Work

956. Imaging of Radiation-Inducible Promoters Using a Naturally Secreted Luciferase from the Marine Copepod Gaussia princeps

Year: 2006

Type: article

Abstract: Ionizing Radiation (IR)-inducible promoters used as genetic switches for cancer therapy are promising tool for controlling therapeutic gene expression. Here we describe the use of a naturally secreted luciferase from the marine copepod Gaussia princeps (Gluc) for monitoring the activity and induction levels of different IR- sensitive promoters. The following promoters were tested: wild-type p53-activated fragment 1 (WAF1, 2.4 kb); early growth response factor (Egr-1, 550 bp); four tandem repeats of the transcriptional factor nuclear factor-kB (4NF-kB, 400 bp); and a combination of both Egr-1 and 4NF-kB promoters. These promoters were cloned upstream of the humanized Gluc cDNA in a plasmid containing the eGFP gene under the control of an immediate-early herpes simplex virus type-1 promoter. Vero2-2 (African green monkey kidney cells) were transfected with each of these constructs and irradiated at different doses of g-rays. Activity of these promoters and their response to radiation was monitored overtime by taking an aliquot of the cell-free conditioned medium, adding coelenterazine, the Gluc substrate, and measuring bioluminescence. At-least two- fold induction was detected with each promoter as observed by bioluminescence imaging of Gluc. This system can be used to monitor, in real-time, the activity as well as the effectiveness of radiation-inducible promoters to drive the expression of toxic genes for cancer therapy. Ionizing Radiation (IR)-inducible promoters used as genetic switches for cancer therapy are promising tool for controlling therapeutic gene expression. Here we describe the use of a naturally secreted luciferase from the marine copepod Gaussia princeps (Gluc) for monitoring the activity and induction levels of different IR- sensitive promoters. The following promoters were tested: wild-type p53-activated fragment 1 (WAF1, 2.4 kb); early growth response factor (Egr-1, 550 bp); four tandem repeats of the transcriptional factor nuclear factor-kB (4NF-kB, 400 bp); and a combination of both Egr-1 and 4NF-kB promoters. These promoters were cloned upstream of the humanized Gluc cDNA in a plasmid containing the eGFP gene under the control of an immediate-early herpes simplex virus type-1 promoter. Vero2-2 (African green monkey kidney cells) were transfected with each of these constructs and irradiated at different doses of g-rays. Activity of these promoters and their response to radiation was monitored overtime by taking an aliquot of the cell-free conditioned medium, adding coelenterazine, the Gluc substrate, and measuring bioluminescence. At-least two- fold induction was detected with each promoter as observed by bioluminescence imaging of Gluc. This system can be used to monitor, in real-time, the activity as well as the effectiveness of radiation-inducible promoters to drive the expression of toxic genes for cancer therapy.

Source: Molecular Therapy

DOI: 10.1016/j.ymthe.2006.08.1048

Authors: Christian E. Badr, Pierre Zalloua, Xandra O. Breakefield, Bakhos A. Tannous

Institutions: Harvard University, Massachusetts General Hospital, American University of Beirut

Language: English

Topic: bioluminescence and chemiluminescence research

Subfield: Molecular Biology

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

SDG: Good health and well-being

Open Access status: hybrid

Estimated APC paid: $4,500