Bioluminescence imaging after HSV amplicon vector delivery into brain.
BACKGROUND
Firefly luciferase (Fluc) has routinely been used to quantitate and analyze gene expression in vitro by measuring the photons emitted after the addition of ATP and luciferin to a test sample. It is now possible to replace luminometer-based analysis of luciferase activity and measure luciferase activity delivered by viral vectors directly in live animals over time using digital imaging techniques.


METHODS
An HSV amplicon vector expressing Fluc cDNA from an inducible promoter was delivered to cells in culture and into the mouse brain. In culture, expression of Fluc was measured after induction in a dose-dependent manner by a biochemical assay, and then confirmed by Western blot analysis and digital imaging. The vectors were then stereotactically injected into the mouse brain and Fluc expression measured non-invasively using bioluminescence imaging.


RESULTS
Rapamycin-mediated induction of Fluc from an HSV amplicon vector in culture resulted in dose-dependent expression of Fluc when measured using a luminometer and by digital analysis. In mouse cortex, a single injection of an HSV amplicon vector (2 microl, 1x10(8) transducing units (t.u.)/ml) expressing Fluc from a viral promoter (CMV) was sufficient to detect robust luciferase activity for at least 1 week. Similarly, an HSV amplicon vector expressing Fluc under an inducible promoter was also detectable in the mouse cortex after a single dose (2 microl, 1x10(8) t.u./ml) for up to 5 days, with no detectable signal in the uninduced state.


CONCLUSIONS
This HSV amplicon vector-based system allows for fast, non-invasive, semi-quantitative analysis of gene expression in the brain.
Pike
Lisa
L
Department of Neurology, Massachusetts General Hospital and Neuroscience Program, Harvard Medical School, Charlestown, MA 02129, USA.
Petravicz
Jeremy
J
Wang
Samuel
S
D000818:Animals; D001921:Brain / Q000201:enzymology*; D002460:Cell Line; D002522:Chlorocebus aethiops; D015870:Gene Expression / Q000187:drug effects; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors; D006801:Humans; D049409:Luciferases, Firefly / Q000235:genetics* / Q000378:metabolism; D049449:Luminescence; D008297:Male; D051379:Mice; D008819:Mice, Nude; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D018139:Simplexvirus / Q000235:genetics*; D020123:Sirolimus / Q000494:pharmacology; D014709:Vero Cells
D011994:Recombinant Proteins; D049409:Luciferases, Firefly; D020123:Sirolimus