Background: Detection of enzyme activity or transgene expression offers potential insight into developmental biology, disease progression, and potentially personalized medicine. Historically, the lacZ gene encoding the enzyme beta-galactosidase has been the most common reporter gene and many chromogenic and fluorogenic substrates are well established, but limited to histology or in vitro assays. We now present a novel approach for in vivo detection of beta-galactosidase using optical imaging to detect light emission following administration of the chemiluminescent 1,2-dioxetane substrate Galacto-Light PlusTM.
Methodology and principal findings: B-gal activity was visualized in stably transfected human MCF7-lacZ tumors growing in mice. LacZ tumors were identified versus contralateral wild type tumors as controls, based on two- to tenfold greater light emission following direct intra tumoral or intravenous administration of reporter substrate. The 1,2-dioxetane substrate is commercially available as a kit for microplate-based assays for beta-gal detection, and we have adapted it for in vivo application. Typically, 100 microl substrate mixture was administered intravenously and light emission was detected from the lacZ tumor immediately with gradual decrease over the next 20 mins. Imaging was also undertaken in transgenic ROSA26 mice following subcutaneous or intravenous injection of substrate mixture.
Conclusion and significance: Light emission was detectable using standard instrumentation designed for more traditional bioluminescent imaging. Use of 1,2-dioxetane substrates to detect enzyme activity offers a new paradigm for non-invasive biochemistry in vivo.