skimming Biophotonics International

I have just received my September print copy of Biophotonics International, a monthly magazine that would collect "photonic solution for Biotechnology and Medecine". I usually skim it in order to find some news about my well-beloved reporter genes.

• So, at page 14 I learn about opto-acoustic small-animal imaging devices and I wonder if there will be a "photoacustic" genetic probe in the near future.
• At page 19 I read that by 2013 the winner DNA diagnostic technology (in terms of $ millions market) would be the stale and rusty PCR and probably not some DNA microarrays or Lab-on-a-Chip Diagnostics. This lead me to remember a strange PCR-reporter application that I found very promising.
• At page 24 I promise myself once again to try to understand what is really optical coherence tomography (OCT) and check its relevancy for reportergenomics. Does any OCT-wise wish to giving me a primer submitting a post?
• At page 33 finally a found mentioned a reporter gene: the yellow fluorescent protein used for two-photon imaging and validated with some patch clamp (not really a news for us isn'it?)
• What I really like it is usually put in the last two pages: the Post Script. In this column, the best photonics pictures recapitulate a strange application of photonics. This month it's featured an Actin Coral: a mix of GFP + quantum dots approach by Diane Lidke that unravels dynamic labyrinths of actin at the cell membrane.

I forget to mention that I got my FREE subscription to Biophotonics International (and other interesting magazines) at http://reportergene.tradepub.com You can try to apply for your own free subscriptions, and so giving a little help to Reportergene (strongly suggested: it works).

Blogging science to allow evidence-based decisions

Concerning research blogging, I previously wondered how it will influence research dynamics and maybe kill some boring press releases. Little things compared to the fine writing skills of those guys. Read how they start:

Scientific discovery occurs in the lab one experiment at a time, but science itself moves forward based on a series of ongoing conversations, from a Nobel Prize winner's acceptance speech to collegial chats at a pub. When these conversations flow into the mainstream, they nurture the development of an informed public who understand the value of funding basic research and making evidence-based voting decisions. It is in the interests of scientists and academic institutions alike to bring these conversations into the public sphere.

Of course, they are talking about blogging. You can read the full article by Shelley, Nicholas and Tara at PLoS Biology.

[movie] Dual-luciferase for membrane biogenesis

Jove is featuring a movie which explains how to study the coordination of membrane biogenesis by a luciferase-based reporter gene approach using the Dual-Glo Luciferase Assay System from Promega. As usual, Jove provides a step-by-step protocol that can be commented for asking clarifications.

Zhang S Jove.com

In my humble opinion, this is definitively the new revolutionary way to make science and I'm quite surprised to get replies from big seniors wondering only about Jove's impact factor.

Shaochong Zhang, Axel Nohturfft (2008). Studying Membrane Biogenesis with a Luciferase-Based Reporter Gene Assay Journal of Visualized Experiments

Protein evolution switch-off fluorescent proteins (and on)

Canonical fluorescent proteins (FPs) emit light once excited by appropriate irradiation. In a peculiar group of them, the reversibly switchable fluorescent proteins (RSFPs), irradiation converts the protein from the fluorescent on-state to the non fluorescent off-state. Then, a subsequent exposure with UV light brings back the protein to the on-state. Intriguingly, switchable fluorescence allow for sub-diffraction resolution microscopy (nanoscopy) as previously explained, so any RSFP is important for new microscopy development. Unfortunately, known RSFPs share more or less the same switching features and properties: this is a limitation for scientists aimed at developing new imaging schemes with different RSFPs.

After seven rounds of directed molecular evolution from the RSFP Dronpa, Martin Andresen and colleagues from the Max Plank, report in the September issue of Nature Biotechnology, the generation of two new bright RSFP with unique absorption and switching characteristics. These new proteins, called Padron and bsDrompa will allow for the implementation of monochromatic, multilabeled imaging and dual color far-field fluorescence nanoscopy.

Let me give a personal perspective: I bet in a near future someone will exploit the on/off properties of those reporters to get a tomographical solution of fluorescence (and bioluminescence) imaging in living mice.

Martin Andresen, Andre C Stiel, Jonas Fölling, Dirk Wenzel, Andreas Schönle, Alexander Egner, Christian Eggeling, Stefan W Hell, Stefan Jakobs (2008). Photoswitchable fluorescent proteins enable monochromatic multilabel imaging and dual color fluorescence nanoscopy Nature Biotechnology, 26 (9), 1035-1040 DOI: 10.1038/nbt.1493

Why do you care about kinetic imaging?

A new optical imaging device has been released from Caliper, the IVIS Kinetic. Compared to older imaging workstations this new product allows acquisition of biological events within milliseconds by fluorescence or bioluminescence imaging both in sleeping or conscious animals. That machine, exploiting a highly sensitive EMCCD camera, will be mainly suited for monitoring real-time functional events like perfusion dynamics and pharmacokinetis, or rapid events like calcium transients or immunological reactions. I'm in doubt whether this one will be a significant advantage toward reportergenomics maturity. Speaking about in vivo reporter gene imaging, both luciferase and GFP family suffer from low sensitivity due to:

1. relatively low light-emission - this explains why there is low time-resolution (you need to integrate signals over minutes, no hope to have a millisecond resolution);
2. scattering and absorption by tissues - this explains why there is low space-resolution (you get a planar image and no satisfactory 3D solutions have been made so far).

