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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March 28, 2011
06:12 AM
1,073 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 al. took fresh or frozen testes from 2- or 3-day-old GFP transgenic mice and grew some organ slices at 34 °C for about a month in a agarose petri dish with media (alpha-MEM) containing KnockOut serum replacement (KSR), B-27, AlbuMAX, HGF, Activin A, FSH, testosterone, BMP-4 and BMP-7 recombinant proteins, and bovine pituitary extracts. Actually, for the study they took advantage of two different GFP reporter mice as testes donor: Gsg2-GFP and Act-GFP. In vitro, the spermatogenesis looked fine both in terms of meiosis (Gsg2-GFP imaging) and in terms of generation of haploid cells (Act-GFP imaging) and eventually mature and functional sperm was produced. Sperm could be extracted, artificially inseminated in female mice, and healthy pups were generated. I'm sure the technique will further improve and diffuse, as it might be an attractive path for diagnostic and therapeutic techniques for male infertility. By the way, I think having the stem-to-sperm cell in the organ culture to be stably transfected with a foreign gene, could be quite simple to obtain. To make a transgenic mouse you would just need to by some spermfectamine. As Bill is saying, a transgenic in every bench.
--- ---/ link to primary source /--- ---
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

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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
1,078 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 chronic bronchopneumonia, but this time they skip commenting on.This study does not make dogs easier to spot at night, so fewer will get hit by cars, but illustrates the possibility to obtain conditional expression of a given gene in dogs, after administration of the drug doxycicline (the switch ON). This could be helpful for the generation of dog models of dominant genetic diseases (i.e., by inserting the disease gene). Honestly, I have some problems to think at dogs as 'research tools'. However, the proof of technology described in this paper could be useful on other fields. I'm thinking at the interface between man and dog relations, what about dogs with super-noses to better alert avalanche victims, gas spills or truffles (i.e., using olfactory receptor genes from other species that are less social like fruitflies)? This could made biomedical research useful for the society on branches other than the healthcare.

--- / original paper /--- --- ---
Kim, M., Oh, H., Park, J., Kim, G., Hong, S., Jang, G., Kwon, M., Koo, B., Kim, T., Kang, S., Ra, J., Ko, C., & Lee, B. (2011). Generation of transgenic dogs that conditionally express green fluorescent protein genesis DOI: 10.1002/dvg.20737

... 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
898 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 recombination of a BAC clone can help on this.

Collagen can be seen by histological staining (i.e., Tricrome Masson),
however this techniques is poor in rendering the space-temporal
development of cartilage maturation. By contrast, fluorescent imaging
with reporter mice identifies spots of cartilage maturation without
need to kill animals for dissection diagrams.
During cartilage maturation, the chondrocytes produce different types of collagen. Collagen, type 2, alpha 1 (Col2a1) is abundantly expressed in immature chondrocytes, but then is down regulated during maturation. In contrast, Collagen, type 10, alpha 1 (Col10a1) expression is absent in immature chondrocytes, but becomes highly expressed in mature hypertrophic chondrocytes. By following in real-time the expression of the two collagens in the developing bone, one could have an idea of the dynamics of cartilage maturation. This approach has been taken by Peter Maye and colleagues from the School of Dental Medicine (University of Connecticut) who developed a double transgenic reporter mouse in which collagen 2a1 promoter drives the expression of a cyan fluorescent protein (Col2a1-ECFP), and collagen 10a1 promoter drives the expression of a red fluorescent protein (Col10a1-mCherry). In a report published on Genesis, the authors illustrate how the fluorescence of the two reporters can be spectrally discriminated in vivo to efficacely monitor cartilage development. In the picture of a developing foetal mouse, long bones are maturing with extremities being the main center of ossification, while in the middle of the two extremities, cartilage is already differentiated. Conversely, the ribs are maturing following a dorsoventral pattern. Increasing the microscope magnification, it is possible to monitor single cartilage cells and identify the exact time at which an intermediate region switches its differentiation program.
--- --- / original reference / --- --- ---
Maye, P., Fu, Y., Butler, D., Chokalingam, K., Liu, Y., Florer, J., Stover, M., Wenstrup, R., Jiang, X., Gooch, C., & Rowe, D. (2011). Generation and characterization of Col10a1-mCherry reporter mice genesis DOI: 10.1002/dvg.20733

