Thermostability of Firefly Luciferases Affects Efficiency of Detection by in Vivo Bioluminescence

Brenda Baggett,Robert J Gillies,Shafinaz Momen,Laurence Tisi,Rupali Roy,Sherif Morgan,David Morse

doi:10.1162/15353500200403178

Brenda Baggett, Robert J Gillies + Show 5 more

Open Access

https://doi.org/10.1162/15353500200403178

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Abstract

Luciferase from the North American firefly (Photinis pyralis) is a useful reporter gene in vivo, allowing noninvasive imaging of tumor growth, metastasis, gene transfer, drug treatment, and gene expression. Luciferase is heat labile with an in vitro halflife of approximately 3 min at 37 degrees C. We have characterized wild type and six thermostabilized mutant luciferases. In vitro, mutants showed half-lives between 2- and 25-fold higher than wild type. Luciferase transfected mammalian cells were used to determine in vivo half-lives following cycloheximide inhibition of de novo protein synthesis. This showed increased in vivo thermostability in both wild-type and mutant luciferases. This may be due to a variety of factors, including chaperone activity, as steady-state luciferase levels were reduced by geldanamycin, an Hsp90 inhibitor. Mice inoculated with tumor cells stably transfected with mutant or wild-type luciferases were imaged. Increased light production and sensitivity were observed in the tumors bearing thermostable luciferase. Thermostable proteins increase imaging sensitivity. Presumably, as more active protein accumulates, detection is possible from a smaller number of mutant transfected cells compared to wild-type transfected cells.

Highlights

In the current study, we test the hypothesis that thermostabilization will lead to a higher accumulation of firefly luciferase in human tumor cells and that this will improve the lower detection limit of luciferase expressing cells in whole animals
The most widely used luciferase reporter gene is that of the North American firefly, Photinis pyralis
A high turnover rate of the reporter would be advantageous in time-sensitive studies, such as those to determine the response to an environmental toxin, infection, or induction or repression of gene expression

Summary

Introduction

We test the hypothesis that thermostabilization will lead to a higher accumulation of firefly luciferase in human tumor cells and that this will improve the lower detection limit of luciferase expressing cells in whole animals. A high turnover rate of the reporter would be advantageous in time-sensitive studies, such as those to determine the response to an environmental toxin, infection, or induction or repression of gene expression. It is proposed that a lower turnover rate provided by thermostabilization will lead to higher accumulation of active luciferase molecules, and that this will lead to higher light output on a per-cell basis and improve detection. This would be advantageous for studies that are not time-sensitive, such as monitoring tumor growth or colonization of metastases. This is especially important in investigating metastases, as there would be significant interest in the fate of micro-metastases, which might otherwise escape detection due to their small size

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