Probably these limitations less influence imaging of bright chemical probes (not reporter-genes) explaining some marketing decisions from Caliper. Or maybe, really this is a revolutionary machine with surprising performances even for reporter imaging. Let it be.

New calcium reporter for two-photon imaging in vivo

Neural networks evolve their functional features over time. At present, no techniques allow detailed neuron recording over repeated experimental session: the only method to repeatedly recording single cell activity in vivo is by means of chronically implanted electrodes. Unfortunately, with electrophysiology, cell death and gliosis give some uncertainty in monitoring the same neuron over months, while imaging techniques may solve such problem allowing unequivocal identification of the neural cell. Fluorescent contrast labels suffer, during longer recording, from some leakage and to load new indicator is quite difficult. Reporter genes, as spotlighted by the Brainbow mouse, are an appealing approach even if quite immature. In fact, at present, genetically encoded sensors for brain imaging are almost represented by fluorescent proteins. Those GFP-like reporters still suffer from narrow linearity and relatively poor brightness, hampering enough space resolution to track in living animals followed over time, the connectomic studies precognized by the Brainbow's dead brain slices.

Marko Mank and colleagues started filling this gap by means of mutagenesis on the TnC calcium biosensor. These efforts increased overall signal strength and sensitivity in the regime of physiologically relevant calcium concentrations leading to a new biosensor, the TN-XXL, that is functional in vivo in flies and mice and, according to the authors, can be used to obtain tuning curves of neurons in visual cortex using in vivo two-photon imaging. As a perspective, the new reporter will be useful to get more insigths about calcium role into plasticity and degeneration.

Marco Mank, Alexandre Ferrão Santos, Stephan Direnberger, Thomas D Mrsic-Flogel, Sonja B Hofer, Valentin Stein, Thomas Hendel, Dierk F Reiff, Christiaan Levelt, Alexander Borst, Tobias Bonhoeffer, Mark Hübener, Oliver Griesbeck (2008). A genetically encoded calcium indicator for chronic in vivo two-photon imaging Nature Methods, 5 (9), 805-811 DOI: 10.1038/NMETH.1243

R&D efficacy revision?

An ongoing LinkedIN conversation is wondering about the steady decrease in the discovery of drugs that would eventually reach the marketplace worldwide per year per billion dollars R&D investment. What does it mean? Basically, even if we daily develop new techniques, we are not discovering new drugs (Note, we are talking about Discovery, not Market).

Michael Gold, VP at GlaxoSmithKline spotlights:

Advances in molecular biology and molecular genetics made it pretty simple to isolate and characterize novel receptors, peptides etc. These technical advances were coupled with a belief that if you dissected a disease deep enough, we'd find a single root cause..some defective receptor or mis-folded protein. This led us to neglect cell physiology or as some call it now "systems biology" and become almost unable to deal with disorders that involve physiological systems and cascades.

Really, we don't need more cell lines.

surprise in my shared readings

Some months ago, I've started to share my google reader readings. Because there is a feed of it, I put it in the wordle claws. It seems that I'm a very interested person in fluorescence and microscopy.



I can not recognize myself, I was quite sure to be a supporter of luciferase and in vivo imaging.

don't crack down bioluminescence imaging

The laboratory that discovered leptin have recently reported a BAC transgenic mouse line that express luciferase under the control of leptin promoter elements. They showed that bioluminescence imaging faithfully recapitulates regulation of leptin mRNA in different condition of fasting/fed regimens in normal/obese background with/out leptin withdrawal. So, by in vivo imaging they now strive to characterize the still unknown cellular program responsible for restoration of adipose tissue after weight loss. Best wishes. I'm a strong supporter of reporter imaging in animal research (here, here and here). However, this latter model pose me some doubts. Even if I recognize the finest quality to study gene expression chronology in a living animal, I wonder about the necessity and the economy to make research with surrogate markers (luciferase) of a single gene expression (leptin). Signalling networks are so robust and redundant!

In my humble opinion, luciferase reporter mice are better exploited to track responsive elements of critical transcription factor, in that case the photon emission would be a surrogate marker of the final result of one/more signalling cascades and not just the mirror of one of our 30,000 genes. That's much informative. Of course, then you need some deconvolution, but you can reasonably get the dissection of your pathway by crossing your reporter mouse into a KO background. Definitively, to study mRNA expression in a tissue, I prefer a hierarchical clustering of quantitative PCR dataset (of different genes), chiefly with new technologies (here and here). Yes, this is just my personal naive opinion, feel free to fill my gaps.


