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

Abstract

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, redshifted spectrum, tissue light penetration, and signal quantification, justifying further optimization of protein expression and enzymatic activity.

New imaging probes to track cell fate: reporter genes in stem cell research

“…Modifications of various luciferases have been developed to advance in vivo cell tracking via BLI [65], the most common being the modified firefly luciferase Luc2 optimized for expression in mammalian cells [40]. …”

In Vivo Cell Tracking with Bioluminescence Imaging

“…The higher photon output of the modified FLuc, which is codon-optimized for mammalian cells, has been used prevalently for in vivo cell tracking. FLuc, with a red-shifted spectrum, has recently been investigated for achievement of better tissue penetration and resolution [26]. Light emitted from FLuc is in the yellow-green region, with a peak at 562 nm in basic media (range, 550-570 nm) [27].…”

Pre- and postmortem imaging of transplanted cells

“…94 This imaging modality relies on stable expression of the reporter gene in the target cells, and such expression can be induced using one of the well-established molecular biology techniques, eg, lentiviral transduction, or with the use of primary cells isolated from transgenic, light-producing animals. [95][96][97][98][99][100] Several BLI reporter genes have been isolated, including the North American firefly (Photinus pyralis; FLuc), jellyfish (Aequorea), sea pansy (Renilla; RLuc), corals (Tenilla), the click beetle (Pyrophorus plagiophthalamus), and several bacterial species (Vibrio fischeri, Vibrio harveyi); however, for cell tracking purposes, FLuc is, by far, most widely used. 101 The variety of currently available luciferase types with different substrate specificities opens a new possibility to observe two or more cell lines simultaneously.…”

“…The potential strategies include developing reporter genes with red-shifted emission profiles of photons known to have better tissue penetration, or developing new generations of substrates, such as CycLuc1, which offers significantly better pharmacokinetics and effectively results in an improved imaging signal. 96,105 Another technique with the potential to improve detection of luciferase-expressing cells is based on the modification of …”