A Transgenic Mouse Model for Imaging Single RNA Dynamics In Vivo

We present a novel mouse model for studying the dynamics of mRNA in live cells and tissues. The MS2-GFP labeling technique has been widely used to visualize single RNA in living cells. We extend the utility of the technique for single molecule imaging in the context of a whole-animal system. The MS2-GFP technique exploits the high affinity binding of the MS2 bacteriophage capsid protein (MCP) to the MS2 RNA binding site (MBS). MCP is fused with green fluorescent protein (GFP), and 24 repeats of MBS are inserted into the RNA of interest. In order to apply the technique for live tissue imaging, we genetically engineered two mouse models: MCP-GFP transgenic mouse, Tg(MCP-GFP), and Actb-MBS mouse. In the Actb-MBS mouse, MBS cassette is knocked into the 3’ untranslated region (3’ UTR) of the essential β-actin gene (Lionnet et al., Nature Methods, 8, 165, 2011). By crossing Tg(MCP-GFP) mouse and Actb-MBS mouse, we generated a hybrid mouse expressing fluorescent RNA in every cell and tissue where β-actin is present. The homozygous mouse is viable and exhibits no gross abnormalities despite the addition of 1.2 kbp MBS cassette and approximately 1.2 MDa of MCP-GFP to the essential β-actin mRNA. These results suggest that this technology could be applied to a wide range of genes with minimal perturbation. Using this mouse, we are investigating the dynamics of β-actin mRNA transport in acute brain slices using multiphoton microscopy. In addition, single-molecule tracking of mRNA is employed to understand the mechanism of mRNA localization in hippocampal neuron cultures. The mouse model combined with high-resolution imaging can provide important insights into the dynamic regulation of an endogenous gene in its native tissue environment.This work was supported by NIH-GM86217, GM84364, and NIH-F32-GM87122.