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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
722 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 hepatic estrogen receptor, hence explaining the high luminescence into the liver. This was particularly evident when animals were fed with soy-milk: surprisingly, we discovered that soy-phytoestrogens genistein and daidzein were playing a minor role in the liver, but something else in the food matrix was responsible for ER activation (Rando et al., Toxicol Appl Pharmacol. 2009 237(3):288-97). What was going on? Consider that soymilk is a high-protein nutrient. In a paper published yesterday in Cell Metabolism, we demonstrate that dietary amino-acids regulate the activity of the estrogen receptor (ER). As a result of ER activation, hepatic IGF-1 secretion in the blood is increased. IGF1 plays a key role in uterine physiology. We observed that calorie restriction or selective ablation of ER in the liver decreased blood IGF1 to levels inadequate for the correct progression of the estrous cycle in the uterus. In a few words, our results suggest that the liver connects feeding to reproduction by sensing the availability of dietary proteins, an essential building block for the growth of the fetus and the production of the milk.

Looking at the calendar, we think the moon is the
fertility gatekeeper. However, in mice, the ovulation
calculator to getting pregnant is more based
on dietary proteins (the cheese) sensed in the liver
like 'estrogenic hormones'.
Our data challenge the well established paradigm on how the nutritional status affects reproductive activity. It is current belief that the organs responsible for energetic homeostasis signal the nutritional status to the neuroendocrine system, which, in turn, regulates the activity of reproductive organs via gonadotropin synthesis and release (i.e., the hypothalamus-hypofisys-gonad axis). We have described a more direct signaling system where the estrogen receptor present in liver is the sensor of nutrient availability and controls, via IGF-1 synthesis, the progression of the estrous cycle in reproductive organs. In this context, the trigger of ER activity isn't its cognate hormone (the estrogen) but are some dietary proteins: in case of severe malnutrition, protein shortage cannot induce liver ER activity and its permissive signalling to reproductive organs. This can help to eludidate why anorexic women and women with liver disease have also troubles in their menstrual cycles. You can read an interview of my former boss on Science daily, but beware: the reporter says that proteins influence the expression of ER: this is not true, they influence its activity not its expression.
--- References to the original research --- ---

Rando G, Ramachandran B, Rebecchi M, Ciana P, & Maggi A (2009). Differential effect of pure isoflavones and soymilk on estrogen receptor activity in mice. Toxicology and applied pharmacology, 237 (3), 288-97 PMID: 19361539

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

... 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
1,034 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., dietary lipids), can activate liver gene expression by binding to cognate 'nuclear receptors' (NRs). In fact, nuclear receptors can modulate the transcriptional rates of genes involved in a broad range of metabolic response programs. Because i) liver gene expression is different between females and males, and ii) the liver metabolic landscape is sex dimorphic, one can argue NRs having some gender-dimorphic activities. However, in most studies only one sex is used (the male for some authors, the female for others), this introduces a bias in our knowledge and makes the sex differences pass unobserved.

Fat liver (steatosis or 'foie gras') is an hallmark of severe diseases including liver cancer. Derangements of nuclear receptor regulation affect lipid metabolism and contribute to the pathogenesis of liver diseases. This places nuclear receptors into the frontline for novel therapeutic approaches for a broad range of hepatic disorders. Detailing the extent of NR-mediated sex-dimorphic actions may be pivotal in preventive medicine and in the design of safer gender-specific therapeutic approaches.

