Expression patterns of green fluorescent protein (GFP) after the intravenous injection with adenovirus vector in mouse organs / 대한내과학회지

ABSTRACT

BACKGROUND: The green fluorescent protein (GFP) from jelly fish, Aequorea victoria, has become a versatile reporter for monitoring gene expression in a variety of cells and organisms. Using GFP as a marker protein we studied whether there are any differencies in the expression patterns among organs in mouse after intravenous injection of adenovirus vectors with GFP gene. METHODS: Recombinant E1, E3-defective type 5 adenovirus vectors (2x10(8)/mouse) with CMV promoter and GFP gene were injected into mice via tail vein. On 3, 6, 9, 14, 21, 28 days after gene transfer, 5 mice per experiments were sacrificed by cervical dislocation and obtained liver, lung, heart, kidney, spleen, small intestine and bone. Half of them were examined by optical microscope after H-E stain. Another half were examined by fluorescent microscope after frozen section. Western blottings were done for each samples with anti-GFP monoclonal antibody and obtained GFP bands were quantitatively compared using Gel-Doc (Bio-Rad, USA) image analyzer. RESULTS: In all organs that we obtained, expression of GFPs are noticed 3 days after gene transfer and reached a maximum around 9th to 14th days, after then the intensities are slightly decreased but maintained until 28th days as determined by Western blotting. On fluorescent microscopic examination, GFPs are well and most frequently expressed on lung among all the examined organs. There are little expression of GFPs on liver parenchymal area around the sinusoids and central veins, although patchy expression of GFPs are observed along the liver capsules. GFPs are highly expressed around the splenic trabecula area but splenic pulp area, it is very sparsely expressed. GFPs are more frequently and highly expressed around the renal tubular area than gromerular area in kidneys. In small intestine, GFPs are expressed on mid portion of microvilli. GFPs are not expressed on myocardium except scanty expression on endocardium. Bone marrow showed GFPs but precise localization is difficult because bony spicules mashed bone marrow during the preparation of frozen section. No specific pathologic lesions possibly related with adenovirus administration are observed on microscopic examination of H-E stained specimens. CONCLUSIONS: GFPs can be detected in cells without the fixing and staining and a good marker to studying the kinetics and persistence of adenovirus mediated gene therapy. And there are different GFP expression patterns according to the organs after intravenous injection of adenovirus vectors with GFP gene in mouse.