Fiber-optic monitoring coupled with confocal microscopy for imaging gene expression in vitro and in vivo.
Detection of fluorescent signals in living cells is a common and powerful technique used to monitor gene expression for multiple biomedical applications. A disadvantage of this approach in vivo, is the limited accessibility for long-term monitoring of the fluorescent signals within organs in living animals. Because of the multiple applications of gene expression monitoring through fluorescent signals, innovative methods for readout are required. We developed a strategy combining gene transfer, fiber-optic or endoscope monitoring, and confocal microscopy for the brain interstitial or cavitary endoscopic visualization of the efficacy of gene delivery and expression in vivo. The approach is also effective in vitro and can be applied to multiple organs in vivo. We show an example of the detection of green fluorescent protein (GFP)-emitted fluorescence following the administration of recombinant GFP-expressing adenovirus or implantation of rat C6 glioblastoma cells infected with the recombinant GFP adenovirus into the rat hippocampus of chronically cannulated rats. The results show that fiber-optic monitoring coupled with confocal microscopy in gene transfer studies is a practical approach that results in a direct, efficient, rapid, and sensitive visualization of fluorescent signals in the brain. This allows for the continuous real-time in vitro or in vivo brain monitoring of gene expression, accurate anatomical localization, multiple experimental manipulations in the same subject or preparation, while no sacrifice of the animal is required to monitor the efficacy of gene transfer and/or expression.
Ilyin
S E
SE
Division of Molecular Biology, School of Life and Health Sciences, University of Delaware, Newark, DE 19716-2590, USA> silyin@prius.jnj.com
Flynn
M C
MC
Plata-Salamán
C R
CR
D000818:Animals; D005336:Fiber Optic Technology / Q000295:instrumentation / Q000379:methods*; D015870:Gene Expression / Q000502:physiology*; D018014:Gene Transfer Techniques / Q000295:instrumentation*; D005822:Genetic Vectors; D005909:Glioblastoma; D049452:Green Fluorescent Proteins; D007202:Indicators and Reagents / Q000032:analysis; D008164:Luminescent Proteins / Q000032:analysis; D008297:Male; D018613:Microscopy, Confocal / Q000295:instrumentation / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods; D055100:Optical Fibers; D051381:Rats; D017208:Rats, Wistar; D014407:Tumor Cells, Cultured / Q000821:virology
D007202:Indicators and Reagents; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins