75 posts · 74,432 views
To master molecular mechanisms we need to know how they works within the whole organism. My vision is that new developments in genetic engineering will branch new applications for reporter genes, not necessarily confined to report transcriptional regulation. In my blog I trend advances in such a 'reportergenomics', a discipline at the crossroad between synthetic and system biology.
96well
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- September 9, 2008
- 02:55 AM
- 1,812 views
Long introns delay transcriptional time
by 96well in Reportergene
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 flu........ Read more »
I. A. Swinburne, D. G. Miguez, D. Landgraf, & P. A. Silver. (2008) Intron length increases oscillatory periods of gene expression in animal cells. Genes , 22(17), 2342-2346. DOI: 10.1101/gad.1696108
I. A. Swinburne, D. G. Miguez, D. Landgraf, & P. A. Silver. (2008) Intron length increases oscillatory periods of gene expression in animal cells. Genes , 22(17), 2342-2346. DOI: 10.1101/gad.1696108
- January 30, 2008
- 12:21 AM
- 1,771 views
Tango Assay: Rubik is simpler.
by 96well in Reportergene
Look what have they engineered at Howard Hughes Medical Institute: you need 3 transgenes. Transgene #1 encodes a reporter gene under transcriptional control of an exogenous transcription factor; transgene #2 encodes such transcription factor (#2a) linked to a endogenous receptor (#2b) by an aminoacid linker targeted by an exogenous protease; transgene #3 encodes such endogenous protease (#3a) linked to a protein (#3b) known to bind the (#2b) receptor when it is activated by their ligands. This........ Read more »
G Barnea, W Strapps, G Herrada, Y Berman, J Ong, B Kloss, R Axel, & K Lee. (2008) From the Cover: The genetic design of signaling cascades to record receptor activation. Proceedings of the National Academy of Sciences, 105(1), 64-69. DOI: 10.1073/pnas.0710487105
- April 9, 2009
- 11:27 AM
- 1,702 views
Ruppy, the first fluorescent-dog
by 96well in Reportergene
A Korean team report the generation of a RFP-transgenic beagle. Dogs exhibits 224 genetic diseases similar to those found in humans making them one of the closest known models for various human hereditary diseases. However, experimentation with animal -which should be at the service of the whole mankind - raises strong and acute ethical challenges, particularly if the experimental model is a pet. Although still prototypical, the concept of "reporter animal" arguments toward a new use of a........ Read more »
Hong, S., Kim, M., Jang, G., Oh, H., Park, J., Kang, J., Koo, O., Kim, T., Kwon, M., Koo, B.... (2009) Generation of red fluorescent protein transgenic dogs. genesis. DOI: 10.1002/dvg.20504
- October 27, 2008
- 09:44 AM
- 1,695 views
just another protein-interaction model
by 96well in Reportergene
Andrea Pichler and colleagues from Mallinkrodt Institute of Radiology describe the generation of a transgenic Gal4-luc reporter mouse (G4F) that expresses the Firefly luciferase (from pGL3, Promega) under the regulatory control of a concatenated (5x) Gal4 promoter. The Gal4-luc strain, would allow noninvasive bioluminescence imaging of protein-protein interaction in vivo, a feature of real interest since traditional biochemical techniques often miss the detection of weak and transient interactio........ Read more »
A. Pichler, J. L. Prior, G. D. Luker, & D. Piwnica-Worms. (2008) Generation of a highly inducible Gal4-Fluc universal reporter mouse for in vivo bioluminescence imaging. Proceedings of the National Academy of Sciences, 105(41), 15932-15937. DOI: 10.1073/pnas.0801075105
- May 11, 2009
- 11:04 AM
- 1,678 views
introducing infrared fluorescence protein IFP
by 96well in Reportergene
In vivo optical imaging of deep tissues in animals is most feasible between 650 and 900 nm because such wavelengths minimize the absorbance by hemoglobin, water, and lipids, as well as light-scattering. Roger Tsien, last year's Nobel Prize in chemistry for his research on fluorescent proteins, introduced in a Science report, a modified version of the Deinococcus radiodurans phytochrome turned to be a infrared fluorescent protein (IFP). Carrying IFP into the mouse liver through an adenovirus-vect........ Read more »
