MicroPET imaging of Cre-loxP-mediated conditional activation of a herpes simplex virus type 1 thymidine kinase reporter gene.
Site-specific recombination tools such as the Cre-loxP system are used to create animal models where conditional gene deletion/activation studies are required. In the current proof of principle study, we have demonstrated that a PET reporter gene (PRG), the herpes simplex virus type 1 thymidine kinase (HSV1-tk), can be made to remain silent and can be activated by Cre-loxP-mediated recombination in cell culture and in living mice. An adenovirus carrying a silent HSV1-tk was tail-vein injected (1 x 10(9) PFU) in six transgenic mice that express Cre recombinase in their liver (Cre+) and in four control mice (Cre-). The liver-specific expression of the PRG in Cre+ mice was detected in the microPET following injection of the reporter probe, 9-[4-fluoro-3-(hydroxymethyl)butyl]guanine ([(18)F]-FHBG). The [(18)F]-FHBG accumulation in the liver in terms of percent-injected dose per gram of tissue was 7.72+/-1.13 for the Cre+ mice and 0.10+/-0.02 for the Cre- mice (P<0.05) 48 h after adenoviral injection. These results were further validated by quantitative RT-PCR, western blotting and by in vitro assays for herpes simplex virus type 1 thymidine kinase enzyme activity. Thus by using the Cre-loxP system it is possible to modulate a PRG and noninvasively monitor the extent of Cre-loxP-mediated gene activation by imaging in a microPET scanner.
Sundaresan
G
G
Crump Institute for Molecular Imaging, and Department of Molecular and Medical Pharmacology, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA.
Paulmurugan
R
R
Berger
F
F
Stiles
B
B
Nagayama
Y
Y
Wu
H
H
Gambhir
S S
SS
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D002460:Cell Line; D017353:Gene Deletion; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D018259:Herpesvirus 1, Human / Q000201:enzymology*; D019426:Integrases / Q000235:genetics*; D008099:Liver / Q000000981:diagnostic imaging / Q000378:metabolism*; D051379:Mice; D008822:Mice, Transgenic; D023421:Models, Animal; D051381:Rats; D011995:Recombination, Genetic; D013937:Thymidine Kinase / Q000235:genetics*; D014055:Tomography, Emission-Computed; D015533:Transcriptional Activation; D014161:Transduction, Genetic / Q000379:methods; D014764:Viral Proteins / Q000235:genetics*
D014764:Viral Proteins; D013937:Thymidine Kinase; C045073:Cre recombinase; D019426:Integrases