75 posts · 74,636 views

I'm a molecular pharmacologist and a fan of genetically-encoded assays. I'm a postdoc interested in dissecting how Nuclear Receptors regulate the crossroads between reproduction, nutrition and aging.

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September 9, 2008
02:55 AM
1,814 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 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... 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

January 30, 2008
12:21 AM
1,775 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 is Tango Assay: when your lead drug in exam binds your target #2b recep... 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

Biology

April 9, 2009
11:27 AM
1,706 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 animal experimentation based on the generation of a knowledge based on the non-invasive observation of physiological events in living animals at molecular detail. This vision is still in its infancy and several ameliorements steps need to be undertaken. One of them, is the development of better transgenic abilities to safely introduce a genetically-encoded reporter into mammals. Due to the technical difﬁculty in obtaining fertilizable eggs and the unavailability of embryonic stem cells, no transgenic dog has been generated so far. Hong et al., report now the use of Somatic Cell Nuclear Transfer (SCNT) to generate, from a stably-transfected fibroblast, a dog carrying the red-fluorescent protein. This will be probably a debated proof of concept.Hong, S., Kim, M., Jang, G., Oh, H., Park, J., Kang, J., Koo, O., Kim, T., Kwon, M., Koo, B., Ra, J., Kim, D., Ko, C., & Lee, B. (2009). Generation of red fluorescent protein transgenic dogs genesis DOI: 10.1002/dvg.20504... 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
1,700 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 interactions and, more often, return false positives. So, how does it work?Protein interaction studies with Gal4-luc reporter mouseActually I'm quite frightened about this model: let I want to know whether protein X (bait) and Y (from library) interact in vivo. In order to detect XY interaction with the G4F mouse, I need to tag X with Gal4BD (get a X-Gal4BD expression vector) and then tag Y with VP16 (get a Y-VP16 expression vector). Then I have to make two more transgenics (and deal about positional effects) and, once I get them, backcrossing them in the G4F background and, like a juggler, breed and genotype three markers together. Three years are enough? Of course, an alternative would be to make transfections in vivo (i.e. with hydrodynamic somatic gene transfer or viral delivery) but even this job will need some validation (i.e. not seeing an interaction means that the interaction did not appear or it’s a false negative due to problems in vector delivery. In conclusion protein-protein interaction is so hot and so hard matter, and my feelings are similar to those expressed for the Tango Assay:There is a trend to interpret complex systems with complex conceptual design in today's molecular pharmacology. For sure this is geeky, funny, and sexy for mind, so it is worth being mentioned in Reportergene. Nevertheless I don't know whether this would be useful also to understand our inner biology. Managing three transgenes (also transiently) is not a joke. Using strong promoter to express receptors and cognate ones is unphysiological and very, very naive.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... 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
1,683 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-vector, the infrared fluorescence performed better than mKate a red fluorescent protein as imaged by a Maestro spectral imager.This makes me gloomy, since I spoke about mKate in the Reportergene's first post. It was September 2007, does it takes only two years for a reporter gene to be outperformed? It is dramatic: it takes roughly two years to make a transgenic reporter mice, and another two-three years to get data with him! Once you start, you know that you will be outdated at half of your journey.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... 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
1,636 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 Zhang, Axel Nohturfft (2008). Studying Membrane Biogenesis with a Luciferase-Based Reporter Gene Assay Journal of Visualized Experiments... 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
1,593 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 tracking in the liver, which can be followed over time.Modified from Ploemen et al., Plos ONE 2009A 3D approximation is obtained through rotational axis bioluminescence imaging through a CaliperLS workstation: eight imaging views from different advantage points are determined. At each angle view, both the picture of the mouse and its luciferase signal at 3 different wavelengths are acquired. Then, the algorithm tries, from surface data, to reconstruct what is happening inside the mouse by providing an approximate 3D-reconstruction. Last, a digital mouse atlas is overlaid onto the 3D diffuse tomography reconstruction to obtain anatomical reference points. The Caliper algorithm is longer to be perfect (see 2008 post on new developments in 3D-bioluminescence) in fact it is based on the assumption that the mouse is optically homogeneous. However, it seems to perform pretty well at least for a parenchimal organ like the liver (see picture). Indeed, also fluorescence tomography works well in liver (see post on infrared fluorescent protein).In conclusion, at least for liver, it is possible to follow a reporter with enhanced spatio-temporal resolution. Since in liver, contrarily to the whole mouse, genetic engineering can be successfully performed with simple hydrodynamic injections, one would expect lot of progress in the future of molecular hepatology.Ploemen, I., Prudêncio, M., Douradinha, B., Ramesar, J., Fonager, J., van Gemert, G., Luty, A., Hermsen, C., Sauerwein, R., Baptista, F., Mota, M., Waters, A., Que, I., Lowik, C., Khan, S., Janse, C., & Franke-Fayard, B. (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

