75 posts · 61,202 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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- March 28, 2011
- 06:12 AM
- 696 views
DIY yourself transgenic mice
by 96well in Reportergene
Although I'm not conducting active research on it, a question that fascinates me is: it is possible to get a transgenic model in a manner as simply as making cell transfection? My feeling is that it would be simpler to follow a sperm approach, by 'trasfecting' sperm and then use it for artificial insemination. In a previous post, I wrote about some guys electroporating the testis (ugly!). Now, a letter in Nature open a new perspective, although it is not discussed in the paper. Indeed, Sato et........ Read more »
Sato, T., Katagiri, K., Gohbara, A., Inoue, K., Ogonuki, N., Ogura, A., Kubota, Y., & Ogawa, T. (2011) In vitro production of functional sperm in cultured neonatal mouse testes. Nature, 471(7339), 504-507. DOI: 10.1038/nature09850
- March 8, 2011
- 12:28 PM
- 728 views
Fluorescent dogs 2.0 (with switch ON/OFF)
by 96well in Reportergene
Do you remember Ruppy, the red fluorescent puppy? The same team just described a new transgenic dog, this time with inducible fluorescence. The principle is always the TetON/TetOFF. According to the paper publised on Genesis, compared to mice, in dogs you need 10 less the dose of doxycycline (per chilogram) to switch ON the canine GFP, as determined by analyzing GFP expression in several organs. It is less clear how they took the organs: in the previous paper they said that one dog died due to........ Read more »
Kim, M., Oh, H., Park, J., Kim, G., Hong, S., Jang, G., Kwon, M., Koo, B., Kim, T., Kang, S.... (2011) Generation of transgenic dogs that conditionally express green fluorescent protein. genesis. DOI: 10.1002/dvg.20737
- February 28, 2011
- 06:58 AM
- 637 views
cartilage transgenic mouse
by 96well in Reportergene
Cartilage is the flexible connective tissue between our bones, and it is mainly based of cells called chondroblasts that secrete a gel-matrix made of collagen proteins. If cartilage maturation is disrupted, diseases may develop like the painful osteoarthritis and other chondrodystrophies. Therefore, it is important to understand the dynamics of cartilage formation and maturation, and a new reporter mouse made with classic pronuclear injection of the linearized product of a bacterial recombinat........ Read more »
Maye, P., Fu, Y., Butler, D., Chokalingam, K., Liu, Y., Florer, J., Stover, M., Wenstrup, R., Jiang, X., Gooch, C.... (2011) Generation and characterization of Col10a1-mCherry reporter mice. genesis. DOI: 10.1002/dvg.20733
- February 3, 2011
- 07:43 AM
- 493 views
Eat proteins read estrogens, said the liver
by 96well in Reportergene
Is the liver a reproductive organ? As you know, I was working with these glowing-in-the-dark mice, in which the reporter luciferase is expressed under estrogen receptor activity. The liver of these animals was always brighter in the morning than in the afternoon: in fact, mice eat during the night, therefore in the morning they were in a fed state and in the afternoon in a relative fasting state. Indeed, after a while, we discovered that some non-identified non-estrogenic food was activating the........ Read more »
Della Torre S, Rando G, Meda C, Stell A, Chambon P, Krust A, Ibarra C, Magni P, Ciana P, & Maggi A. (2011) Amino Acid-Dependent Activation of Liver Estrogen Receptor Alpha Integrates Metabolic and Reproductive Functions via IGF-1. Cell metabolism, 13(2), 205-14. PMID: 21284987
- January 20, 2011
- 11:47 AM
- 745 views
[review] sex dimorphism in nuclear receptor signaling
by 96well in Reportergene
Men and women have different organs, including the liver. The reasons for this sex difference are not completely clear, but probably relies on some different metabolic needs occurring during the reproduction. This is particularly evident in lay-egg animals like fishes and birds: to make egg nutrients, the liver is stimulated by the estrogen hormone to increase lipid and protein production (vitellogenesis). In mammals also (and humans, ditto) hormones like estrogen, and other nutrients (i.e., die........ Read more »
Wagner, M., Zollner, G., & Trauner, M. (2011) Nuclear receptors in liver disease. Hepatology. DOI: 10.1002/hep.24148
Rando, G., & Wahli, W. (2011) Sex differences in nuclear receptor-regulated liver metabolic pathways. Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease. DOI: 10.1016/j.bbadis.2010.12.023
- October 28, 2010
- 05:24 AM
- 672 views
better protein pharmacokinetics with a tail
by 96well in Reportergene
A combined protein/polymer-engineering approach prolongs protein circulation and enhance drug accumulation in tumours.