Martin Wagner and colleagues, have recently reviewed the wide role of nuclear receptors in regulating liver metabolism. We complete this review with a second one focusing on the current knowledge of hepatic, sex-associated roles of individual NRs. We show that sexual dimorphism affects both the level of individual gene expression and the concerted activities of interconnected genes controlled by nuclear receptors. Finally, we discuss the hypothesis that this sex-dimorphic NR ‘interactome’ targets selected pathways in the liver, and deregulation of these pathways may favor the development of sex-biased diseases.
--- --- ---/ References /--- --- ---

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

... 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
912 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, is the possibility to engineer the drug protein to ameliorate its efficacy. Therefore, one could change few amino-acids to improve some binding activities, combine two protein moieties to obtain a new double-edged drug and so on, depending on the creativity of the medicinal chemist synthetic biologist.
Some proteins like interferon-α2a are effective against cancer, but are toxic also for other cells. Therefore, some engineering could maximize protein delivery to tumours while minimizing its bioavailibility to other organs. Gao and colleagues from Duke University, reasoned about a potential trick to do this job. The microvasculature of tumours is leaky compared with that of most normal tissues, therefore a drug complexed with a 'bulk' polymer would leak slowly from healthy microvessels, but quickly from the microvessels supplying tumours. As a proof of concept, they injected a normal green fluorescent protein in the mouse tail vein and monitored its accumulation in the tumour. Then, compared it with a second GFP in which its C-terminus was used as a docking site to grow a long polymer tail.

A post-translational protein splicing strategy was used to install a polymerization initiator at the C terminus, followed by in situ living methacrylate polymerization. The resulting 20-nm polymer–protein conjugate did indeed experience longer circulation times, and it leaked into tumours in mice 50 times more efficiently than the unmodified protein. In conclusion, the pharmacokinetic profile of therapeutic proteins could be significantly ameliorated by protein engineering.
Synthetic biology has been previously blamed to be nothing but a cool divertimento for junior scientists less serious than the senior medicinal chemist. The work of Gao and colleagues indeed demonstrate that plasmid juggling can provide us with better drugs, lets play the game again and again.
---/ reference /--- --- ---
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

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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
1,088 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.

RNA-binding motifs
UV-light irradiation has been used in the past to isolate RNAs bound to specific binding proteins (RBPs) but the discovery rate was inefficient because of problems in separating signal from background. Haffner and colleagues exploited photoactivable nucleosides as efficient and non-toxic crosslinkers that are well incorporated into the nascent RNA. Cells are grown in the presence of X mM of 4-thiouridine (4SU): 4SU-substituted RNA is 'activated' by UV light at 365 nm and specifically crosslinks to its RNA-binding partner proteins. Interestingly, if crosslinked and de-crosslinked (after immunoprecipitation of the RNA-bound-protein) 4SU is then mis-paired with guanine (G) instead of adenine (A) during the reverse transcription to cDNA. This allows the precise mapping of the binding sites by scoring the G/A transitions in the cDNA sequence. The researchers used this technique to define the binding motifs of 13 RBPs. (Haffner et al., Cell 2010)

Proteasome dynamics
Fluorescent reporters have already been used in the past to monitor proteosomal degradation, but effects on reporter degradation are difficult to separate from effects on reporter synthesis. Hamer and colleagues designed a reporter in which these processes can be easily distinguished. The green-to-red photoconvertible protein Dendra2 is fused to a form of ubiquitin that targets Dendra2 for proteosomal degradation. By observing only the photoconverted red form of the reporer, effects on its synthesis can be ruled out. The researchers used this reporter to monitor the cell type- and age-dependence of ubiquitin proteosome activity in living C.elegans. (Hamer et al., Nature Methods 2010)

---/ citations /--- ---

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

Hamer, G., Matilainen, O., & Holmberg, C. (2010). A photoconvertible reporter of the ubiquitin-proteasome system in vivo Nature Methods, 7 (6), 473-478 DOI: 10.1038/nmeth.1460

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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
474 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-heritable fashion. This new variety of protein opens several avenues for synthetic biology. Here, I spotlight two new non-natural reporter proteins.