K. Birsoy, A. Soukas, J. Torrens, G. Ceccarini, J. Montez, M. Maffei, P. Cohen, G. Fayzikhodjaeva, A. Viale, N. D. Socci, J. M. Friedman (2008). Cellular program controlling the recovery of adipose tissue mass: An in vivo imaging approach Proceedings of the National Academy of Sciences, 105 (35), 12985-12990 DOI: 10.1073/pnas.0805621105

this is another example of BLI crack down

How many building blocks do you expect in a cell?

Expectations that defined variation in the DNA blueprint would serve to pinpoint our whole inner biology was completely unfulfilled by the genome projects. To date, under the light of system biology, we can conceive at least 68 omics disciplines - would say Jamey Marth (HHMI) - DNA is just one character and there are 68 molecules that contribute to the synthesis and primary structures of the 4 fundamental macromolecular components of all cells (nucleic acids, proteins, glycans and lipids).

We need to take care of our 68 players in defining conceptual frameworks for biology. We are writing our periodic table of cell's building blocks.

Jamey D. Marth (2008). A unified vision of the building blocks of life Nature Cell Biology, 10 (9), 1015-1015 DOI: 10.1038/ncb0908-1015

Long introns delay transcriptional time

In a negative feedback loop, does intron lenght affects gene expression? Yan Swinburne and colleagues (Harward) answered this question by engineering a gene network and modifying only intron length between clonal variants. What they observed was such network (with delayed autoinhibition) exhibiting pulses of reporter expression that were correlating with intron length. A successive simulation with mathematical models suggested that fluorescence bursting (Venus fast-maturing variant of yellow fluorescent protein) accumulated during transcription elongation.

Note in the construct diagram the presence of PEST and ARE used to destabilize both protein and mRNA: destabilization is fundamental to limit reporter accumulation and so to gain time-resolution. (Swinburne et al, Genes and Development 2008)

The delay of transcriptional machine by introns may be important in many contexts (somitogenesis during development, NF-kb patterns). Undoubtedly, this work further evidences that reporter genes are instrumental to the goals of system biology.

I. A. Swinburne, D. G. Miguez, D. Landgraf, P. A. Silver (2008). Intron length increases oscillatory periods of gene expression in animal cells Genes & Development, 22 (17), 2342-2346 DOI: 10.1101/gad.1696108

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Dark box? Black box?

I have been gone through a 2007 interview to Mark Roskey (vice president of reagents and applied biology at Caliper Life Sciences) appeared in Nature Methods. According to Roskey:

It should be one box that researchers put sample in and get an answer out.

Mark, are you talking about PhD students?

Balls electroporation?

In my humble opinion, to study functional genomics in vivo, the best would be to place your reporter gene into humanized animals (i.e. making transgenic reporter-humanized mice). Pronuclear DNA micro-injection in the oocyte is the most frequently used technique for generating transgenic mice; unfortunately, pronuclear transfer is expensive, labor-intensive, time-consuming and require coordination of many experimental steps, so personally I feel an urgent need to alternate, cost-effective and more rapid approach to obtain desired transgenic founders.
Recently, I have been occasion to wonder about a male-driven transgenesis approach, and I found interesting literature. Here a few references (feel free to comment missing items).

Suveera Dhup, Subeer S Majumdar (2008). Transgenesis via permanent integration of genes in repopulating spermatogonial cells in vivo Nature Methods, 5 (7), 601-603 DOI: 10.1038/NMETH.1225

Chang KT, Takahashi M (1999). Production of Transgenic Rats and Mice by the Testis-Mediated Gene Transfer Journal of Reproduction and Development, 45 (1), 29-36

Maione B, Lavitrano M, Spafafora C, Kiessling AA (1998). Sperm-Mediated Gene Transfer in Mice Molecular Reproduction and Development, 50, 406-409

reporter gene normalization

Reporter gene normalization on google chrome browser

AKA, how to warp keywords to trap new visitors
(yes, today I'm evil)

Basal expression from inducible transgenes

Of course, a critical drawback of inducible transgene expression is the amount of basal expression (leakage) responsible to making noise in the discrimination of specific transgene patterns analysis (and even, to reduce potentially harmful effects of transgene).

The Australian team of from Stuart M Pitson (University of Adelaide) by incorporating AU-rich mRNA destabilizing elements (ARE) into the 3′ untranslated region (UTR) of inducible constructs, demonstrated that this modification minimized sphingosine kinase 1 (SK1) basal expression from a Tet-inducible vector with only a slight decrease in the maximum level of fully induced SK1. This approach, might improve signal-to-noise ratio and should also be applicable to other inducible expression systems.

On the other hand, if your transgene is a reporter gene, sometimes a minimal basal level expression would be appealing to monitor physiological patterns that could be increased or decreased by your treatments. Isn't it?

Duyen Pham, Paul Moretti, Gregory Goodall, Stuart Pitson (2008). Attenuation of leakiness in doxycycline-inducible expression via incorporation of 3′ AU-rich mRNA destabilizing elements BioTechniques, 45 (2), 155-162 DOI: 10.2144/000112896

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