Mastering transgene expression

Spatiotemporal control of transgene expression is of paramount importance in animal engineering: the exact activation/inactivation of your favorite transgene is a powerful tool to trace the hidden rules of integrative biology, and also to strive gene therapy. In mice, gene targeting can be restricted in a tissue and/or temporal manner through generation of 'premutant' mice (e.g., "floxed" or flanked with loxP sites) which are then breed with transgenic mates that opportunely express the corresponding DNA recombinase (e.g., Cre) allowing the desired conditional gene deletion/expression with respect to space and/or time. The opportune design of the loxP flanking sites may be considered an art, as exemplified by the stochastic combinatorial approach by Livet and Lichtman, however in most instances, is the choice of the Cre expression system to be the fundamental step.

Hundreds of mice have been generated with Cre expressed under control of tissue or cell specific promoters, allowing space/anatomical control of the recombination. And other mice were generated to check the correct recombination. Conversely, relatively few systems allow temporal control. Current solutions include the "activation" of Cre by a ligand given opportunely at selected time. Particularly, I read about:

1. The exploitation of a tetracycline-responsive promoter (St Onge, 1996);
2. The use a fusion protein to obtain modulation of Cre activity by steroids like ecdysone (No et al., 1996) or tamoxifen (Danielian et al., 1998);
3. A dimerizable approach, in which two Cre mojeties complement upon binding to a rapamycin molecule (Jullien et al., 2007).

Even if each of these systems compete for low basal activity and high inducibility, unfortunately none of such ligands affects only Cre activity: tetracycline, steroids and the mTOR-inhibitor rapamycin may be considered somewhat like as potential "metabolic/endocrine distruptors". Hence, particularly in animals, the treatment with the ligand may introduce a bias in subsequent endpoint measurements.

Recently, I read about noninvasive in vivo local heating by means of high-intensity focused ultrasound (HIFU) . Deckers and colleagues, implemented such technique in combination with a heat-inducible promoter [heat shock protein 70 (HSP70)] driving luciferase expression. They monitored local hypertermia with MRI thermometry and evaluated gene induction by bioluminescence imaging. Nice. Really cool. Now I'm wondering how can I convince my boss to buy one HIFU, one MRI and one more BLI workstation instead of just another syringe to inject tetracycline?!


R. Deckers, B. Quesson, J. Arsaut, S. Eimer, F. Couillaud, C. T. W. Moonen (2009). Image-guided, noninvasive, spatiotemporal control of gene expression Proceedings of the National Academy of Sciences, 106 (4), 1175-1180 DOI: 10.1073/pnas.0806936106

fluorescent cognac

I'm so lucky to be paid to do funny things, like playing with stuff like DNA or other glowing things or just sitting in a chair and sketch in my lab-notebook some graphs concerning the behaviour of signals over time. Unfortunately, there is a tax to pay for living such a lifestyle: you will suffer from a persistent susceptibility of getting ideas about your work, every moment of your journey. Sam, like me, is an "every day scientist", and can not taste the mother of brandy, without speculating about fluorescence. What a life!

Unfortunately, cognac fluoresces in the blue-green, so forget about using cognac as a tracer for in vivo experiments involving humans.

A Brainbow with wrist-watch

A series of trans-synaptic pseudorabies viruses (PRVs) encoding fluorescent sensors and time-shifted fluorescent proteins like memCherry, memGFP and memCerulean, were recently proposed to trace several circuits in parallel in order to gain a dissection of the complex architecture of brain regions. The work by Zsolt Boldogkoi and colleagues from Szeged University (Hungary) has been published on the February issue of Nature Methods.



Zsolt Boldogkői, Kamill Balint, Gautam B Awatramani, David Balya, Volker Busskamp, Tim James Viney, Pamela S Lagali, Jens Duebel, Emese Pásti, Dóra Tombácz, Judit S Tóth, Irma F Takács, Brigitte Gross Scherf, Botond Roska (2009). Genetically timed, activity-sensor and rainbow transsynaptic viral tools Nature Methods, 6 (2), 127-130 DOI: 10.1038/NMETH.1292

Other posts about genetically encoded time sensors, here and here.

the italian way

A cartoon by Philippe Tastet illustrates the job we do in Milan with reporter mice. The whole strip collection, including several labs, is available at Crescendo website, a consortium of 22 laboratories that works to reach the goal of using genomic and post-genomic approaches to study processes in development and aging that are mediated by nuclear receptors such as steroid or orphan receptors.
Transgenic Bio Luminescence Imaging

a reporter mice for calcium imaging [guest-post]

Calcium imaging is a technique that is definitely coming to age, and fancier and fancier genetically encoded indicators are constantly being developed.

Different approaches have been taken for in-vivo imaging, some of which rely on the use of an optical fiber, or “fibroscope”, which is then attached to a (generally two photon-) microscope. This is clearly an invasive method and limits the observation to the limited zone of action of the fiber. Another less invasive approach is to use long working-distance objectives to image an exposed region of the body: this has been succesfully used, for instance, to look at dendritic spine plasticity in the neonatal brain over several days through a transparent window in the skull (Gray et al., PlOS Biology 2006). However, this also involves very delicate surgery and allows imaging over a limited area.

