Abstract

A current focus of biological research is to quantify and image cellular processes in living subjects. To detect such cellular processes, genetically-encoded reporters have been extensively used. The most common reporters are firefly luciferase, renilla luciferase, green fluorescent protein (GFP) and its variants with various spectral properties. Herein, novel design of split GFP and split luciferase will be described; the principle is based on reconstitution of the split-reporter fragments (Figure1-(1)); The basic strategy of the reconstitution is to split a reporter protein into two non-active fragments that are fused to a pair of interacting proteins. The interaction between the two proteins brings the two fragments into close proximity, allowing reconstitution of an intact reporter protein. To demonstrate the usefulness of the reconstitution technology, we have applied the reporters for developing a genetic method to identify mitochondrial proteins and their localization (Figure 1-(2)), and imaging dynamics of endogenous mRNA in single living cells (Figure 1-(3)). We have recently used split-luciferase reporters with different spectral characteristics for two pairs of protein interactions and three protein interactions with a third shared protein in living subjects. We have developed another design of reporter proteins; a cyclic luciferase by protein splicing to monitor protease activities in living mice (Figure 1-(4)). Herein, we will focus on recent advances in the imaging technologies and discuss the advantages and disadvantages of the use of the reconstitution analysis of fluorescent and bioluminescent proteins