Comparison of red-shifted firefly luciferase Ppy RE9 and conventional Luc2 as bioluminescence imaging reporter genes for in vivo imaging of stem cells

Abstract

One critical issue for noninvasive imaging of transplanted bioluminescent cells is the large amount of light absorption in tissue when emission wavelengths below 600 nm are used. Luciferase with a red-shifted spectrum can potentially bypass this limitation. We assessed and compared a mutant of firefly luciferase (Ppy RE9, PRE9) against the yellow luciferase luc2 gene for use in cell transplantation studies. C17.2 neural stem cells expressing PRE9-Venus and luc2-Venus were sorted by flow cytometry and assessed for bioluminescence in vitro in culture and in vivo after transplantation into the brain of immunodeficient Rag2-/- mice. We found that the luminescence from PRE9 was stable, with a peak emission at 620 nm, shifted to the red compared to that of luc2. The emission peak for PRE9 was pH-independent, in contrast to luc2, and much less affected by tissue absorbance compared to that of luc2. However, the total emitted light radiance from PRE9 was substantially lower than that of luc2, both in vitro and in vivo. We conclude that PRE9 has favorable properties as compared to luc2 in terms of pH independence, red-shifted spectrum, tissue light penetration, and signal quantification, justifying further optimization of protein expression and enzymatic activity.

Fig. 1

Expression of the two luciferase variants in C17.2 cells. (a) Diagram of lentiviral vector used in this study to express PRE9 or luc2 with Venus as an additional reporter gene. (b, c) Expression of Venus in C17.2 cells transduced by FM-PRE9 (b) and FM-luc2 (c). $\text{bar}=100\mathrm{\mu m}$. (d) Flow cytometry diagram for sorted cells, with a median fluorescent intensity for PRE9 and luc2 expressing cells of 639.2 and 663.6, respectively. The contour histogram of untransfected cells is in blue. (e) Western blot of cell lysates from sorted PRE9 and luc2 expressing cells. (f) Quantification of western blot. Results are expressed as the ratio of luc2 to PRE9 protein density with $\beta$-actin expression as an internal reference.

Fig. 2

BLI signal intensity of PRE9- (solid lines, left $Y$-axis) and luc2 (dashed lines, right $Y$ axis) expressing cells in culture. (a) Time course of a bioluminescent signal in living cells after the addition of luciferin ($15\mathrm{\mu g}/\mathrm{ml}$) in PBS ($n=3$). Left $Y$ axis was for PRE9 cells. Right $Y$ axis was for luc2 cells. (b) Comparison of the magnitude of luminescence from a different density of cells in 96 well plates, 10 min after the addition of luciferin ($15\mathrm{\mu g}/\mathrm{ml}$) in PBS ($n=3$). (c) Quantitative analysis of luminescence from PRE9 and luc2 cells, showing the fold of increase of light output from luc2 cells compared to PRE9 cells.

Fig. 3

In vitro emission spectrum of PRE9- and luc2-transduced cells. (a) Spectrum of luc2 and PRE9 cells in PBS, $\mathrm{pH}=7.4$. (b, c) pH dependence of spectrum for luc2 (b) and (c) PRE9 cells. For both groups, measurements were obtained 10 min after the addition of $15\mathrm{\mu g}/\mathrm{ml}$ luciferin ($n=3$).

Fig. 4

In vivo imaging of transplanted PRE9 and luc2 cells. (a) BLI of cells transplanted in the striatum. Green circles: ROIs for mice ($n=4$) injected with luc2 cells; red circles: ROIs for mice ($n=4$) injected with PRE9-cells. (b) Quantitative analysis of total flux for each ROI, showing a significant difference between the two groups ($p<0.01$).

Fig. 5

Head-to-head comparison of in vitro and in vivo data. (a) Luc2:PRE9 ratio of in vitro and in vivo photon radiance ($1\times {10}^5$ cells each). (b) Emission spectrum for PRE9 cells (in vitro: solid lines, in vivo:dashed lines, $n=3$). (c) Emission spectrum for luc2 cells (in vitro: solid lines, in vivo:dashed lines, $n=3$). (d) In vivo comparison of PRE9 (solid lines) and luc2 (dashed lines) emission spectrum.