Secreted Luciferase from Gaussia Copepod

According to Schätzing's "The Swarm" (2004) the seas and their inhabitants begin a violent revolution against mankind.
On the other corner, according to some researchers, in the ocean there is a Swarm of useful molecules; for instance in reportergene field, Gaussia Luciferase (GLuc) is a pretty new chemiluminescent reporter encoded by the gene isolated from the marine copepod Gaussia princeps.
This luciferase, which does not require ATP, catalyzes the oxidation of the substrate coelenterazine in a reaction that emits light (peak at 470 nm). Compared to more established luciferases like firefly (Fluc) or Renilla (Rluc), GLuc is secreted, so after synthesis doesn’t remain in the cytoplasm. Principal differences of secreted reporter genes versus intracellular reporters are:

• easy access in the cell culture supernatant,
• no requirement for complex cell lysis and sample preparation,
• cheaper profiling of expression kinetics using single culture and
• could serve as model product proteins for generic biopharmaceutical manufacturing scenarios.

There are some interests to translate secreted-reporters also in vivo, where they can be monitored in blood/urine and eventually complement more expensive in vivo bioluminescence imaging approaches (ok, I’m a fun of in vivo imaging so I can not emotionally support very well this trend). Anyway, recently I’ve observed some nice job. For instance, last year in the synthetic biology field, Weber and colleagues from the Swiss Federal Institute of Technology have implemented in a mouse a synthetic time-delay gene circuit (composed of a genetic switch, diode, capacitor, resistor, transistor and lamp) in which the lamp was the secreted SEAP reporter gene. Now, Wurdinger and colleagues from Harvard states in Nature Methods, to be able to assay in just 5 μL of blood (a little drop) the presence of secreted Gaussia Luciferase, demonstrating that Gluc assay is 1000 fold more sensitive than SEAP assay. Does Gaussia GLuc foster advances in cellular circuitries, therapeutic reprogramming (diabetes) and/or manufacturing of protein therapeutics? We are waiting for ocean's tsunami...

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Weber, W., Stelling, J., Rimann, M., Keller, B., Daoud-El Baba, M., Weber, C.C., Aubel, D., Fussenegger, M. (2007). A synthetic time-delay circuit in mammalian cells and mice. Proceedings of the National Academy of Sciences, 104(8), 2643-2648. DOI: 10.1073/pnas.0606398104

Have fun with luciferase

Some weeks ago I was wondering about why to build a dinosaur, blinking at Jurassic Park as the forerunner of DNA-based entertainment companies.

One question now: it is possible to have fun with luciferases? And possibly grow a new market pipeline? Probably YES. "Prolume" is a biotechnology company whose core business is based upon newly discovered genes from deep water marine bioluminescent organisms. This has broad applications for biomedical research, drug discovery, and entertainment. Such company claims to sustain its own research thanks to some patents covering the use of bioluminescent proteins in consumer products, including foods, beverages, toys and novelties. I usually observe lot of brainy researchers complaining about grants starvation. Although I don't know whether Prolume gears (and research) really glow, such machine is a good example of self-sustainability and of proactive behavior.

Two fuddy-duddies joined (on the bummel)

Yeast and LacZ, together. The first one is probably the first model organism: some thousand years ago, a woman has been the first biotechnologist (employing yeast she made the bread), so the referee tried to make some bread in vitro (he got the beer), then other researchers tried to exploit Saccharomices Cerevisiae but only minor applications like two-hybrid assay were reported.

Also the product of the gene LacZ (beta-galactosidase) is still ubiquitous in research, but (it seems) it doesn't work very well with yeast. Now, de Almeida and colleagues from Manchester University claims on Yeast 2008; 25: 71-76 a cell-based quantitative lacZ assay compatible for high-throughput screening. They optimized a procedure just marketed by Promega (Beta-Glo) and Applied Biosystems (Galacto-Lightplus); briefly, the substrate Xgal is substituted by so-called 6-O-beta-galactopyranosyl-luciferin. Such molecule, is cleaved by beta-gal and release D-luciferin. Luciferin, in presence of ATP is oxidized by another reporter (firefly luciferase) and shed light. Results: three order of magnitude and less than 100 yeast cells visualized. Suggested for lazy people that doesn't want to change such fuddy-duddy lacZ with a brighter stand-alone luciferase.

de Almeida, R.A., Burgess, D., Shema, R., Motlekar, N., Napper, A.D., Diamond, S.L., Pavitt, G.D. (2008). ASaccharomyces cerevisiae cell-based quantitative β-galactosidase assay compatible with robotic handling and high-throughput screening. Yeast, 25(1), 71-76. DOI: 10.1002/yea.1570

Where did the scientific method go? Here it is.

A letter to Nature Biotechnology by Noseda and McLean, points the finger to "A lack of documented methodology and information" that is essential to faithfully reproduce the science claimed in the manuscript. Scientific communication still relies on traditional print journals, which can not capture the intricacy of contemporary life science research. Nowadays we live in 2.0 times. Go youtube! I've just discovered JoVE (Journals of Visualized Experiment): a video journal for biological research. Employing visualization to increase reproducibility and transparency in biological sciences, such site is the best response to Noseda and McLean to aid bench-to-bedside translation.