The approach recently described in PlOS One by Rogers and colleagues, instead, is completely non invasive. They generated a transgenic mouse line expressing the calcium sensitive protein GFP-aequorin in an inducible manner (through the use of a floxed-stop approach).
The reporter can therefore be targeted to the cell type of interest just by crossing this mouse with a line expressing Cre in that cell type. In the presence of Ca2+, the aequorin part of the probe can oxidize a substrate called coelenterazine, producing luminescence, that in turn excites the GFP, making it fluoresce. The idea is then to inject coelenterazine and look at the GFP-generated fluorescence. This method allows whole-animal imaging, although this obviously comes at the price of a lower spatio-temporal resolution (which is still pretty good in my opinion).

And indeed, it seems to work quite well! When GFP-aequorin was targeted to the mitochondria and expressed in muscles, it allowed imaging of mitochondrial calcium during muscular contraction. This was tested after an “artificial” contraction following tetanic stimulation, in physiological conditions, such as the spontaneous muscular activity in newborn mice, and during an induced “pathological” condition, kainate-derived seizures. To see the system in action I strongly advice to have a look at the cool videos attached to this open-source paper.

In summary, this approach allows a non-invasive and quantifiable whole animal imaging of physiological and pathological states. It may also be a very interesting indicator for measurement of the metabolic state of a certain tissue, apoptosis and many other processes that depend on calcium. Of course the next step to this would be to have such a system working in freely-moving animals. Are we really so far away from this? I don't think so, expecially as the same group already published an article about it (Roncali et al., J. Biomed. Opt. 2008)!

Kelly L. Rogers, Sandrine Picaud, Emilie Roncali, Raphaël Boisgard, Cesare Colasante, Jacques Stinnakre, Bertrand Tavitian, Philippe Brûlet (2007). Non-Invasive In Vivo Imaging of Calcium Signaling in Mice PLoS ONE, 2 (10) DOI: 10.1371/journal.pone.0000974

Nicola Romanò is a neuroendocrinologist, interested in the analysis of the electrical/calcium activity of hypothalamic neurons governing pituitary hormonal secretion. He presently studies the neuroendocrine control of the prolactin axis. He also writes on the Inside Neuroscience blog (in Italian) on MolecularLab.it. As an "imaging person" he loves photography. His photo portfolio (not science-related) can be found at www.nicolaromano.net

Bioluminescence: from protein structure to biosensing applications

For those interested, the second issue of Photochemical and Photobiological Sciences contains a collection of papers on the theme of bioluminescence guest edited by Vadim Viviani. Several topics are addressed in this issue, including structure and function relationships of luciferases and other accessory proteins of different bioluminescent systems, mechanisms of bioluminescence of Cypridina and firefly luciferases, and biotechnological applications of multicolor luciferases. The front cover shows a larva from the Phrixothrix hiatus, a nice worm expressing two different coloured luciferases. The red-one is particularly appealing for reasons I previously posted.

fluorescent timers: a new biophotonic tool

In standard reporter assays the basal activity of the cloned promoter often results in accumulation of both luciferase mRNA and protein. This “background” activity may be an advantage since, once measured, gives you some numbers that you may use to calculate a fold induction (you can't divide by 0!). However, the slow clearance rate of these pre-existing molecules substantially may delay and dilute the measurable response, hampering the accurate quantification of changes in cell signaling pathways. Hence, in standard assays, transient or relatively minor effects may be hidden and kinetics somewhat inaccurate: this explains why researchers prefer reporters with short half-life, and eventually decrease it with both mRNA and protein destabilizing elements (see for instance the RapidReporter Gaussia luciferase from Activemotif).

Today, another significant quality step in the race toward the 4th dimension (time), have been conquested: new fluorescent 'timers' (FTs) that gradually change colour from blue to red could allow researchers to track the age and dynamic behaviour of proteins in living cells. Previous work suggested that some red fluorescent proteins start out fluorescing blue, but then change to red as the protein is chemically modified over time. Vladislav Verkhusha and his colleagues at the Albert Einstein College of Medicine in New York mutated the red fluorescent mCherry, toward altered maturation rates from blue to red, getting three fluorescent proteins, each with a specific maturation rate. The proteins were used to track newly synthesized proteins in mammalian cells grown in culture, but maybe in future we will have a "flight recorder" inside a cell, to log different molecular events with respect to time.

Fedor V Subach, Oksana M Subach, Illia S Gundorov, Kateryna S Morozova, Kiryl D Piatkevich, Ana Maria Cuervo, Vladislav V Verkhusha (2009). Monomeric fluorescent timers that change color from blue to red report on cellular trafficking Nature Chemical Biology, 5 (2), 118-126 DOI: 10.1038/nchembio.138

small-animal imaging for dummies

Thanks to my complimentary subscription* to the print magazine Biophotonics International, I read with interest the article by Richard Gaughan intitled “Small Animals, Big Promise”, which gives an introductory showcase to some imaging modalities in living mice. There was a box in which experts in the field gives some hints to researchers committed/concerned to incorporate small-imaging workstations into their research protocols, and I agree that:

new users often don't appreciate that in vivo imaging is generally on the macroscopic scale, while they are used to think in terms of what they can look at with a confocal microscope.

In my opinion, given the complementary nature of such different approaches, the compelling advantage to allow longitudinal studies on thath macroscopic “blobby” resolution of the anatomical projections of different organs, may help to define if (and eventually when) proceed to a tissue biopsy to get more on the microscopic scale. This will also help to minimize animal use. Accordingly to this vision, I've recently seen on my free* print-copy of Nature Methods, an advertisement of new solutions for bioluminescence microscopy. To date, not only GFP-, but also luciferase-harboring mice may be imaged at each resolution.

* you can qualify for free-subscriptions to Biophotonics International and Nature Methods, here.