Noninvasive vascular spectral unmixing

Accurate and noninvasive imaging of tumor vascularization in live animals has been done so far with fluorescently labeled vascular probes. Tumor blood vessels, however, often have poor integrity, resulting in the leakage of the vascular probes into the tumor. If the probe half-life is long and there is poor clearance of the probe from the tumor, then the fluorescent signal in the tumor would no longer be restricted to tumor blood vessels. P. Mayes and colleagues in the laboratory of W. El-Deiry at the University of Pennsylvania School of Medicine (Philadelphia, PA) have abandoned vascular probes through the multispectral imaging of reporter genes in fluorescent tumors.

Blood vessels by jedielfqueen

By multispectral imaging they examined a tumor expressing red fluorescent protein (dsRed from Clontech), a clear 10-nm upward shift in wavelength was associated with the spectral emission signature of tumor blood vessels relative to the fluorescent tumor tissue. This spectral unmixing technique could image 10-µm diameter tumor capillaries. In a small pilot study of antiangiogenic therapy on the fluorescent tumors, spectral unmixing detected a decrease in tumor vascularization over one week, which was verified by immunohistochemistry. It is worth to note that such event was not observed with a vascular probe, thereby demonstrating the superiority of spectral unmixing and of genetic markers in general when coupled to noninvasive imaging.

Patrick Mayes, David Dicker, Yingqiu Liu, Wafik El-Deiry (2008). Noninvasive vascular imaging in fluorescent tumors using multispectral unmixing BioTechniques, 45 (4), 459-464 DOI: 10.2144/000112946

related post: imaging of extracellular ATP in cancer microenvironment

just another protein-interaction model

Andrea Pichler and colleagues from Mallinkrodt Institute of Radiology describe the generation of a transgenic Gal4-luc reporter mouse (G4F) that expresses the Firefly luciferase (from pGL3, Promega) under the regulatory control of a concatenated (5x) Gal4 promoter. The Gal4-luc strain, would allow noninvasive bioluminescence imaging of protein-protein interaction in vivo, a feature of real interest since traditional biochemical techniques often miss the detection of weak and transient interactions and, more often, return false positives. So, how does it work?

Protein interaction studies with Gal4-luc reporter mouse

Actually I'm quite frightened about this model: let I want to know whether protein X (bait) and Y (from library) interact in vivo. In order to detect XY interaction with the G4F mouse, I need to tag X with Gal4BD (get a X-Gal4BD expression vector) and then tag Y with VP16 (get a Y-VP16 expression vector). Then I have to make two more transgenics (and deal about positional effects) and, once I get them, backcrossing them in the G4F background and, like a juggler, breed and genotype three markers together. Three years are enough? Of course, an alternative would be to make transfections in vivo (i.e. with hydrodynamic somatic gene transfer or viral delivery) but even this job will need some validation (i.e. not seeing an interaction means that the interaction did not appear or it’s a false negative due to problems in vector delivery. In conclusion protein-protein interaction is so hot and so hard matter, and my feelings are similar to those expressed for the Tango Assay:

There is a trend to interpret complex systems with complex conceptual design in today's molecular pharmacology. For sure this is geeky, funny, and sexy for mind, so it is worth being mentioned in Reportergene. Nevertheless I don't know whether this would be useful also to understand our inner biology. Managing three transgenes (also transiently) is not a joke. Using strong promoter to express receptors and cognate ones is unphysiological and very, very naive.

A. Pichler, J. L. Prior, G. D. Luker, D. Piwnica-Worms (2008). Generation of a highly inducible Gal4-Fluc universal reporter mouse for in vivo bioluminescence imaging Proceedings of the National Academy of Sciences, 105 (41), 15932-15937 DOI: 10.1073/pnas.0801075105

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Automated transgenic sorters?

