Conditional transgenesis reporter mice

The technology of gene inactivation (knockout, KO) has boosted our understanding of the role of a number of genes in vivo. However, following gene deletion, the interpretation of the results may be complicated by the existence of compensatory adaptation at different network levels (i.e., nuclear gene networks, organ-to-organ endocrine network, central nervous synaptic networks and so on). A classic approach to limit some of those problems is to choose a conditional KO strategy in desired tissues or at desired time-points, in order to observe the phenotypic pictures before development of functional adaptations in response to gene inactivation.

The site-specific Cre-loxP system is widely used for various methods of genome engineering, and lot of transgenic lines express Cre recombinase in a tissue-specific manner. Cre-mediated recombination is frequently monitored using reporter lines in which Cre expression activates a reporter gene driven by a ubiquitous promoter. Dated 1999, the most known test line is the Soriano's R26R (Rosa26 lacZ) in which beta-galactosidase is expressed following Cre recombination.

A green central nervous system marked by GFP after
Nestin-Cre-recombinase action on IRG developing mouse

Recently, a new reporter line has been described: the IRG mouse. Compared to R26 reporter, IRG adopt insulators instead of locus strategy (see the previous post where do you express your transgene?). In this line, a red fluorescent protein (RFP) is expressed ubiquitously prior to Cre-mediated recombination and shifted to an enhanced green fluorescent protein (EGFP) following recombination. For instance in the picture, you can see Cre-mediated recombination in CNS (nestin positive cells) of an embryo at day 13.5 of development. Given the distinct advantages of fluorescent reporters over the lacZ staining (no need for exogenous substrates, fluorescence can be visualized in tissues without fixation artifacts and cells can be isolated using FACS sorting), the IRG mouse will bring soon new adepts to the emerging field of optical imaging.

----/ original citations /--- --- ---

• Soriano, P. (1999). Generalized lacZ expression with the ROSA26 Cre reporter strain. Nature Genetics, 21(1), 70-71. DOI: 10.1038/5007
• De Gasperi, R.,[...] Elder, G.A. (2008). The IRG mouse: A two-color fluorescent reporter for assessing Cre-mediated recombination and imaging complex cellular relationships in situ. Genesis, 46, 308-317. DOI: 10.1002/dvg.20400

No more cell lines please

With recent concerns on the effects of lipophilic chemicals or hormones on human health, the establishment of various nuclear receptor-based evaluation methods to assess endocrine disrupting potential is required. Among these methods, mammalian cell reporter gene assays have shown high popularity as lipophilic chemicals are sensed, evaluated, and analyzed directly through a reporter gene within a whole cell. You can find hundreds of reviews like that:

Recent work from the XXX group has established a novel stable reporter cell line via site-specific genomic recombination by integrating a xxxx NR gene, its xxx response element and a luciferase reporter gene into the genome of a host HeLa cell line. The reporter cell established showed high sensitivity, selectivity, stability, and specificity against receptor specific lipophilic chemicals.

Unfortunately, it has been shown that each mammalian cell responds to NR activation in a different fashion, the reasons of such variability are due to different tissue expression of coregolators, adaptor molecules that couple NR to activating or repressive transcriptional machinery. The need is not for new cancer cell lines like HeLa, MCF7 or what else, in particular when engineered to express doubtful physiologic levels of receptors.

The need is for systems able to discriminate in vivo (and possibly in each tissue) activities of multiple nuclear receptors (or even multiple transcription factors) at physiological levels (and possibly without invasiveness).

molecular imaging in drug discovery

Currently up to 10 companies (including biospace lab, caliperLS, kodak, advanced research technologies, hamamatsu, visen medical, olympus, berthold, optosonics, maunakea technologies, and UVP) sell integrated systems for in vivo imaging of optical reporters (luciferase, fluorescent proteins). The measurement of the reporter is captured by charged couple detector (CCD) cameras that are attached in the system. A recent analysis from Frost & Sullivan declares a shift in the market of optical imaging devices. According to the analysis

The optical imaging market had transformed from an emerging market to a growing market.

Reporter genes, coupled with non–invasive molecular imaging technology, will ameliorate the drug development phase, which till date is experiencing immense cost and time pressures.

The more the spending in research and development by pharmaceutical companies across the globe, the more it would drive the optical imaging market.

I'm going to have a talk on bioluminescence principles in a "Molecular Imaging in Drug Discovery" workshop, I'm curious to see how many pharma companies will attend the event.

red fluorescence spectrum from soviet countries?

Despite years of effort since its cloning in 1992, the GFP of the jellyfish Aequorea victoria (the most known and used green fluorescent reporter) has not been turned red... until now.

A team of Russian researchers started from a blueshifted GFP variant and performed a molecular mutagenesis evolution approach to get, not only shift toward red, but also some clues to the chromophore formation theory. Basically they made libraries of millions of mutants and sent them through a Dako cell sorter to select the best red performances that were mutated once again and analyzed iteratively until they achieved the desired shift to the red fluorescence. The results was a protein, R10-3, that has both red and green fluorescence, so a pure red protein has not derived yet (but the evolutive job is still in progress).
It is not the first time that a Russian team turns to red fluorescent reporters: some months ago we were reading the Nature Methods paper about Katushka and mKate. Is a matter of fact that red color is an innate hue in some countries: are there the basis to make a social association study about scientists, countries and reporter colors?

• Mishin, A.S., Subach, F.V., Yampolsky, I.V., King, W., Lukyanov, K.A., Verkhusha, V.V. (2008). The First Mutant of the Aequorea victoria Green Fluorescent Protein That Forms a Red Chromophore. Biochemistry, 47(16), 4666-4673. DOI: 10.1021/bi702130s

In this post you can freshen why red spectrum matter in fluorescence imaging

reportergenomics?

Via researchblogging I found an interesting post on Chance and Necessity blog that suggest that deciphering of the regulatory code requires functional analysis beyond simple sequence alignment. Are the times of reportergenomic?

Do you know who is the author of the book Chance and Necessity: An Essay on the Natural Philosophy of Modern Biology?

Does a blogger influences impact factor?

It is quite common to infer that a paper from a journal with high Impact Factor (IF) would have a stronger scientific value than a study appeared in a minor journal. Basically, citation numbers are used to gauge scientific influence in the same way as google research results depend on numbers of linking pages. Nevertheless, a huge number of scientists criticized with this viewpoint, and new alternative citation metrics, like H-index, are continuously being proposed.

The June editorial of Nature Neuroscience contributes to the overall discussion, noting that probably most references are transcribed from other reference lists rather than from the original source article. If this is true, we can speculate that most authors do not read the papers they cite, and (foremost) this makes citation counting far less significant. So, instead of citations what about downloads? With the (discutible) assumption that everyone downloading the paper is reading it, can one predict how well any particular paper is cited years after publication, based solely on the number of downloads it receives immediately following its appearance online? Actually the correlation appeared to be linear (at least for Nature Neuroscience papers).

In perspective, the Nature Neuroscience editors precognize that new metrics (such as paper downloads and other "web 2.0" technologies) can find a place in a compilation of aggregated stats, painting a more accurate and informative picture of manuscript influence. In this context, I put the Researchblogging community. Are we bloggers ready to influence citation metrics?