[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

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

ResearchBlogging.orgNeural 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

don't crack down bioluminescence imaging

ResearchBlogging.orgThe 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?

ResearchBlogging.org 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

ResearchBlogging.orgOf 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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