Tape your luciferase assay

When I was undergraduate, I spent months performing hundreds of luciferase assays in individuals 1.5 mL tubes. Such situation induced me to evolve in 96 wells and became a microplate supporter. I developed a sort of fet1sh for high-throughput system, and my secret dream was one flexible robotic harm to continously process my assays. The Douglas Scientific Nexar array tape-based automation instrument it's even better, in fact such platform replaces individual microplates with wells embossed into a continuous plastic tape. The instrument is composed of quick-release interchangeable modules in order to combine precision liquid handling, array sealing, and assay reading into one flexible tape based platform. Tape it! www.global-array.com

Ancient gene resurrection

Two months ago I was wondering why to build a dinosaur? In a more general way the problem can be stated as: Why to resurrect an extinct species? Even if I don't have yet anwers, some scientists started really to deal with a sister question: How to resurrect an extinct gene? Obviously to do that, they used also reporter gene technology (lacZ). I don't write more about it because there is yet in the blogosphere a good review. Read tasmanian park at the blog of Giovanna Di Sauro.

Niche blog i like in science #1

Reportergene aims to be a niche where passionates in quantitative biology meet and share their opinion about the trends of recent research. I like niches because only there it is possible to find out what are you looking for, OUT OF THE NOISE. Let me share with you a niche blog in science that share the spirit of Reportergene.

blog.khymos.org (dedicated to molecular gastronomy)

Deep tissue imaging and optical windows

It is well known that the in vivo measure of optical (fluorescent and bioluminescent) reporters is hampered by tissue absorption at wavelenghts minor than 600 nm. Today, the most important trend to minimize tissue absorption is to design new red-shifted reporters and even infrared reporters. Another complementary approach is to "window" some tissue and gain access to desired cells. Sometimes this could appear very cruel, like in case of the skin flap approach proposed by Huang and collaborators in 2007. Now, a new eclectic approach is presented by Stephan Speier et colleagues in the May issue of Nature Medecine.

In their technical report, the swedish scientists from Karolinska, isolated pancreatic islets from transgenic mice expressing enhanced GFP under control of the rat insulin-1 promoter, then transplanted and engrafted the islets in the anterior chamber of the eye in order to noninvasively image vascularization, beta cell function and death at cellular resolution (microscope and confocal scanner from Leica Microsystem). In a few words, the researcher used the cornea as a natural body window that has the further advantage of being an immune-privileged site. The eye window and more classic skin windows protocols are available at Nature protocols

• Speier, S., Nyqvist, D., Cabrera, O., Yu, J., Molano, R.D., Pileggi, A., Moede, T., Köhler, M., Wilbertz, J., Leibiger, B., Ricordi, C., Leibiger, I.B., Caicedo, A., Berggren, P. (2008). Noninvasive in vivo imaging of pancreatic islet cell biology. Nature Medicine, 14(5), 574-578. DOI: 10.1038/nm1701
• Huang, Q., Acha, V., Yow, R., Schneider, E., Sardar, D.K., Hornsby, P.J. (2007). Bioluminescence measurements in mice using a skin window. Journal of Biomedical Optics, 12(5), 054012. DOI: 10.1117/1.2795567
  

Reporter gene imaging of stem cells [movie]

The discovery of human embryonic stem cells (hESCs) has dramatically increased the tools available to medical scientists interested in regenerative medicine. However, direct injection of hESCs, and cells differentiated from hESCs, into living organisms has thus far been hampered by significant cell death, teratoma formation, and host immune rejection. Understanding the in vivo hESC behavior after transplantation requires novel imaging techniques to longitudinally monitor hESC localization, proliferation, and viability.

Wilson Kitchener and colleagues from Stanford University School of Medicine cooked a 10 minutes movie in which they show how in each stem cell, transcription and translation of luciferase into bioactive light-emitter was detected with sensitive, noninvasive instrumentation (CCD cameras from Caliper) directly in alive, sleeping animals.

• Kitchener, W., Yu, J., Lee, A., Wu, J. (2008). In vitro and in vivo bioluminescence reporter gene imaging of human embryonic stem cells. Journal of Visualized Experiments, 2008(14) DOI: 10.3791/740

Luciferase sheds light on DNA sequencing

DNA sequencing is one of the most important platforms for the study of biological systems today, so Reportergene decided to devote its 50th post to a curious technology rebound of reporter genes. Sequence determination is most commonly performed using dideoxy chain termination technology. Recently, pyrosequencing has emerged as a new sequencing methodology. Pyrosequencing is a DNA sequencing technique that is based on the detection of released pyrophosphate (PPi) during DNA synthesis. In a cascade of enzymatic reactions, visible light is generated that is proportional to the number of incorporated nucleotides. The cascade starts with a nucleic acid polymerization reaction in which inorganic PPi is released as a result of nucleotide incorporation by polymerase. The released PPi is subsequently converted to ATP by ATP sulfurylase, which provides the energy to luciferase to oxidize luciferin and generate light. Because the added nucleotide is known, the sequence of the template can be determined in a pyrogram.

The technique and methodology of Pyrosequencing from Methods in Molecular Biology, is available for free at the Journal of Visualized Experiment, the "youtube" of biologist we previously reviewed.