Reportergenomics

In genetic engineering, a reporter gene is one that codes for a product that is easy to measure by recent technology. For instance, luciferase is easily detectable because it is luminescent.

The reason why reporter genes are worth blogging is that it is now quite simple to "copy and paste" single genes with known function(s) in and out of most any organism via recombinant DNA. Thus, combining reporter genes with other cellular elements, allows contextual, system-level, space-temporal measurements of molecular functions in living cells/organisms. The art of reportergenomic patchwork might facilitate understanding of our inner biology in the context of normal physiology as well as in disease. The very limitation of reporter design is the creativity of the researcher.

As technology evolves, the reporter toolbox increase in terms of new proteins and new detection methods. This blog collects updated information from separate fields like optics, physics, pharmacology, chemistry and medicine. More than 50 peer-reviewed journals are scrutinized to distill new knowledge and promote the development of this new field.

If you wish to get involved, or share any hot news about genetically-encoded knowledge, you can play the referee game, add your comment, put me in your blogrool/tweet, submit a guest post, or directly contact me at:info@reportergene.com.

Non-invasive in vivo optical imaging of the lacZ and luc gene expression in mice

The bacterial lacZ gene encoding for beta-galactosidase (beta-gal) is a common reporter gene used in cell cultures, and in the last years also in transgenic mice. Nonetheless, the absence of fluorigenic substrates usable in live animals greatly hampered the non-invasive follow-up of this reporter gene expression.

In the september issue of gene theraphy, the journal thath highlight the latest research into genetic and cell-based technologies to treat disease, Josserand V et al. from INSERM (the french institut of health), used far-red fluorescence for imaging beta-Gal expression in live cells in vitro or in vivo.
A selected substrate (beta-D-galactopyranoside) was used to monitor beta-Gal expression as a reporter of tumor growth, or of the physiological levels of an endogenous gene or of gene transfer in lung. A quantitative evaluation of this method as well as a comparison of its sensitivity with Firefly Luciferase-based bioluminescence was also performed. In vivo measurements showed that just only 1000 beta-Gal tumor cells located under the skin were detectable. In deeper organs like lung, as little as 5 ng of beta-Gal or Luciferase enzymes per mg of proteins were measured, confirming that both techniques reached similar sensibilities.

Nonetheless, quantitative comparison of beta-Gal levels measured with far-red imaging or with a standardized enzymatic evaluation after killing revealed that the 2D-fluorescent reflectance imaging method is submitted to a color-dependent disparity of the organs and cannot supply quantitative measurements but that a simple correction can be applied.

Red Russian Katushka

For deep imaging of animal tissues, the optical window favorable for light penetration is in near-infrared wavelengths, which requires proteins with emission spectra in the far-red wavelengths. In the 4th issue of Nature Methods, the journal that publish significant improvements to tried-and-tested techniques in the life sciences, Dmitry Shcherbo and colleagues from Moscow University, report a far-red fluorescent protein, named Katushka, which is brighter compared to the spectrally close HcRed or mPlum, and is characterized by fast maturation as well as a high pH-stability and photostability.
These unique characteristics should make Katushka a good reporter for visualization in living tissues. A monomeric version of Katushka, named mKate, was also generated: mKate is characterized by high brightness and photostability, and should be an excellent fluorescent label for protein tagging in the far-red part of the spectrum.

About

This is a very small blog advocating the advantages of genetically encoded approaches in the study of biomolecular processes. This site is not affiliated with any company, and the "blogger in chief" is an Italian postdoc working with nuclear receptors in European academic settings. You can read more about reportergene's philosophy or enjoy a cartoon illustrating how the blogger plays with luciferases and fluorescent proteins to better understand life from a molecular point of view.