a reporter for infrared spectroscopy
The non-natural amino-acid p-azido-l-phenylalanine (azF) vibrates in a window which is free from other protein vibration, this feature has been exploited to create a new infrared protein probe by incorporating azF into a G-coupled Protein Receptor (Rhodopsin). This allow to study GPCR's conformational changes by Fourier-transform infrared (FTIR) analysis. Upon the receptor activation, early conformational changes were observed.
---/ citation /--- --- ---
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

non-natural red bioluminescence
The non-natural amino-acid 4-methoxy-phenylalanine has been incorporated into the position F82 of the reporter aequorin, resulting in a significant (44 nm) red-shift of the aequorin bioluminescence.
---/ citation /--- --- ---
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 DOI: 10.1021/cb9002909

... 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
955 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 days of hormone replacement therapy with 10 different Selective Estrogen Receptor Modulators (SERMs).
This is a 2006: I checked the accuracy of the algorithm measuring the area under the curve by weighing small pieces of paper. My bench-mates are still laughing...We found that each SERM caused tissue-specific oscillations on ER transcriptional activity which were predictive of the drug structure (after rational extraction of meaningful molecular descriptors).

For more than one century, the measure of drug structure-activity relationships has been based on mathematical equations describing the interaction of the drug with its biological receptor. This is now obsolete, as 'binding' does not necessarily spell 'function'. Here, a systematic study of spatio-temporal effects is proposed as a measure of drug efficacy for the classification of pharmacologically active compounds.We conclude that the use of reporter mouse facilitates the identification of SERMs able to mimic the physiological estrous cycle and anticipates the possibility of a reverse approach in medicinal chemistry where the space-temporal plot of target activity drives the identification of subtle structure-activity relationships.

--- / citation /--- --- ---
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
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
Rando G, Arca S, Casiraghi E, Campadelli P & Maggi A (2009). Automatic Segmentation of Mouse Images. Proc 10th European Congress of International Society for Stereology, Bologna, Italy, 2009 link: abstract

... 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
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

February 17, 2010
01:11 PM
1,087 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 invasive and prone to promptly activate compensatory mechanisms in the newtork redundancy program, making the phenotypic interpretation a pain in the ASS. To better understand networks, one should be less invasive and knock-down edges other than nodes.
This new 'molecular edge surgery' can be done now, thanks to a reporter gene: the yeast Cytosine Deaminase (yCD) and to Po Hien Ear and Stephen Michnick and their Nature Methods paper. The yCD reporter can be succesfully splitted and used in classic protein-fragment complementation assays (PCA). The measure of reporter activity in this case is life or death, as yCD allows both positive and negative selection in yeast. So, if protein X interacts both with Y and Z, the molecular edge surgery is done performing first the selection with a library of mutant alleles X* screened against Z (selected for death: disruption of X-Z interaction), the same mutants are then screened against Y (selected for growth: retaining X-Y interaction).
At the end of the streamlined selection one should obtain 'edgetic' mutants preserving the X-Y edge but eliminating the X-Z edge of the protein-protein interaction network. The yCD-PCA assay should provide more granular insights into the relationship between loss of a specific edge on a network and the Phenotype and eventually trace those consequences back to the protein structure. This seems also a beautiful technique for prototyping drugs able to antagonize one 'bad' protein-protein interaction, while maintaining the 'good' one.

---/ citation /--- --- ---
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

... 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
1,018 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 nonscience. This at first may seem to eliminate one quarter of the time for talking science, but in the long term, gains from increased motivation more than make up for any losses.I'm a little bit skeptical about reading psychologicalities to increase self-motivation and productivity, as I think that to get things done the best is stopping reading this and making something concrete. However, I was in my bed after a long productive day and Molecular Cell is Molecular Cell period. The essay is also well-written. The author does not explain how to recognize symptoms of non-motivation, so I add here my personal thermometer: the measure of my degree of motivation is the laziness taken to duly complete my lab-book.

Post scriptum:
research blogging is a way to increase social connectedness in my case, but after a while I realized that also my competences were increased: I remember more the studies I blog.