Shu, X., Royant, A., Lin, M., Aguilera, T., Lev-Ram, V., Steinbach, P., & Tsien, R. (2009) Mammalian Expression of Infrared Fluorescent Proteins Engineered from a Bacterial Phytochrome. Science, 324(5928), 804-807. DOI: 10.1126/science.1168683
- September 24, 2008
- 08:15 AM
- 1,633 views
[movie] Dual-luciferase for membrane biogenesis
by 96well in Reportergene
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.comIn 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........ Read more »
Shaochong Zhang, & Axel Nohturfft. (2008) Studying Membrane Biogenesis with a Luciferase-Based Reporter Gene Assay. Journal of Visualized Experiments. DOI: http://www.jove.com/index/details.stp?id
- November 25, 2009
- 08:27 AM
- 1,588 views
Malaria in 3D: bioluminescence imaging
by 96well in Reportergene
In a recent Plos One paper, Ploemen and colleagues (Nijmegen Medical Centre) use previously generated luciferase-bearing malaria parasites (PbGFP-Luccon) to study the spatio-temporal development of malaria infection in liver of living infected mice. The final aim of the paper is to propose 3D-imaging to explore the effect of drug and vaccines on P. falciparum infection without surgery and other invasive methodologies in the mouse. Interestingly, they report good tri-dimensional plasmodium tracki........ Read more »
Ploemen, I., Prudêncio, M., Douradinha, B., Ramesar, J., Fonager, J., van Gemert, G., Luty, A., Hermsen, C., Sauerwein, R., Baptista, F.... (2009) Visualisation and Quantitative Analysis of the Rodent Malaria Liver Stage by Real Time Imaging. PLoS ONE, 4(11). DOI: 10.1371/journal.pone.0007881
- September 15, 2008
- 04:17 AM
- 1,572 views
New calcium reporter for two-photon imaging in vivo
by 96well in Reportergene
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 cel........ Read more »
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.... (2008) A genetically encoded calcium indicator for chronic in vivo two-photon imaging. Nature Methods, 5(9), 805-811. DOI: 10.1038/NMETH.1243
- March 10, 2008
- 06:06 PM
- 1,493 views
GFP a mercury biosensor
by 96well in Reportergene
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 nee........ Read more »
R Chapleau, R Blomberg, P C Ford, & M Sagermann. (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
- September 11, 2009
- 01:15 PM
- 1,480 views
engineering photo-activable proteins
by 96well in Reportergene
Plants contain proteins subjected to conformational changes in direct response to light irradiation. Moieties of those proteins, like the LOV2 domain from the Avena sativa Phototropin1 can be used to introduce light-operated switches onto other functional proteins. In a recent letter to Nature, Yi Wu and colleagues (Carolina University) poked at the Stratagene Quickchange kit to obtain a constitutive active Rac protein that was coupled to the vegetable LOV2 light switch using an overlapping PC........ Read more »
Wu, Y., Frey, D., Lungu, O., Jaehrig, A., Schlichting, I., Kuhlman, B., & Hahn, K. (2009) A genetically encoded photoactivatable Rac controls the motility of living cells. Nature, 461(7260), 104-108. DOI: 10.1038/nature08241
- March 23, 2009
- 10:25 AM
- 1,471 views
Predicting enhancer activity [guest post]
by 96well in Reportergene
Less than 2% of genomic DNA codes for protein. The remaining noncoding portions have been dismissively referred to as junk. Junk implies that because the DNA doesn’t code for proteins, it isn’t functional. In recent years, researchers showed that so-called junk DNA contains regulatory regions, promoters and enhancers that regulate gene expression. Identifying and cloning a gene is one thing, but knowing when and where it’s expressed is crucial to understand how organisms develop and functi........ Read more »
Visel, A., Blow, M., Li, Z., Zhang, T., Akiyama, J., Holt, A., Plajzer-Frick, I., Shoukry, M., Wright, C., Chen, F.... (2009) ChIP-seq accurately predicts tissue-specific activity of enhancers. Nature, 457(7231), 854-858. DOI: 10.1038/nature07730
- January 13, 2010
- 06:10 PM
- 1,400 views
EXTassays, toward maturity of RNA reporters
by 96well in Reportergene
I read with some interest a recent Nature Methods paper appeared this January. Anna Botvinnik and colleagues from Max Planck Institute, conceived a new reporter system able to measure receptor activation (receptor dimerization), downstream signaling (adapter recruitment) and subsequnent cis-regulatory responsive elements transactivation efficacies by...