... 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
1,575 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 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... 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
1,498 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 needed for its detection in a living organism. With the introduction of a cysteine residue at position 205, Ch... 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

Biology

September 11, 2009
01:15 PM
1,483 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 PCR approach. The result of such a cut and paste was genetically encoded into HeLa, HEK93 and MEF/3T3 cell lines. Then, by irradiating whole cells or even localized micro-spot on the cell surface, PA-Rac1 was sufficiently photo-activated to generate polarized cell movements. In other words, light was controlling the motility of living cells via photoactivable Rac. Structural studies indicate that the Rac-LOV2 interface can be engineered to cage other proteins. Engineering and Biology are getting married. If you are a kick ass engineer, consider hacking biology at Ginkgo BioWorks, they are hiring.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

... 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
1,475 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 function. Identifying regulatory regions, however, has been a challenge. Promoters tend to be located adjacent to the genes they control, but enhancers are scattered throughout the genome, sometimes 1 million bases of DNA away from the gene they regulate.Axel Visel and his colleagues found a way to identify when enhancers are regulating genes. A protein called p300, expressed throughout the body, is found in many enhancer-associated protein complexes. p300 is also required for embryonic development, a crucial time when activated genes are literally building the body. Visel dissected forebrain, midbrain and limb tissue from more than 150 mouse embryos, cross-linked the DNA and protein, and digested the DNA (pieces of DNA bound to protein are protected). Using antibodies, Visel purified only those pieces of DNA bound to p300 and then sequenced that DNA and identified it as a possible enhancer by alignment to the mouse genome. This technique, called chromatin immunoprecipitation coupled to massively parallel sequencing (ChIP-seq), is not new, but using p300 as the bait was a clever twist.To confirm these regions of DNA regulate gene expression in vivo, Visel identified orthologous regions from human DNA. Candidate enhancers, average size 2.4 kb, were cloned upstream of mouse minimal hsp68 promoter and lacZ, a reporter vector used previously by this and other groups. Candidate enhancers were not cloned in any particular orientation. Vectors were injected into fertilized mouse eggs, the eggs were implanted and at embryonic day 11.5 embryos were harvested for whole-mount X-gal staining. Only similar staining patterns observed in three different embryos (representing three independent transgene integrations) were considered valid. If ChIP-seq identified an enhancer active in the limb but not in the brain, then the human orthologue of the enhancer should turn only the mouse’s limbs blue. In most cases, that’s what happened. (See photo.)Overall, 87% (75 out of 86) of the enhancers produced expression patterns in mice that agreed with the ChIP-seq results – a tremendous improvement over the same researchers’ previous prediction method (47%, 246 out of 528), in which enhancers were identified based on evolutionary conservation and tested using the same reporter assay in transgenic mice. The p300 ChiP-seq method is especially good because it’s large-scale, enabling scientists to study thousands of enhancers throughout the genome from any tissue during any time in an animal’s life. Visel and colleagues identified thousands of enhancers active in the brain and limbs of mouse embryos and verified 75 using transgenic F0 embryos. Using this technique, future studies can identify in vivo enhancers from additional anatomic regions, embryonic (or adult) stages, and from mouse models of human disease.As my graduate adviser used to say, if you put junk in, you get junk out. Clearly junk DNA is anything but.Visel, A., Blow, M., Li, Z., Zhang, T., Akiyama, J., Holt, A., Plajzer-Frick, I., Shoukry, M., Wright, C., Chen, F., Afzal, V., Ren, B., Rubin, E., & Pennacchio, L. (2009). ChIP-seq accurately predicts tissue-specific activity of enhancers Nature, 457 (7231), 854-858 DOI: 10.1038/nature07730Daniel Gorelick is a neuroscientist who is currently taking a year off from his postdoctoral fellowship to serve as a AAAS Science & Technology Policy fellow in the U.S. Department of State. He writes the Science Planet blog and covers science and technology for www.America.gov, a State Department Web site. E-mail: scienceblog@state.gov... 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
1,403 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 multiple reporter into a library ready to transfect. In this paper, the authors just coupled each reporter with a unique expressed oligonucleotide tag, transfected the library, isolated the reporter mRNAs as a pool and analyzed it by hybridization to microarray. In brief, this is EXTassay. Wondering about transfection efficiency? Owing to its small scale, plasmid DNA was not denaturated by Trizol and was localized in the acqueous phase (they said), so it can be isolated and hybridized to serve as calibrator.

