Gene expression imaging by enzymatic catalysis of a fluorescent probe via membrane-anchored beta-glucuronidase.
Development of nonimmunogenic and specific reporter genes to monitor gene expression in vivo is important for the optimization of gene therapy protocols. We developed a membrane-anchored form of mouse beta-glucuronidase (mbetaG) as a reporter gene to hydrolyze a nonfluorescent glucuronide probe (fluorescein di-beta-D-glucuronide, (FDGlcU) to a highly fluorescent reporter to assess the location and persistence of gene expression. A functional beta-glucuronidase (betaG) was stably expressed on the surface of murine CT26 colon adenocarcinoma cells where it selectively hydrolyzed the cell-impermeable FDGlcU probe. FDGlcU was also preferentially converted to fluorescent probe by (betaG) on CT26 tumors. The fluorescent intensity in betaG-expressing CT26 tumors was 240 times greater than the intensity in control tumors. Selective imaging of gene expression was also observed after intratumoral injection of adenoviral betaG vector into carcinoma xenografts. Importantly, mbetaG did not induce an antibody response after hydrodynamic plasmid immunization of Balb/c mice, indicating that the reporter gene product displayed low immunogenicity. A membrane-anchored form of human betaG also allowed in vivo imaging, demonstrating that human betaG can be employed for imaging. This imaging system therefore, displays good selectivity with low immunogenicity and may help assess the location, magnitude and duration of gene expression in living animals and humans.
Su
Y-C
YC
Faculty of Biomedical Science and Environmental Biology, Kaohsiung Medical University, Kaohsiung, Taiwan.
Chuang
K-H
KH
Wang
Y-M
YM
Cheng
C-M
CM
Lin
S-R
SR
Wang
J-Y
JY
Hwang
J-J
JJ
Chen
B-M
BM
Chen
K-C
KC
Roffler
S
S
Cheng
T-L
TL
D000818:Animals; D002384:Catalysis; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000201:enzymology*; D005434:Flow Cytometry; D005452:Fluoresceins / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism*; D015870:Gene Expression; D017930:Genes, Reporter; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000378:metabolism; D005966:Glucuronidase / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D008856:Microscopy, Fluorescence; D009374:Neoplasms, Experimental
D005452:Fluoresceins; D005456:Fluorescent Dyes; C041357:fluorescein glucuronide; D005966:Glucuronidase