...no, you don't need a 64-milion new-generation machine, you need Trizol!
As I reviewed in my first 2010 post, there is a trend to develop mu........ Read more »
Botvinnik, A., Wichert, S., Fischer, T., & Rossner, M. (2010) Integrated analysis of receptor activation and downstream signaling with EXTassays. Nature Methods, 7(1), 74-80. DOI: 10.1038/nmeth.1407
- February 3, 2009
- 03:43 AM
- 1,336 views
fluorescent timers: a new biophotonic tool
by 96well in Reportergene
In standard reporter assays the basal activity of the cloned promoter often results in accumulation of both luciferase mRNA and protein. This “background” activity may be an advantage since, once measured, gives you some numbers that you may use to calculate a fold induction (you can't divide by 0!). However, the slow clearance rate of these pre-existing molecules substantially may delay and dilute the measurable response, hampering the accurate quantification of changes in cell signaling pa........ Read more »
Fedor V Subach, Oksana M Subach, Illia S Gundorov, Kateryna S Morozova, Kiryl D Piatkevich, Ana Maria Cuervo, & Vladislav V Verkhusha. (2009) Monomeric fluorescent timers that change color from blue to red report on cellular trafficking. Nature Chemical Biology, 5(2), 118-126. DOI: 10.1038/nchembio.138
- November 23, 2008
- 04:46 AM
- 1,312 views
omics for protein stability
by 96well in Reportergene
A new genetic technology has been developed at Harvard HHMI that allow tracking the stability of individual proteins within a complex (omics) mixture. Sherry Yen and colleagues, introduced in Science magazine the Global Protein Stability (GPS), a parallel multiplexing strategy to simultaneously measure the half-lives of thousands of proteins in mammalian cells. Of course, the core is based on two fluorescent reporters (DsRed and EGFP), so we can provocatively name it reportergenomics. How does i........ Read more »
H.-C. S. Yen, Q. Xu, D. M. Chou, Z. Zhao, & S. J. Elledge. (2008) Global Protein Stability Profiling in Mammalian Cells. Science, 322(5903), 918-923. DOI: 10.1126/science.1160489
- February 25, 2008
- 07:00 AM
- 1,307 views
How to improve reporter activity
by 96well in Reportergene
Got a weak promoter in exam and your reporter signal is very close to background? Welcome to the club. Discovery companies tried to approach this problem designing for you more stable reporters (that accumulate over time), but they ask you to sacrifice any dynamic curiosity about what is going to happen over time. So what you can do?
In 1988 some viral sequences were described to initiate ribosome binding and translation in a cap-independent manner, they were named IRES (Internal Ribosome Entry........ Read more »
Bouabe, H., Fassler, R., & Heesemann, J. (2008) Improvement of reporter activity by IRES-mediated polycistronic reporter system. Nucleic Acids Research, 36(5). DOI: 10.1093/nar/gkm1119
- March 2, 2010
- 06:01 PM
- 1,266 views
A darwinian legacy OR Why we need fluorescent rabbits
by 96well in Reportergene
My post about fluorescent rabbits is gaining a momentum on the Flickr group 'Bunny Lovers Unite' and in the Rabbitmatch's blog. Most people ask itself: WHY making fluorescent bunnies? And others feel outraged.
