Introducing a new reporter gene, membrane-anchored Cypridina luciferase, for multiplex bioluminescence imaging

Abstract

Bioluminescence reporter gene imaging is a robust, high-throughput imaging modality that is useful for tracking cells and monitoring biological processes, both in cell culture and in small animals. We introduced and characterized a novel bioluminescence reporter—membrane-anchored Cypridina luciferase (maCLuc)—paired with a unique vargulin substrate. This luciferase-substrate pair has no cross-reactivity with established d -luciferin- or coelenterazine-based luciferase reporters. We compare maCLuc with several established luciferase-based reporter systems (firefly, click beetle, Renilla , and Gaussia luciferases), using both in vitro and in vivo models. We demonstrate the different imaging characteristics of these reporter systems, which allow for multiplexed-luciferase imaging of 3 and 4 separate targets concurrently in the same animal within 24 h. The imaging paradigms described here can be directly applied for simultaneous in vivo monitoring of multiple cell populations, the activity of selected signal transduction pathways, or a combination of both constitutive and inducible reporter imaging.

Brightening up Biology: Advances in Luciferase Systems for in Vivo Imaging

“…67 In 2021, Moroz et al demonstrated quadruple-BLI in vivo with a slightly different approach, using light wavelengthblocked filters to separate signals. 69 They multiplexed RLuc, CBRLuc, CBGLuc, and a new reporter named membraneanchored Cypridina luciferase (maCLuc), which is paired with the vargulin substrate, to visualize a metastatic tumor model.…”

Whole-Body Imaging to Assess Cell-Based Immunotherapy: Preclinical Studies with an Update on Clinical Translation

“…A membrane-anchored form of the Gaussia luciferase shows higher signal compared to D-luciferin-based firefly luciferase and coelenterazine-based Renilla and the original Gaussia luciferases in detecting T cells and visualized CAR-T cells targeting tumor [52]. While signals from coelenterazine-based luciferases, including membraneanchored Gaussia luciferase, start to "fade" within a few minutes, firefly luciferase and a recently developed vargulin-based membrane-anchored Cypridina luciferase show stability over 15 min [53]. Multiplex BLI is possible by using different luciferase/substrate combinations.…”

Expanding luciferase reporter systems for cell-free protein expression

“…The most commonly used substrates are D-luciferin (for Firefly luciferase) and coelenterazine (for Renilla luciferase). Vargulin was recently reported for an additional no-cross-reactive substrate with Cypridina luciferase 17 .…”