Consider insulin and you will have no doubts that a protein could be a drug. As proteins are coded by a corresponding DNA, the application of recombinant DNA has been largely exploited in clinics to provide patients with therapeutic 'recombinant' proteins. Among the advantages of owing a potential pharmacon on a plasmid, and growing such a marketable molecule in cell cultures, i........ Read more »
Gao, W., Liu, W., Christensen, T., Zalutsky, M., & Chilkoti, A. (2010) In situ growth of a PEG-like polymer from the C terminus of an intein fusion protein improves pharmacokinetics and tumor accumulation. Proceedings of the National Academy of Sciences, 107(38), 16432-16437. DOI: 10.1073/pnas.1006044107
- June 28, 2010
- 12:26 PM
- 794 views
Photoactivation through the central dogma of molecular biology
by 96well in Reportergene
Photoactivation is the property of a molecule of being capable of pronounced changes in its chemical properties in response to irradiation with light of a specific wavelength and intensity. This feature provides unique possibilities for the design of new strategies aimed at the spatio-temporal deciphering of molecular pathwhays occuring in living cells, organelles and intracellular molecules. Here, I spotlight two recent applications: from photoactivable nucleotides to photoactivable proteins.
........ Read more »
Hafner, M., Landthaler, M., Burger, L., Khorshid, M., Hausser, J., Berninger, P., Rothballer, A., Ascano Jr., M., Jungkamp, A., & Munschauer, M. (2010) Transcriptome-wide Identification of RNA-Binding Protein and MicroRNA Target Sites by PAR-CLIP. Cell, 141(1), 129-141. DOI: 10.1016/j.cell.2010.03.009
- April 29, 2010
- 12:34 PM
- 359 views
non-natural reporter proteins unleashed to the red
by 96well in Reportergene
After the pioneering works of Schultz's group and Chamberlin's group in 1989, bio-synthetic incorporation of non-natural amino-acids into proteins has been largely explored in chemical biology. Direct evolution of new tRNAs able to carry 'new' amino-acids, combined with codon extension to the 4th base has provided us with the ability to expand the chemical functionality of proteins by introducing new chemical moieties into their backbone. More importantly, we can do this in a genetically-heritab........ Read more »
Ye, S., Zaitseva, E., Caltabiano, G., Schertler, G., Sakmar, T., Deupi, X., & Vogel, R. (2010) Tracking G-protein-coupled receptor activation using genetically encoded infrared probes. Nature, 464(7293), 1386-1389. DOI: 10.1038/nature08948
Rowe, L., Ensor, M., Mehl, R., & Daunert, S. (2010) Modulating the Bioluminescence Emission of Photoproteins by Site-Directed Incorporation of Non-Natural Amino Acids . ACS Chemical Biology, 2147483647. DOI: 10.1021/cb9002909
- March 30, 2010
- 03:52 PM
- 724 views
toward a better drug classification
by 96well in Reportergene
Just a bit of self-promotion about my last paper. Despite the superiority of longitudinal vs cross-sectional studies, the dynamics of drug action are poorly explored in pre-clinical studies. Little is known about how drugs affect the activity of their intended target over time. Here, we used a longitudinal imaging approach to accurately follow the state of transcriptional activity of one drug target (the estrogen receptor) in 8 anatomical areas of living ERE-luc reporter mice over 21 consecutive........ Read more »
Rando, G., Horner, D., Biserni, A., Ramachandran, B., Caruso, D., Ciana, P., Komm, B., & Maggi, A. (2010) An Innovative Method to Classify SERMs Based on the Dynamics of Estrogen Receptor Transcriptional Activity in Living Animals. Molecular Endocrinology, 24(4), 735-744. DOI: 10.1210/me.2009-0514
- March 2, 2010
- 06:01 PM
- 979 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
- February 17, 2010
- 01:11 PM
- 762 views
Molecular surgery: playing with network edges
by 96well in Reportergene
Protein X interacts with protein Y, what are the phenotypic consequences? And what is the impact of the X-Y partnership in the whole protein-protein interaction network? To address this question, scientists often remove specific network nodes by eliminating (knock-out) or downregulating (knock-down) the gene encoding one protein product (i.e. X). This is a poor strategy, because usually X interacts not only with Y, but also with P, Q, R, S, T, U, V, W and Z. Thus, X-KO strategy is too much invas........ Read more »
Ear, P., & Michnick, S. (2009) A general life-death selection strategy for dissecting protein functions. Nature Methods, 6(11), 813-816. DOI: 10.1038/NMETH.1389
- February 11, 2010
- 01:43 PM
- 714 views
Are you a motivated labmate?