The trick to probe receptor dimerization relies in the genetic manipulation of the receptor gene, and it is quite similar to the Tango assay. Other than being solely a receptor, the protein in exam is coupled in frame with partial TEV recombinase and a GV transcription factor. Upon receptor dimerization (activation) follows reconstitution of a TEV protease and cleavage of GV, which goes in the nucleus where it can find a corresponding GV-reporter.

Despite I'm still uncomfortable with the idea to interrogate receptor activity by receptor transfection, I start feeling RNA-reporters being on the road toward maturity, and I admit: probably I'have been too much severe with Tango assays in January 2008. I should think at this when planning my future experiments, there is more other than luciferase in 2010.

---/ Citation /--- --- --- ---
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

... 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
1,339 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 pathways. Hence, in standard assays, transient or relatively minor effects may be hidden and kinetics somewhat inaccurate: this explains why researchers prefer reporters with short half-life, and eventually decrease it with both mRNA and protein destabilizing elements (see for instance the RapidReporter Gaussia luciferase from Activemotif). To date, another significant quality step in the race toward the 4th dimension (time), have been conquested: new fluorescent 'timers' (FTs) that gradually change colour from blue to red could allow researchers to track the age and dynamic behaviour of proteins in living cells. Previous work suggested that some red fluorescent proteins start out fluorescing blue, but then change to red as the protein is chemically modified over time. Vladislav Verkhusha and his colleagues at the Albert Einstein College of Medicine in New York mutated the red fluorescent mCherry, toward altered maturation rates from blue to red, getting three fluorescent proteins, each with a specific maturation rate. The proteins were used to track newly synthesized proteins in mammalian cells grown in culture, but maybe in future we will have a "flight recorder" inside a cell, to log different molecular events with respect to time.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... 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
1,319 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 it works?Take a strong promoter like CMV and make a library coding the first reporter (DsRed)alone and the second one (EGFP) fused in frame within one of the 8000 human proteins X (take the X library from the hORFeome cDNA library). Of course, you need viral IRES to let the ribosome translate two protein from the same mRNA. Then, express the library in your favourite cell line. Once protein X is degradated, if you "believe" also EGFP will be degradated, then you can FACS the EGFP/dsRED ratio obtaining 7 population of cells containing only stable--unstable proteins in your vector. Make 7 microarrays opportunely designed to find the DNA of your vector and identify which proteins (ORF) are stable/unstable. Give the results to a team of bioinformatics/system biologists and mine the dataset to find correlation between protein stability and roles/pathways/amino acid composition/astrological signs and what else you can get statistically significant. Submit the manuscript to Science.Take care! It would be possible that the EGFP fusion with X protein, generates a protease site. In that case, the EGFP/dsRED ratio may not be a reliable readout of protein stability. If you don't want to take such risk, hire a PhD student and ask him to made 8000 western blots with 8000 working antibodies. Then, submit the manuscript to Reportergene** in 2019, when your PhD student will give you the results, Reportergene will have the biggest impact factor.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... 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
1,314 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 Site) and they opened the way for bicistronic vectors. So, lot of scientist make construct like this:
PROMOTER-GENE1-IRES-GENE2-polyA
One single promoter drive a double transcript containing two messengers; then the IRES is supposed to drive to the ribosome the GENE2 alone with an efficiency ranged between 20% and 50% of the cap-dependent GENE1. This constitute the major disadvantage of bicistronic reporter.