---/ citation /--- --- ---
Alon, U. (2010). How to Build a Motivated Research Group Molecular Cell, 37 (2), 151-152 DOI: 10.1016/j.molcel.2010.01.011

... 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
981 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 changes in the chromophore environment. The camel advantage is that, in contrast to conventional antibodies, small, stable Nanobodies are functional in living cells. Exploting this feature, a beautiful experiment was done: a NB 'Enhancer' able to shift the absorption spectra of GFP was first confined into nucleus by means of a lamin-B1 tag. This transformed it in a ratiometric biosensor for subsequent nuclear traslocation of GFP-'transcription factors'. In conclusion, the NB ability to modify the conformation of proteins was demonstrated in living cells anticipating new opportunities for bioimaging studies of cell signaling.

---/ citation /--- --- ---

Kirchhofer, A., Helma, J., Schmidthals, K., Frauer, C., Cui, S., Karcher, A., Pellis, M., Muyldermans, S., Casas-Delucchi, C., Cardoso, M., Leonhardt, H., Hopfner, K., & Rothbauer, U. (2009). Modulation of protein properties in living cells using nanobodies Nature Structural & Molecular Biology, 17 (1), 133-138 DOI: 10.1038/nsmb.1727... 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
1,100 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 rabbits are promising models for cardiovascular research.... 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
1,403 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

January 4, 2010
12:00 AM
1,245 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 blogged about a new method to detect more than 30 reporters simultaneously: instead of coding for a protein, each reporter was coding for a very similar mRNA. The difference between each mRNA was just a 5-nt lenght: i.e. reporter mRNA #1 was 100 nt long, mRNA #2 was 105 nt, mRNA #3 was 110 nt and so on. Once transfected, the library of reporters was reverse-transcribed with the same primer pairs and then analyzed and semi-quantitated by capillary electrophoresis. Today, forget electrophoresis, and think parallel sequencing. The same plasmid backbone can be coupled to an oligo barcode! The work of Patwardhan et al. open this way: at Washington, they were bothering about the effect of each possible mutation in a promoter, one base at a time. By means of synthetic saturation mutagenesis, mutant promoters were obtained by parallel solid synthesis and then released into solution, resulting in a complex library. The trick was that each oligo in the library was designed to include a unique barcode sequence downstream of the promoter's transcription start site. One library aliquot was used in an in vitro transcription reaction to make RNA from the promoter (subsequently reversely transcribed). The second aliquot was amplified using PCR only. High-throughput illumina sequencing has been carried out and quantitative comparison between the RNA barcode and the DNA barcode was used to give a measure of promoter effectiveness in a way that seems to me more clean than our classical beta-gal normalization. Ok, at the moment the technology is applied only in vitro, but the concept is clear: next generation sequencing can help the reportergenomic community. Give me a barcode plasmid and I'm going to make the reporter mouse.... 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
1,091 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 one tooth side, will transfer more energy than bacteria impacting on the other side. And here comes the magic: motion!

By conventional photolitography, Sokolov and colleagues from Argonne National Laboratory, report on PNAS the design of microscopic gears (6 ug mass) able to extract useful work from swimming Bacillus subtilis sworms. According to their calculations, few hundred bacteria work together in order to turn the gear at a velocity of 1-2 rpm, generating femtowatt power.

Here the concept is that brownian motion can generate directed motion (in non-equilibrium conditions). Since Middle Age, oxen, horses and other animals have been used to generate power to move mills and other machines (in non-equilibrium conditions). Bacteria, and perhaps other floating cells/molecules might provide new energy sources for new micro-machines. Applications are wide and may impact also molecular and synthetic biology. What about designing proteins with asymmetrical teeth? Merry Christmas.

--------citation----------
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

... 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
863 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 University of Texas Southwestern Medical Center in Dallas shows that when stem cells in the rat testes are at developmental crossroad, they have two choices: procrastinate becoming again stem cells or differentiate into sperm. The researchers found that when grown in the same culture medium, stem cells gave rise to more stem cells that sperm. Thus, if we want for a while being fascinated by predestination, it is conceptually possible that both the female oocyte and the male stem-to-sperm cell that initiated our life were waiting each other from the very beginning of our parent's life.... 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
1,593 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

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