Fluorescence activated cell sorters (FACs) are well-established machines able to separate different cell populations on the basis of their inner fluorescence. Although technical challenging, new strategies strive to adopt FACs architecture to the automated isolation of fluorescent organisms, like worms (recently appeared on Nature Methods) and Xenopus tadpoles (recently patented by WatchFrog). To date, with mice we have still to run boring PCR or setup tedious Southern blots to get out our offspring genotype. Maybe in future we will have an automated mice sorter, to date I found only an automated rodent trap patented in 1920.

disillusion #2 - presenting a poster in Italy

I've recently been to a conference supposed to network endocrine disruptor (ED) research laboratories in Italy. I got a poster aimed at explaining the opportunities to use in vivo reporter animals to better study EDCs. I experienced odd abstract submission that was initially refused by the scientific secretary.

We cannot accept your abstract in the present form.

The problem was that I submitted an abstract written in English language.

Please send us your contribution in Italian language.

I wonder how can be possible to effectively communicate scientific ideas in other languages other than English. Moreover I discovered one speaker getting accepted one abstract (Italian) about politics instead of science. He claimed that “it make not sense to speak about science when science is compromised by law”.

I recognize that scientists need to deal with political management of public money to get funded research. Anyway I hardly accepted that an english scientific abstract was refused and an italian political abstract was accepted in a so-called SCIENTIFIC meeting. But I'm a naive man.

I usually share the bench with foreigner students (from Chile, India and Poland). Do you guess they were surprised when they saw me getting back to reduce refine and replace the abstract and the poster? Actually not. They work every day with italian computers because the english license is not available in our University (ok, that's another disillusion story).

Disclaimer: even if I got again my yearly disillusion, I'm a satisfied italian scientist, the meeting was organized by the great “National Institute of Heath” of Italy and was finally helpful to network collaborations and expertise. Finally here it is a review I've been recently involved, that try to address why reporter animals may be important for environmental risk assessment as an alternative toxicology approach (written in english this time).

stem cell imaging research

If you previously read and appreciated imaging of stem cells #1 and Luciferase and stem cells #2, and you started making your own experiment to address the regeneration conquest by such boring so-called stem cells, maybe you will interested to contribute to this symposium

Stem cells research: the role of imaging techniques

on Decembre 8 and 9 at the Timone hospital in Marseilles.

Registrations are open and more details are available at the following url:http://indico.cern.ch/conferenceDisplay.py?confId=42807 where you will find a provisional program of the workshop.

GFP nobelized

Two gold medals for reportergenomics! Last year was the gene targeting in mouse (knock-in, knock-out). This year is the GFP reporter protein. By combining animal engineering with molecular imaging techniques it has become possible to conduct dynamic studies on specific molecular processes in living animals. Synergism is more than two Nobels. Great!

Luciferase and stem cells #2

As previously posted, bioluminescence imaging (BLI) can be helpful to longitudinally monitor stem cells localization, proliferation, and viability. To date, Alessandra Sacco and colleagues from Stanford, illustrate in Nature that reporter genes can be useful also to longitudinally monitor self-renewal and differentiation of luciferase-expressing muscle stem cells (MuSCs) after transplantation in mice. This results has been realized by mating Myf5-nLacZ mice (a reporter mouse for myogenic transcription) with a constitutive firefly luciferase mice (a reporter mouse for assessing cell number).

Bioluminescence by jon.nelson

Of course, that is a technological advance, since it seems now possible to follow the dynamics of stem cell behavior in a manner not possible with retrospective histological analysis. We need now to develop new cognitive skills (and a little bit of statistic also) to understand this unprecedented "real time" picture, since it is quite common for generation 1.0 scientists to wonder only about "end-point" results, without considering the bulk of information revealed by longitudinally imaging in the same experimental subject.

Alessandra Sacco, Regis Doyonnas, Peggy Kraft, Stefan Vitorovic, Helen M. Blau (2008). Self-renewal and expansion of single transplanted muscle stem cells Nature DOI: 10.1038/nature07384