When Bouabe and colleagues from Munich University comes to the club of weak promoter, they wondered: "Why don't put another copy of the same reporter after the IRES?".
PROMOTER-REPORTER-IRES-REPORTER-polyA
Such approach is described in Nucleic Acid Research and constitutes a simple technology that allows improvement of reporter gene activity using multiple IRES-Reporter linked in tandem. Basically IRES allow the same weak promoter to express more reporter without interfering with its transcriptional dynamics. Fluorescent proteins like EGFP (that to date, need to be at least 10,000-100,000 molecules/cell in order to be visualized) are probably the reporter that would benefit more of such improvement, without the need to touch their stability.... 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
1,270 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 little exposure to the non-science world to say why WE CAN make better animal research.

From a modern mechanical and operational perspective, life relies on the interaction between a sworm of molecules and their hierarchical organization into structures in which we distinguish a functional unity (i.e., organelles, cells, tissues, organs and systems). It is current hope and belief that a whole, systematic decoding of such minute, living, interacting building blocks that are bio-molecules, will grant a better comprehension of life mechanisms. From more than one century, we learn how to identify and discriminate those molecules, by means of iterative, multidimensional separation techniques (i.e., 2D electrophoresis, tandem mass spectrometry). This provided us with the awareness of the vast compilation of molecules and their distribution among different single cells and, ultimately, into the whole biome.

Laboratory animals were the main source of specimens to put under our microscopes. However, from the slides, we progressively realized that the picture we were painting was actually a static still-life, without any motion. This is not surprising, given the fact that most of the research conducted so far have been based on post-mortem analyses. Worst, such frozen picture renders difficult to discriminate causes from consequences. Animal sacrifice raises scientific, ethical and economical concerns, moreover the paradox of studying life from dead samples, may reasonably induce to wonder about the artefactual side of the knowledge generated so far. Not only terminal euthanasia, but each animal distress poses the same doubts: are we acquiring a distressed knowledge?

Before the molecular age, giants like Darwin and Mendel revolutionized life sciences with a systematic observation of animal and vegetal living specimen in their natural environment (the galapagos) with minor to null human intervention other than observation. The point is: shall we systematically observe (and understand) molecular life into a living, awake, freely moving animal in its natural environment? Can we imagine a day in which our preferred pet, in the security of our home, would allow to make scientific discoveries because he/she possess a radio-gene able to communicate its physiological and molecular status to our wireless router which will promptly share this information to the scientific community online?

I'm dreaming about normal happy pets, that can be turned into research contributors just through a simple injection. This is science-fiction but we aren't very far from such a goal. Recent advances in molecular imaging led us to dream a clean future for animal research. Take the simplest molecule: H2O. Its diamagnetism led clinicians to monitor water in space and time by means of magnetic resonance imaging (the same can be done for fat). In 2003, we were able to monitor something more elegant than the space-temporal profile of a single molecule in a healthy living mouse: by means of optical bioluminescence imaging, my PhD mentor was able to observe in cycling female mice, not a molecule, but its molecular activity (specifically, the activity of the estrogen receptor, a hormone-regulated transcription factor).

