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

The rainbow and the brain

Recently, we met two very bright rodent brains: one is the Ratatouille mouse by Pixar, the other one is Brainbow (Rainbow Mouse Brain) by Jean Livet and colleagues from Harvard University. In November 2007, Nature published spectacular, colourful pictures of neurons and their axon and dendrites. In the brainbow mouse, each neuron can randomly dress 90 different colors. Each color is generated by the combination of 3-4 GFP variants following the same mechanism that your iPad use to obtain a wide color space just by mixing three primary channels (red, green and blue), but in the rodent, the pixels are actually neurons and the primary channels are fluorescent proteins.
How to make a Brainbow. Basically, a transgenic mouse expressing Cre recombinase, is mated with another transgenic mouse in which a CMV/thy1 promoter expresses an array of 3 or 4 GFP color 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). The random recombination event generates a panel of neurons that massively express one, or two, or three fluorescent proteins of different colors. Finally, the colored light of each GFP protein merges generating roughly 90 different hues, which can be observed by fluorescence imaging.

Which progress would bring that mouse to neuroscience? Why one scientist should use this mouse to track single neurons in a brain section when a black stain, the Golgi-Cajal stain, is already in use since 1873? The answer can be found in large-scale systems biology: with the Brainbow colors it is possible to follow each neuron (and possibly each connection that thousands of neurites belonging to a single neuron can develop). By doing so, we could try to obtain "connectomic maps" of the brain. In addition, since the color of the neuron remains the same, why do not better explore brain development with some highly-rendered lineage analysis? What about probing individual regenerative events after spinal cord injuries? The spectra of investigational opportunities seems quite large considered that we are just dealing with an old reporter drived by a common CMV promoter. And, in case I'm wrong, the Brainbow mouse remains a memorial to the best GFP imaging ever.

---/ Source. The Brainbow mouse has been first described in: 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

Follow up updates

A Brainbow with wrist-watch. In Hungary, time-shifted GFP colors have beed added to a sort of Brainbow-like mice. 4D connectomics in space and time is now possible. -->
Not only mice: Flybow fruit-flies (D. melanogaster) are out now, read the news at Brainwindows. Some other beautiful Brainbow pictures freely available in a Cell Picture Show.
Multicolor Zebrafish. Lineage analysis with Brainbow Zebrafish reveals stem-cell clonal selection possibilities for organ development.
Brainbow mice are used to prototype 3-photon microscopy to image deeper in the brain (see labrigger notice)

Half reporter gene is better than one

ResearchBlogging.orgIn 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