Where do you express your transgene?

Transgenic mice are an effective model to study (not only) gene function in vivo. Whenever you will be so lucky/unlucky to get a gene to study in vivo, a big double-question remain: where and where are you going to express such transgene? The first "where" addresses the position on genome in which you wish to place the transgene expression cassette, the second "where" entails cells/tissues/organs that you want (or not) to be the expression places. Due to biological complexity, often it is impossible to distinguish between the two side of the same question, and it is a fact that conventional transgenesis leaves to the chance the "2 where" question. Because of that, conventional transgenesis results in random integration of transgenes into the mouse genome, and basically this means NO CONTROL. Several attempts have been made to gain a more efficient transgenesis: concerning the genome position some authors choose to surround the expression cassette by insulator sequences (i.e. MAR ), that buffer transgene from (often tissue-specific) enhancer/silencer effects, and from (often tissue-specific) chromatin silencing; other authors tried to find a good place (locus) in the genome in which perform gene-targeting (i.e. Rosa26).
Recently, the lab of Bernd Kinzel (Novartis), published a technology report in Genesis (vol.45), in which the locus of beta-actin was identified as a good dock for gene expression. Beta-actin is a cytoskeletal building-block expressed in almost every mammalian cell, and it is necessary for life, so only heterozygous transgenic can be developed. To better approach the tissue question, they further engineered the locus by placing a floxed-STOP cassette between the beta-actin promoter and the reporter gene (EGFP). What they observed is that transgene expression was efficiently repressed by STOP, but become activated after Cre-mediated excision of the floxed STOP cassette. Obviously several Cre-mice are available in order to drive the spatio-temporal expression of Cre recombinase. In conclusion, reporter genes can be adopted to make new models to facilitate predictable transgene expression in a spatially and temporally controlled manner.

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Jägle, U., Gasser, J., Müller, M., & Kinzel, B. (2007). Conditional transgene expression mediated by the mouse β-actin locus genesis, 45 (11), 659-666 DOI: 10.1002/dvg.20342

Technicolor fluorescence nanoscopy

As seen for the brainbow mouse, microscopic imaging of molecules that have different hues, reveals their spatial correlation. But diffraction hampers standard microscopy and implies that biomolecules cannot be located more precisely than within ~200 nm. Different approaches (as reviewed last month) tried to broke the Abbé limit, and another one (Resonance Energy Transfer) leads to explore tight proximities (~5 nm). Bates et al. claims now on Science, the possibility to probe 25 nm fluorescence microscopy (nanoscopy!) in a modular system that can accomodate also nine color channels. Such thunderclap introduces STORM (STOchastic Reconstruction Microscopy) that is based on activation and deactivation of isolated molecules. Each probe consists of a photo-switchable "reporter" fluorophore that can be cycled between fluorescent and dark states, and an "activator" that facilitates photo-activation of the reporter. This on/off cycle produce tiny focal spots so sparsely that they coordinates can be easily tracked by the camera, and the cycle is repeated until the tick-marcks form an image. Using this approach, authors showed multicolor imaging of DNA model samples and mammalian cells with 20- to 30-nanometer resolution. This technique will facilitate direct visualization of molecular interactions at the nanometer scale.

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Bates, M., Huang, B., Dempsey, G., & Zhuang, X. (2007). Multicolor Super-Resolution Imaging with Photo-Switchable Fluorescent Probes Science, 317 (5845), 1749-1753 DOI: 10.1126/science.1146598

Renilla Reniformis luciferase structure

The 3D structure of Renilla reniformis luciferase (RLuc) is solved. The tertiary structure was succesfully determined from high resolution (1.4 A) crystallographic analysis as illustrated in the volume 378 of the Journal of Molecular Biology by the group of Gambhir at the Stanford Molecular Imaging Program. Since the cloning of the gene for RLuc in 1991, this kind of luciferase from Sea pansy has been widely used in molecular biology, mainly to normalize reporter gene assays. Indeed, the widest application consists in the dual luciferase assay (DLA) in which the Renilla (driven by a basal promoter) normalizes the output of Firefly luciferase (tested promoter). Recently, work emerged also utilizing Rluc for in vivo imaging, to create novel imaging probes by fusing the luciferase to engineered antibodies, and to generate self-illuminating quantum dots. In this blog, protein interaction studies using Renilla were previously reviewed.

Now, structural data freely available in the protein data bank site (PDB) can help scientists to find more effective way to employ the luciferase (i.e., screening potential steric indrances prior to the creation of fusion protein constructs). Moreover, the crystallographic model (a classic alpha/beta hydrolase fold) confirms many amino-acid residues that are important for spectral and luminescence properties. In the same paper, the structure of the Renilla GFP (RrGFP), that together with the luciferase is responsible for the green light in the Sea pansy (via resonance energy transfer), has been also described.

