R26R-GR: A Cre-Activable Dual Fluorescent Protein Reporter Mouse

You-Tzung Chen,Silvia K Nicolis,Richard R Behringer,Wei-Le Wang,I-Shing Yu,Shuo-Ting Yen,Ke-Han Huang,Fu-Ju Chou,Ming-Shian Tsai,Chuan-Wei Jang,Rebecca Favaro,Hao Fan ,Chung‐Liang Chien ,Kang‐Yi Su ,Chang‐Ching Lin ,Amy T Ku ,Tsung‐Lin Yang ,I‐Chang Su ,Yu‐Chen Hsu ,Cheng‐Yen Huang ,Shu‐Wha Lin ,Jin‐Bon Hong

doi:10.1371/journal.pone.0046171

Abstract

Green fluorescent protein (GFP) and its derivatives are the most widely used molecular reporters for live cell imagining. The development of organelle-specific fusion fluorescent proteins improves the labeling resolution to a higher level. Here we generate a R26 dual fluorescent protein reporter mouse, activated by Cre-mediated DNA recombination, labeling target cells with a chromatin-specific enhanced green fluorescence protein (EGFP) and a plasma membrane-anchored monomeric cherry fluorescent protein (mCherry). This dual labeling allows the visualization of mitotic events, cell shapes and intracellular vesicle behaviors. We expect this reporter mouse to have a wide application in developmental biology studies, transplantation experiments as well as cancer/stem cell lineage tracing.

Highlights

Green fluorescent protein (GFP), which was first isolated from jellyfish, is among the most widely used molecular markers in contemporary molecular, cellular and developmental biology [1,2]
Generation of the R26R-GR Allele in the Mouse To generate a general reporter mouse, we targeted an inducible dual fluorescent protein reporter cassette to the R26 locus using a previously described strategy [19] (Figure 1A)
The H2B-enhanced green fluorescence protein (EGFP) encoded a histone 2B protein fused with an enhanced green fluorescent protein which allows the observation of chromatin structure in the nucleus, providing cell cycle information including mitosis [21]

Summary

Introduction

Green fluorescent protein (GFP), which was first isolated from jellyfish, is among the most widely used molecular markers in contemporary molecular, cellular and developmental biology [1,2]. Different from the vital dyes, GFP is a gene product. The GFP gene can be fused with other gene sequences to generate fusion proteins so that subcellular protein localization and dynamics can be visualized in live cells. The development of organelle-specific fluorescent proteins (FPs) by fusing FPs with other proteins or peptides that target them to different organelles provides a way to follow the dynamic cellular changes in more detail [3]. The development of FP color variants with different excitation or emission wavelengths makes it possible to simultaneously monitor more than one target protein or organelle [4,5,6]. It is possible to express multiple organelle-FP variants in the same cell [7,8,9,10]. Combinations of emission colors from FPs create codes to increase labeling diversity for description of complicated systems such as neuronal cell synaptic connections in the brain or the stem cell clonal competitions in the intestine [11,12]

Methods

Results

Conclusion