by 96well in Reportergene
I went through this little gift from Uri Alon: its essay appeared in Molecular Cell which aims to conjugate psychological principles to the every-day lab routine for improving motivation. How Uri Alon improves the motivation of his lab? He try to balance three fundamental needs of any scientist: competence, autonomy and social connectedness, for instance:I make our weekly group meeting an event that enhances social connectedness. The first half hour of the two hour meeting is devoted to nonscien........ Read more »
Alon, U. (2010) How to Build a Motivated Research Group. Molecular Cell, 37(2), 151-152. DOI: 10.1016/j.molcel.2010.01.011
- January 20, 2010
- 02:04 PM
- 743 views
Camel vs Jellyfish: a battle with green fluorescence
by 96well in Reportergene
Does the spectral properties of GFP can be modulated by antibody-derivatives? To explore this hypothesis, Axel Kirchhofer and colleagues from Munich Center for Advanced Photonics have designed a number of NanoBodies (NBs) to bind to GFP. (Nanobodies? They are small, antigen-binding, single-domain polypeptides derived from some camelid antibodies). The authors found NBs could increase or decrease GFP fluorescence: in fact, co-chystallization of GFP-NB complexes revealed NBs inducing subtle chan........ Read more »
Kirchhofer, A., Helma, J., Schmidthals, K., Frauer, C., Cui, S., Karcher, A., Pellis, M., Muyldermans, S., Casas-Delucchi, C., Cardoso, M.... (2009) Modulation of protein properties in living cells using nanobodies. Nature Structural , 17(1), 133-138. DOI: 10.1038/nsmb.1727
- January 19, 2010
- 11:00 PM
- 937 views
Green Fluorescent Rabbit
by 96well in Reportergene
A new study demonstrates the feasibility of using a lentiviral approaches to create transgenic rabbits with more efficiency than classical pronuclear injection transgenesis developed in rabbits two decades ago.
Tracing Ruppy, the reporter-dog, the enhanced transgenic bunny carries a green fluorescent protein. Rabbits are still used as laboratory animals as they are genetically closer to primates and are large enough to allow safe and secure blood sampling compared to mice. Thus, transgenic rabb........ Read more »
Hiripi, L., Negre, D., Cosset, F., Kvell, K., Czömpöly, T., Baranyi, M., Gócza, E., Hoffmann, O., Bender, B., & Bősze, Z. (2010) Transgenic rabbit production with simian immunodeficiency virus-derived lentiviral vector. Transgenic Research. DOI: 10.1007/s11248-009-9356-y
- January 13, 2010
- 06:10 PM
- 1,154 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
- January 4, 2010
- 12:00 AM
- 945 views
2010: luminometers OR sequencers?
by 96well in Reportergene
Next generation sequencing (solexa, illumina, 454) is offering a new opportunity for the design of multiplexed reporter assays. With the notable 2007 exception of Brainbow (in which however, it was not possible to discriminate the origin of the 90 or more observed fluorescent colors because they come from random recombination), simply the co-detection of more than three fluorescent proteins is very challenging in real life because of spectral overlapping and other shortcomings. Early in 2008, I ........ Read more »
Patwardhan, R., Lee, C., Litvin, O., Young, D., Pe'er, D., & Shendure, J. (2009) High-resolution analysis of DNA regulatory elements by synthetic saturation mutagenesis. Nature Biotechnology, 27(12), 1173-1175. DOI: 10.1038/nbt.1589
- December 23, 2009
- 08:11 AM
- 985 views
bacteria towing santa's wagon?
by 96well in Reportergene
Bacteria swim in water solution with a random movement resembling brownian motion. Imagine they while impacting randomly on a micro-gear. They will transfer some energy to the gear, but due to random movements the resultant will not provide any directed motion. However, you should remember from physics101 that a principle for the conservation of the force does NOT exist: imagine each tooth of the gear as a lever arm, and do design gears with asymmetric teeth. In this way, bacteria impacting on o........ Read more »
Sokolov, A., Apodaca, M., Grzybowski, B., & Aranson, I. (2009) Swimming bacteria power microscopic gears. Proceedings of the National Academy of Sciences. DOI: 10.1073/pnas.0913015107
- November 28, 2009
- 07:50 AM
- 623 views
Sorry, I'm experiencing a temporal drift toward metaphysics
by 96well in Reportergene
As we know it, our molecular life as individuals starts with a fusion between a female oocyte and a male sperm cell. When our mother was born, she got already in her ovary that small not-matured oocyte that than contributed to our first half cell at the time of ovulation several years later. Conversely, it is believed that our father at the time of conception, just donate our second half cell by means of a sperm cell (randomly) produced de novo.
Now, Zhuoru Wu and her colleagues at the Universi........ Read more »
Wu, Z., Luby-Phelps, K., Bugde, A., Molyneux, L., Denard, B., Li, W., Suel, G., & Garbers, D. (2009) Capacity for stochastic self-renewal and differentiation in mammalian spermatogonial stem cells. The Journal of Cell Biology, 187(4), 513-524. DOI: 10.1083/jcb.200907047
- November 25, 2009
- 08:27 AM
- 1,279 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
- October 28, 2009
- 01:35 PM
- 682 views
Chromophores, a new class of reporters
by 96well in Reportergene
A new Nature letter has the potential to abnormally extend (until extinction) the whole spectrum of reporter genes. So far, "reporters" were those genes coding for an easily detectable product (i.e., those coding for fluorescent or luminescent proteins). Wei Min and other Harvard's colleagues introduced a new technique, namely stimulated emission microscopy, that seems able to turn into mini-lasers any non-fluorescent light-absorbing molecule. It means that several chromophores, such as haemog........ Read more »
Min, W., Lu, S., Chong, S., Roy, R., Holtom, G., & Xie, X. (2009) Imaging chromophores with undetectable fluorescence by stimulated emission microscopy. Nature, 461(7267), 1105-1109. DOI: 10.1038/nature08438
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