Animal research is long debated, and my hope is that the development of new reporter probes would allow to reconsider current research protocols while increasing the scientific significance of the experiments done, this is the focus of my current research. Here, a take opportunity of this........ Read more »
Ciana, P., Raviscioni, M., Mussi, P., Vegeto, E., Que, I., Parker, M., Lowik, C., & Maggi, A. (2002) In vivo imaging of transcriptionally active estrogen receptors. Nature Medicine, 9(1), 82-86. DOI: 10.1038/nm809
Maggi A, & Rando G. (2009) Reporter mice for the study of intracellular receptor activity. Methods in molecular biology (Clifton, N.J.), 307-16. PMID: 19763513
- April 11, 2009
- 04:03 PM
- 1,265 views
Might a machine win a Nobel prize?
by 96well in Reportergene
In 1997, the IBM computer Deep Blue wins a chess-game vs Garry Kasparov. This is considered a milestone in Artificial Intelligence research. Now, a second milestone dates April the 3rd, 2009 with Science publishing two reports on automating science. In the first one, Schmidt and Lipson (Cornell) propose a computational approach for detecting physical laws from experimentally collected data. As a principle for the identification on non-triviality, they first numerically calculate partial derivati........ Read more »
Schmidt, M., & Lipson, H. (2009) Distilling Free-Form Natural Laws from Experimental Data. Science, 324(5923), 81-85. DOI: 10.1126/science.1165893
King, R., Rowland, J., Oliver, S., Young, M., Aubrey, W., Byrne, E., Liakata, M., Markham, M., Pir, P., Soldatova, L.... (2009) The Automation of Science. Science, 324(5923), 85-89. DOI: 10.1126/science.1165620
- March 25, 2008
- 06:09 PM
- 1,261 views
Novel molecular bar-coding tech
by 96well in Reportergene
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 t........ Read more »
Gary Geiss, Roger E Bumgarner, Brian Birditt, Timothy Dahl, Naeem Dowidar, Dwayne L Dunaway, H Perry Fell, Sean Ferree, Renee D George, Tammy Grogan.... (2008) Direct multiplexed measurement of gene expression with color-coded probe pairs. Nature Biotechnology, 26(3), 317-325. DOI: 10.1038/nbt1385
- March 17, 2009
- 06:05 AM
- 1,255 views
define gene
by 96well in Reportergene
If we want to come grips with new "reporter genes" we need to operationally know what is a gene. Giving a gene definition is a complex trivial task. For our purposes, wikipedia is not exactly so strictly operational:A gene is the basic unit of heredity in a living organism.More helpful is probably the definition recently proposed by Graziano Pesole:A gene is a discrete genomic region whose transcription is regulated by one or more promoters and distal regulatory elements and which contains the i........ Read more »
PESOLE, G. (2008) What is a gene? An updated operational definition. Gene, 417(1-2), 1-4. DOI: 10.1016/j.gene.2008.03.010
- February 17, 2009
- 08:02 AM
- 1,241 views
A Brainbow with wrist-watch
by 96well in Reportergene
A series of trans-synaptic pseudorabies viruses (PRVs) encoding fluorescent sensors and time-shifted fluorescent proteins like memCherry, memGFP and memCerulean, were recently proposed to trace several circuits in parallel in order to gain a dissection of the complex architecture of brain regions. The work by Zsolt Boldogkoi and colleagues from Szeged University (Hungary) has been published on the February issue of Nature Methods.Zsolt Boldogkői, Kamill Balint, Gautam B Awatramani, David Balya,........ Read more »
Zsolt Boldogkői, Kamill Balint, Gautam B Awatramani, David Balya, Volker Busskamp, Tim James Viney, Pamela S Lagali, Jens Duebel, Emese Pásti, Dóra Tombácz.... (2009) Genetically timed, activity-sensor and rainbow transsynaptic viral tools. Nature Methods, 6(2), 127-130. DOI: 10.1038/NMETH.1292
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