Abstract
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 β-glucuronidase (mβG) as a reporter gene to hydrolyze a nonfluorescent glucuronide probe (fluorescein di-β-D-glucuronide, (FDGlcU) to a highly fluorescent reporter to assess the location and persistence of gene expression. A functional β-glucuronidase (βG) 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 (βG) on CT26 tumors. The fluorescent intensity in βG-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 βG vector into carcinoma xenografts. Importantly, mβG did not induce an antibody response after hydrodynamic plasmid immunization of Balbβc mice, indicating that the reporter gene product displayed low immunogenicity. A membrane-anchored form of human βG also allowed in vivo imaging, demonstrating that human βG 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.
| Original language | English |
|---|---|
| Pages (from-to) | 565-574 |
| Number of pages | 10 |
| Journal | Gene Therapy |
| Volume | 14 |
| Issue number | 7 |
| DOIs | |
| Publication status | Published - Apr 2007 |
| Externally published | Yes |
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ASJC Scopus subject areas
- Genetics
Cite this
Gene expression imaging by enzymatic catalysis of a fluorescent probe via membrane-anchored β-glucuronidase. / Su, Y. C.; Chuang, K. H.; Wang, Y. M.; Cheng, C. M.; Lin, S. R.; Wang, J. Y.; Hwang, J. J.; Chen, B. M.; Chen, K. C.; Roffler, S.; Cheng, T. L.
In: Gene Therapy, Vol. 14, No. 7, 04.2007, p. 565-574.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Gene expression imaging by enzymatic catalysis of a fluorescent probe via membrane-anchored β-glucuronidase
AU - Su, Y. C.
AU - Chuang, K. H.
AU - Wang, Y. M.
AU - Cheng, C. M.
AU - Lin, S. R.
AU - Wang, J. Y.
AU - Hwang, J. J.
AU - Chen, B. M.
AU - Chen, K. C.
AU - Roffler, S.
AU - Cheng, T. L.
PY - 2007/4
Y1 - 2007/4
N2 - 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 β-glucuronidase (mβG) as a reporter gene to hydrolyze a nonfluorescent glucuronide probe (fluorescein di-β-D-glucuronide, (FDGlcU) to a highly fluorescent reporter to assess the location and persistence of gene expression. A functional β-glucuronidase (βG) 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 (βG) on CT26 tumors. The fluorescent intensity in βG-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 βG vector into carcinoma xenografts. Importantly, mβG did not induce an antibody response after hydrodynamic plasmid immunization of Balbβc mice, indicating that the reporter gene product displayed low immunogenicity. A membrane-anchored form of human βG also allowed in vivo imaging, demonstrating that human βG 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.
AB - 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 β-glucuronidase (mβG) as a reporter gene to hydrolyze a nonfluorescent glucuronide probe (fluorescein di-β-D-glucuronide, (FDGlcU) to a highly fluorescent reporter to assess the location and persistence of gene expression. A functional β-glucuronidase (βG) 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 (βG) on CT26 tumors. The fluorescent intensity in βG-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 βG vector into carcinoma xenografts. Importantly, mβG did not induce an antibody response after hydrodynamic plasmid immunization of Balbβc mice, indicating that the reporter gene product displayed low immunogenicity. A membrane-anchored form of human βG also allowed in vivo imaging, demonstrating that human βG 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.
UR - http://www.scopus.com/inward/record.url?scp=33947388445&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=33947388445&partnerID=8YFLogxK
U2 - 10.1038/sj.gt.3302896
DO - 10.1038/sj.gt.3302896
M3 - Article
C2 - 17235292
AN - SCOPUS:33947388445
VL - 14
SP - 565
EP - 574
JO - Gene Therapy
JF - Gene Therapy
SN - 0969-7128
IS - 7
ER -