Tango Assay: Rubik is simpler.

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 receptor, it interacts with the #3b partner protein bringing its twin protease #3a. The protease cuts its cognate aminoacid motif in #2. As a results of such artificial signaling cascade (that emulates Drosophila's Notch receptor) you get the release of a exogenous transcription factor #2a that drives the expression of your favorite #1 reporter gene. This is "The genetic design of signaling cascades to record receptor activation" Barnea et al.

It seems to me. 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.

Barnea, G., Strapps, W., Herrada, G., Berman, Y., Ong, J., Kloss, B., Axel, R., Lee, K.J. (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

Launched!

After up to three months the beta version expires today.
Wellcome Reportergene!

The blog Reportergene efforts to illustrate how biotechnology can exploit recombinant DNA to employ reporter genes as genetic engineering tools, by reviewing research highlights and discussing limitations and advantages of today's molecular biology. Moreover, at the end of the week, an "outlier" post will propose hints to have fun with those genomic fireworks.

Who writes this blog?
96well, is a 28 year old molecular pharmacologist that daily deals with reporter genes and reporter animals. He likes Risotto and Chianti red wine.

Lab-Life meets Thrillers

Reportergene is read by biotechnology professionals. This is what seems to tell me Google analytics: in effect during weekends visits drop down. Right. No posts about dynamic ranges on Friday evening. Relax yourself. I'm going to read again a novel written more than 10 years ago: The Third Twin, by Ken Follett. Why? Because I'm coming to apply to Roche's Imaginality the opportunity to win a thriller movie every month. January is The Third Twin. I discovered KF when I was very young, good novels for spare time. Young scientist Jeannie Ferrami discovers two young men, law student Steve and convicted murderer Dan, appear to be identical twins. Yet they were born on different days, to different mothers. Jeannie investigates, but shadowy forces retaliate and Steve is accused of a terrible crime...

Toward 3D luciferase imaging: bioluminescence tomography (BLT)

2008 is reportergenic! Take volume 13 of Frontiers in Bioscience of January 1, 2008. Ge Wang and colleagues of Virginia Tech, offer a nice overview of development of 3D bioluminescence tomography.
From pionieristic work in 2002, BLT aims to extend the 2D view of bioluminescent imaging toward a third direction: the deep. In such overview, the authors explains the history of bioluminescence imaging (BLI) from Berthold to Xenogen, elaborate on multispectral extensions of BLT and describes associated image unmixing and normalization techniques.

Wang, G. (2008). Overview of bioluminescence tomography-a new molecular imaging modality. Frontiers in Bioscience, 13(13), 1281. DOI: 10.2741/2761

Noninvasive Bioimaging of Laboratory Animals

2008 is reportergenic! The first volume of the year of the ILAR Journal is focused on laboratory animal imaging. According to the introduction:

the primary objective of this issue [...] is to provide readers with a compendium of reviews that describe the methods, the limitations, and a few examples of the most common applications of small animal imaging to human disease.

The ILAR Journal is the quarterly, peer-reviewed publication of the Institute for Laboratory Animal Research (ILAR). The table of contents is very well garnished: Design and Setup of Animal Imaging Centers, Anesthesia, Rodent Cardiac Imaging, Imaging with Magnetic Resonance Microscopy, Small Animal PET Imaging, Multiphoton Microscopy. And this are only the first five papers, then we can find also Multiplexing with Multispectral Imaging, Brain Imaging, Noninvasive Bioluminescence Imaging and High-Resolution fMRI Maps. If you are wonder what are the relations with reporter genes, you need seriously to read such issue: to date reportergenes for fluorescence, bioluminescence and PET are well known, while synthethic and biological reporter genes for MRI are on the road of development. Putting one reporter gene in one mouse you obtain a reporter mouse. With molecular imaging, you can observe reporter gene expression noninvasively in vivo. The imaging modalities available for use with laboratory animals provide a means to explore the molecular mechanism of several diseases, minimize many of the limitations of static tissue-based techniques, and, most importantly, decrease the numbers of animals required. In fact, depending on the application, it is possible to reduce the number of animals required per study by as much as 80% to 90%. I think this is a concrete and viable scenario for Reducing, Replacing and Refining the use of animals in research.

Why to build a Dinosaur?

Some years ago, when I was starting my education in genetic engineering, I read Jurassic Park. I remember clearly the billionaire John Hammond (CEO of InGen) precognizing that the best molecular biologist will be hired from an entertainment company and not from some big pharma. I asked myself how a nucleic acid could amuse the big masses, and I hypothized a DNA gem. Then naively I refused my idea: how to dress such sticky long chain? Probably I'm so stupid, because in the same period I was studying some boring crystal forms like B-DNA, A-DNA, Z-DNA...

Nowadays, while reading Haunted by C.Palahniuk, I meet Lady Baglady: she has got a diamond ring, and such diamond is a CRYSTAL from her died husband! Just because the truth overcomes the fiction, now you can buy online DNA jewel made with (your own | your husband | your pet) deoxyribonucleic acid. If the best biotechnology professional will be employed in the entertainment field, to date I'm not such one.

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