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(PP162) Uneven distribution of green fluorescence positive cells within multicell spheroids following adenoviral transduction.
Rhee, Juong*,1, Gu, Yeun2, Lee, Yong3, 1 Radiation Oncology, Baltimore, MD, U.S.A.2 Graduate School of Helath Science, Suzuca, Mie, Japan3 Surgery, Pittsburgh, PA, U.S.A.
ABSTRACT- We studied the efficiency of adenoviral transduction of a green fluorescence protein (GFP) and examined its spatial distribution within multicell spheroids. We used an adenoviral backbone plasmid (pAdEasy-1) that contains a GFP gene and produced a proliferation-deficient adenovirus. DU145 cells were grown into spheroids using a spinner flask and then infected with the adenovirus. Expression of GFP was monitored with either a confocal fluorescence microscope using live spheroids or an epi-fluorescence microscope using cryostat sections of fixed spheroids. Expression of GFP was observed only in the superficial layer of spheroids while inner layers were unaffected, resulting in low transduction efficiency. This low efficiency was hardly improved by several attempts. Instead we observed an interesting phenomenon in the spheroids. The spatial distribution of GFP-positive cells within the spheroids gradually became altered as time passed. Utilizing a Hoechst stain, a known spatial marker used for localizing cells within a spheroid, we confirmed the aforementioned alteration of cell positioning. We also found evidence that stained cells in the superficial layer gradually became mixed with cells in inner layers that had been stained weekly. It is likely that cells in spheroids constantly re-localize within the spheroid and alter their spatial positions within a spheroid.
Key words: Adenoviral transduction, Transduction efficiency, Multicell spheroid, Cell relocalization
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