This goal, was achieved by the conceptualization of so-called reporter mice: models engineered in order to allow the external non-invasive monitoring of any selected molecular mechanism in the full respect of animal’s dignity. This is, in other words, the ability to observe the molecular life into a living whole organism. A Darwinian legacy.

The most interesting feature of longitudinal imaging with living animal systems, however, is its excellent potential to Reduce the number of animals because animal sacrifice is NOT needed and each animal is its own control. We need now to improve this technology (toward the radio-gene) to Refine current methods by providing the opportunity to study molecular circuits systematically in 4 dimensions, furthermore new technology will abolish the pain for the animals (and voluntary humans). What we need now, are engineers providing multiplexing abilities and better resolutions (in space and in time). Molecular imaging should be considered a very valid Replacement alternative, and we need to shift mathematical engineers and bioinformaticians from still-life omics data set, to living data set. We need to hope and belief to alternative ways to conduct animal experimentation. Animals aren't humans. We need to develop safer, safest technologies to study gene networks in animals in order to validate the safety and to eventually apply this 'radio-genes' to humans, to ourselves. Nosce te ipsum (know thyself, said Socrates). This blog tales recent advances toward this vision, the vision of a clean, efficient, innocent biomedical research.

---/ citation /--- --- ---
Ciana, P., Raviscioni, M., Mussi, P., Vegeto, E., Que, I., Parker, M., Lowik, C., & Maggi, A. (2003). 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.), 590, 307-16 PMID: 19763513

Rando G, Biserni A, Ciana P, & Maggi A (2010). Profiling of drug action using reporter mice and molecular imaging. Methods in molecular biology (Clifton, N.J.), 602, 79-92 PMID: 20012393

... 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

March 25, 2008
06:09 PM
1,266 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 to a particular target mRNA and contains also a short common sequence coupled to an affin... 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

Biology

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 derivatives between variables from the data, then they generate candidate symbolic functions by randomly combining (and iteratively re-combining) mathematical building blocks. They finally compare the derivative expressions with the derivate data and score the best pairs according to parsimony criteria.Given the dimensionality and the complexity of current “omics” data, the computation time required to detect solutions is probably near to 1000-10,000 hours, however the algorithm’s search seems highly parallelizable and very appealing for distributed approaches. What is more astonishing, is the following step. In a second report King and colleagues (Aberystwyth University) extend the concept of “artificial scientist” by generating ADAM: this is a physically implemented laboratory automation system that […] executes cycles of scientific experimentation. (ADAM) automatically originates hypotheses to explain observations, devises experiments to test these hypotheses, physically runs the experiments by using laboratory robotics, interprets the results and then repeats the cycle.As a proof of concept, they applied Adam to the identification of genes encoding orphan enzymes in the yeast. Despite the abundance in data, theoretical gaps still exist in systems biology and integrative physiology, automatic science can potentially increase the rate of scientific progress. At the end of this provocative paper, the authors wonder: Might this process diminish the role of future scientists?Quite the opposite: does chess-software diminished the number of chess-players?Schmidt, M., & Lipson, H. (2009). Distilling Free-Form Natural Laws from Experimental Data Science, 324 (5923), 81-85 DOI: 10.1126/science.1165893King, R., Rowland, J., Oliver, S., Young, M., Aubrey, W., Byrne, E., Liakata, M., Markham, M., Pir, P., Soldatova, L., Sparkes, A., Whelan, K., & Clare, A. (2009). The Automation of Science Science, 324 (5923), 85-89 DOI: 10.1126/science.1165620... 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 17, 2009
06:05 AM
1,258 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 information for the synthesis of functional proteins or non-coding RNAs, related by the sharing of a portion of genetic information at the level of the ultimate products (proteins or RNAs)The take-home lesson is that a genetically-encoded assay should not necessarily rely on expressed proteins.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... 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

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