The rainbow and the brain

Mankind recently have known two rodent brains really very bright: one belongs to Ratatouille mouse by Pixar, the other one belongs to Jean Livet and colleagues from Harvard University and was named Brainbow (Rainbow Mouse Brain). Spectacular, colourful pictures of neurons and their axon and dendrites impacted the November 1 issue of Nature. In the brainbow mouse, each neuron can randomly dress one of the 90 colors generated by the combination of 3-4 GFP variants (XFP) can originate from stochastic recombination of Cre recombinase, mimicking the same mechanism that allows a TFT monitor to encode a wide colour space just by mixing three primary channels (red, green and blue), but in rodent case the pixel are neurons and the primary channels are fluorescent proteins!

Basically, a transgenic expressing Cre recombinase (induced by a ligand like tamoxifene), is mated with

a transgenic in which a CMV/thy1 promoter express an array of 3 or 4 GFP reporters variants placed between

different sets of incompatible lox sites. In each neuron (Thy1 promoter) of the progeny, Cre recombinase is

forced to choose between two mutually exclusive excision events (lox).

As a consequence of the random recombination event, a panel of neuron that massively express one,

or two, or three different fluorescent proteins is generated.

Finally, the signal of each GFP protein merges generating many different hues (at least 89).

Now, the question is: which progress would bring that mouse to science? Why one scientist could be interested in tracking single neurons just in an anatomical context? The answer is in large-scale system biology: tracking each neuron (and possibly each connections that thousands of neurites belonging to a single neuron can develop), we could describe the "connectomic maps" of the brain (actually not completely rendered). But also lineage analysis could take advantage of such system, and why don't better explore brain development? What about probing individual regenerative events in spinal cord injury? The field of opportunities seems to me quite large considered that we are just dealing with an old reporter drived by a common CMV promoter. And, in case I'm wrong, rainbow mouse remains a memorial to the best GFP imaging ever. Listen the dedicated Nature Neuroscience Podcast here.

Update 2011. Brainbow fruit-flies (D. melanogaster) have been generated, read the news at Brainwindows.

2nd Update 2011. Other beautiful brainbow pictures freely available in a Cell Picture Show.

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Livet, J., Weissman, T., Kang, H., Draft, R., Lu, J., Bennis, R., Sanes, J., & Lichtman, J. (2007). Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system Nature, 450 (7166), 56-62 DOI: 10.1038/nature06293

Half reporter gene is better than one

In the previous post, I introduced you Resonance Energy Transfer techniques to study protein-protein interaction (briefly, RET technology exploit two reporter genes coupled to two cognate proteins in exam; once proteins interact, one reporter transfer its energy to the second one that start glowing, "reporting" the interaction otherwise invisible). One can wonder «why using two reporters instead of cutting in half a single one?», this is exactly what argued E. Stefan and S. Michnick at Montreal University during their research on the G protein-coupled receptors (GPCRs), a protein superfamily very probed in the pharmacological field (currently targeted by >30% of approved drugs). In a recent volume of PNAS, the authors report a protein-fragment complementation assay (PCA), that is based only on the reporter enzyme Renilla reniformis luciferase (Rluc).

Binding of the two proteins of interest brings the unfolded fragments [of luciferase] into proximity, allowing for folding and reconstitution of measurable activity of the reporter protein.

Although PCAs assay are not so new (they were used also for pionieristic studies on operon lacZ), the innovation of Stefan's work consists in having designed that PCAs to be reversible: it means that not only the assay mirrors protein interactions, but once this interaction gets off, the two half-reporter split once again stopping to report! Accounting for dissociation-association kinetics these assay could be useful for reporting drug-induced dissociations (drived by antagonists in some models or agonists in others). To be honest, reversible characteristic is shared also by RET techniques, but in contrast to RET...

Rluc-PCA is a [direct] readout for absolute values of protein complexes.

So, advantages of the tecnique are clear: there are some limitations? The assay is blind versus inverse agonism: by definition, inverse agonists stabilize the receptor in its inactive conformation (it means that signal will persist although the dimer is not active). To don't forget, all the time you fuse a protein with a second foreign protein, you should be sure that this "tag" doesn't affect significantly both expression and trafficking of the protein in the physiological cellular context. Saved these points, the choice of bioluminescence (high signal-to-backround ratio) and the viability of the assay to be probed both by microscopic bioluminescence imaging (not so trivial) and by high-throughput plate-luminometers, suggests that Rluc-PCA sensor meets several requirements to study cell biological aspects of signal trasmission in living cells.

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Stefan, E., Aquin, S., Berger, N., Landry, C., Nyfeler, B., Bouvier, M., & Michnick, S. (2007). Quantification of dynamic protein complexes using Renilla luciferase fragment complementation applied to protein kinase A activities in vivo Proceedings of the National Academy of Sciences, 104 (43), 16916-16921 DOI: 10.1073/pnas.0704257104

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