Reporter gene sheds light

In Nature Network, you can find Stripped Science: the webcomic on life science. With a new strips on every Tuesday and Friday, Viktor Poór (University of Pécs), explains also the advantages of using a reporter gene.

Novel molecular bar-coding tech

Geiss and other 19 colleagues from NanoString describes on Nature Biotechnology a multiplexed technology which captures and counts individual mRNA transcripts: the NanoString.
The technology uses molecular barcodes and single molecule imaging to detect and count hundreds of unique transcripts in a single reaction without any enzymatic reaction. In brief, a probe library is made with two sequence-specific probes for each gene of interest...

• The first probe is a capture probe that is complementary to a particular target mRNA and contains also a short common sequence coupled to an affinity tag such as biotin.
• The second probe, the reporter probe, contains a second sequence complementary to mRNA and a color-coded tag (with >16000 codes) that provides the detection signal.
• All probes are mixed toghether with total RNA in a single hybridization reaction that results in the formatio of tripartite structures (probe#1-RNA-probe#2).
• After capture on the surface, an applied electric field extends and orients each tripartite structure in the same direction, and then the aligned frame is imaged and analyzed.
• Each target molecule of interest is identified by the color code generated by the ordered fluorescent segments (the barcode) and the level of expression is measured by counting the numbers of codes for each mRNA.

The new platform was compared to microarrays, TaqMan® PCR, and SYBR®Green real-time PCR and results demonstrated that the nCounter system is more sensitive than microarrays and is similar in sensitivity to real-time PCR. The pdf paper is available for free at the Nanostring site.

Geiss, G.K., Bumgarner, R.E., Birditt, B., Dahl, T., Dowidar, N., Dunaway, D.L., Fell, H.P., Ferree, S., George, R.D., Grogan, T., James, J.J., Maysuria, M., Mitton, J.D., Oliveri, P., Osborn, J.L., Peng, T., Ratcliffe, A.L., Webster, P.J., Davidson, E.H., Hood, L. (2008). Direct multiplexed measurement of gene expression with color-coded probe pairs. Nature Biotechnology, 26(3), 317-325. DOI: 10.1038/nbt1385

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One reporter for multiple profiling of TFs

Some days ago I was challenging the concept that a reporter need to be a protein. Imagine my surprise in reading the March issue of Nature Methods, where Romanov and colleagues from Attagene Inc, describe a novel method for simultaneously reporting on the activity of a large panel of transcription factors. Here the reporter is a synthethic sequence that isn't translated, but only transcribed, an example of RNA-based reporters. Moreover, the paper breaks the dogma 'one promoter - one reporter' by analyzing activities of multiple TFs with just 'one' reporter sequence.

The trick is that they cloned TFs binding sites in front of a reporter sequence with an enzymatic cleavage site in a separate position for each TF, so that after these constructs are transfected within a cell, digest of the reporter species produced spectrum of DNA fragments that mirrored TF activities profile.

To be more technical, for multiplexed detection of transcription factors the "Factorial(TM)" 30 TF reporter library is introduced into cells of interest by using standard transfection procedure. Some time after transfection, total cellular RNA is isolated and reversely transcribed. The reporter cDNAs are amplified by PCR using a pair of primers that are common for all reporters. The PCR products are labeled with a fluorescent dye and digested by the HpaI. The digestion produces a spectrum of fluorescently labeled DNA fragments of different lengths that are resolved by capillary electrophoresis (CE), detected as separate fluorescent peaks and then quantified.

Although CE is limited by a narrow dynamic range, and the assay is challenged by the management of up to 30 sequence transfection in the same cell culture, this novel technique offers a valuable alternative for TF activity fingerprinting that outclasses classic TF binding ELISAs for significance and elegance.

---/ citation /--- --- ---
Romanov, S., Medvedev, A., Gambarian, M., Poltoratskaya, N., Moeser, M., Medvedeva, L., Gambarian, M., Diatchenko, L., & Makarov, S. (2008). Homogeneous reporter system enables quantitative functional assessment of multiple transcription factors Nature Methods, 5 (3), 253-260 DOI: 10.1038/nmeth.1186

GFP a mercury biosensor

Heavy metals like cadmium and mercury are "invisible" poisons that can accumulate and disrupt both metabolic and endocrine homeostasis of humans and livestock. While traditional analytical methods allow for highly accurate measurements of these metal concentrations, they commonly do not allow for time-dependent or location-specific in vivo measurements. As the uptake and distribution of this extremely toxic metal are not yet understood, highly sensitive and non-invasive methods are needed for its detection in a living organism. With the introduction of a cysteine residue at position 205, Chapleau and colleagues from the University of California, illustrate in Protein Science how the green fluorescent protein (GFP) from Aequorea victoria was converted into a highly specific biosensor for mercury:

• the mutant protein exhibits a dramatic absorbance and fluorescence change upon mercuration at neutral pH also in presence of metal binding agents like EDTA;
• these changes exhibit sigmoidal binding behavior with respect to the metal concentration with a detection limit in the low nanomolar range;
• time-resolved binding studies indicate rapid subsecond binding of the metal to protein;
• crystal structure and mass spectrometry further characterize this novel biosensor.

The aim of the authors is to provide a biosensor that, in presence of mercury, decrease (or anyway changes) its own fluorescence. Although (until now) the only "in vivo" application claimed in the title is the "GFP"-sensor expression in bacteria challenged with mercury 20 micromolar, the use of GFP as a mercury-specific noninvasive biosensor is a first example of a real-time biosensor that would enable the direct imaging of the uptake of heavy metals by a living cell, or (better) a living animal.

Chapleau, R.R., Blomberg, R., Ford, P.C., Sagermann, M. (2008). Design of a highly specific and noninvasive biosensor suitable for real-time in vivo imaging of mercury (II) uptake. Protein Science DOI: 10.1110/ps.073358908

What’s fun about biotech?

Much of the progress of civilization has turned on the artifice of harnessing nature’s engines of creation. In the last ten or twenty years, there’s been a quantum leap in this artifice and biotechnology became a molecular discipline with lot of opponents, namely BioLuddites. But other people thought molecular biotechnology should have got a cheerleader or two, heroes, they start identifying, collecting and cataloguing new developments they think were amusing, cool, noteworthy, and/or earth-shaking. As an attempt to make Biotechnology interesting and accessible to a larger, (relatively) non-technical audience was born Aminopop, what’s fun about biotech.

It's time for revision?

«A reporter gene is one that codes for a protein easily detectable in a protein background»

Such classic definition need to be revised when studying transcription factors (TFs): who really needs the protein (that may be unpredictably affected by irrelevant post-transcriptional mechanisms)? What are your feelings?

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