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.
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.
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