of IFP-expressing mouse liver. White box: normalized excitation (blue) and emission (red) spectra of IFP1.4 (Shu et al., 2009)

Actually, this interesting advance makes me a little gloomy, since I spoke about mKate in the Reportergene's first post. It was September 2007, does it takes only two years for a reporter gene to be outperformed? It is dramatic: it takes roughly two years to make a transgenic reporter mice, and another two-three years to get data with him! Once you start, you know that you will be outdated at half of your journey.

Posted in labels: GFP protein, in vivo, molecular imaging, researchblogging 0 referee

Darwin and Mendel mainly worked with mere observation, and now it is possible to do it at molecular level. To support RG, you can contribute diffusing the concept that molecular biology can be studied in living, healthy life species (i.e., fluorescent dogs). Please, candidate Reportergene at Google's Blog of Note until May the 14th. It is just a matter of one click. Click it!

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Reportergene results 14th in the top Researchblogging contributors with 60 posted articles and >12500 views. Interestingly I'm aware of only 54 posts (as you can see on my left sidebar). ResearchBlogging, scroll me down! I'm not so productive :-)

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Traditionally, responsive promoter sequences on DNA have been considered only passive docking sites for a pletora of DNA-binding proteins supposed to play the active hard role of gene expression. Several proteins have been pulled-down according to their ability to bind DNA sequences (i.e., far western blotting) and lot of plasmids were generated carrying any responsive DNA element upstream of a reporter gene to mainly study the activity of such proteins (i.e., transcription factors) and eventually discover new drugs. In other words, reporter assay data have been mainly queried to address gene expression from the protein stand-point (trans-action).

It is worth to note, that protein activity is longly known to be allosterically regulated by the binding of ligands or cofactors outside the protein’s active site. Now, Sebastian Meijsing and colleagues from the Yamamoto Lab, are shifting the balance toward cis-acting factors (the DNA itself). They propose in a Science report that DNA is a sequence-specific allosteric ligand for the nuclear receptor GR (glucocorticoid receptor). GR may be considered a ligand-activated transcription factor (i.e., it is activated by cortisol or dexametasone). The Yamamoto group exploited a classic luciferase assay to test the activity resulting from the interaction between GR (protein) on its GREs (DNA) during dexametasone stimulation.

Molecular gymnast by:Elio Abbondanzieri

Interestingly, odd differences were found in their elegant 2x2 experimental scheme in which either single-poing mutations on the receptor or on the responsive elements were combinatorially tested to finally postulate that DNA topology actively directs transcription similarly to an allosteric regulator. Gene expression , crystallographic, gel-shift and ChIP assays corroborate this intuition. Pay attention to your plasmids: DNA is more than a passive docking site.

Posted in labels: basics, luciferase, researchblogging 0 referee

In 1997, the IBM computer Deep Blue wins a chess-game vs Garry Kasparov. This is considered a milestone in Artificial Intelligence research. Now, a second milestone dates April the 3rd, 2009 with Science publishing two reports on automating science. In the first one, Schmidt and Lipson (Cornell) propose a computational approach for detecting physical laws from experimentally collected data. As a principle for the identification on non-triviality, they first numerically calculate partial derivatives between variables from the data, then they generate candidate symbolic functions by randomly combining (and iteratively re-combining) mathematical building blocks. They finally compare the derivative expressions with the derivate data and score the best pairs according to parsimony criteria.

Given the dimensionality and the complexity of current “omics” data, the computation time required to detect solutions is probably near to 1000-10,000 hours, however the algorithm’s search seems highly parallelizable and very appealing for distributed approaches. What is more astonishing, is the following step. In a second report King and colleagues (Aberystwyth University) extend the concept of “artificial scientist” by generating ADAM:

this is a physically implemented laboratory automation system that […] executes cycles of scientific experimentation. (ADAM) automatically originates hypotheses to explain observations, devises experiments to test these hypotheses, physically runs the experiments by using laboratory robotics, interprets the results and then repeats the cycle.

As a proof of concept, they applied Adam to the identification of genes encoding orphan enzymes in the yeast.

Despite the abundance in data, theoretical gaps still exist in systems biology and integrative physiology, automatic science can potentially increase the rate of scientific progress. At the end of this provocative paper, the authors wonder:

Might this process diminish the role of future scientists?

Quite the opposite: does chess-software diminished the number of chess-players?

Posted in labels: basics, researchblogging 5 referee

A Korean team report the generation of  a RFP-transgenic beagle. Dogs exhibits 224 genetic diseases similar to those found in humans making them one of the closest known models for various human hereditary diseases. However, experimentation with animal -which should be at the service of the whole mankind -  raises strong and acute ethical challenges, particularly if the experimental model is a pet. Although still prototypical, the concept of "reporter animal" arguments toward a new use of animal experimentation based on the generation of a knowledge based on the non-invasive observation of physiological events in living animals at molecular detail. This vision is still in its infancy and several ameliorements steps need to be undertaken. One of them, is the development of better transgenic abilities to safely introduce a genetically-encoded reporter into mammals. Due to the technical difficulty in obtaining fertilizable eggs and the unavailability of embryonic stem cells, no transgenic dog has been generated so far. Hong et al., report now the use of Somatic Cell Nuclear Transfer (SCNT) to generate, from a stably-transfected fibroblast, a dog carrying the red-fluorescent protein. This will be probably a debated proof of concept.

Hong, S., Kim, M., Jang, G., Oh, H., Park, J., Kang, J., Koo, O., Kim, T., Kwon, M., Koo, B., Ra, J., Kim, D., Ko, C., & Lee, B. (2009). Generation of red fluorescent protein transgenic dogs genesis DOI: 10.1002/dvg.20504

Posted in labels: GFP protein, in vivo 0 referee

Pharmacokinetics and pharmacodynamics should include in future opto-kinetic and opto-dynamic disciplines. At Stanford, Raag Airan and colleagues developed opsin-receptor chimaeras (the optoXR family) as a new class of retinal-based tools. In a Nature letter, they show that the class of OptoXRs can be functionally expressed in vivo, to permit differential photoactivable control of intracellular cascades with significant impact on the phenotype (i.e., behavior when light was targeted in brain via 200um optical fibers). A green light stimulation (~500 nm, 7 mW/mm2) was sufficient to increase intracellular concentrations of (cAMP, IP3, Ca2+) at levels comparable to those induced by pharmacological stimulation. The use of light, instead of drugs, to modulate network physiology, would be of substantial interest given the versatility of optics to precisely define relevant stimulation times with great control of pulsatile versus tonic modulation. This is also of major concern for synthethic biology, given the wide portfolio of photo-proteins and appropriate light-responsive elements.

Airan, R., Thompson, K., Fenno, L., Bernstein, H., & Deisseroth, K. (2009). Temporally precise in vivo control of intracellular signalling Nature DOI: 10.1038/nature07926

Posted in labels: in vivo, researchblogging 0 referee