Author(s): You-Tzung Chen 1 , 2 , Ming-Shian Tsai 3 , Tsung-Lin Yang 4 , Amy Tsu Ku 1 , Ke-Han Huang 1 , Cheng-Yen Huang 5 , Fu-Ju Chou 1 , Hsiang-Hsuan Fan 1 , Jin-Bon Hong 1 , Shuo-Ting Yen 1 , Wei-Le Wang 1 , Chang-Ching Lin 1 , Yu-Chen Hsu 3 , 6 , Kang-Yi Su 6 , 7 , I-Chang Su 2 , Chuan-Wei Jang 8 , 9 , Richard R. Behringer 8 , 9 , Rebecca Favaro 10 , Silvia K. Nicolis 10 , Chung-Liang Chien 11 , Shu-Wha Lin 3 , 12 , * , I-Shing Yu 13 , *
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 , . Different from the vital dyes, GFP is a gene product. When the GFP reporter gene is introduced within a transgenic construct or an endogenous locus, its expression pattern reflects the end result of the complex modulating activities of the transcriptional regulatory elements. The GFP gene can also be fused with other gene sequences to generate fusion proteins so that subcellular protein localization and dynamics can be visualized in live cells. For example, 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 . 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 -. It is also possible to express multiple organelle-FP variants in the same cell -. 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 , .
The R26 locus was first identified in a gene trapping experiment in mouse embryonic stem cells . There was a ß-galactosidase and neomycin phosphotransferase fusion reporter (ß-geo ) gene trap construct inserted into this locus that resulted in high level, ubiquitous expression throughout development. Subsequent study revealed that although the insertion of the gene trap cassette disrupted two alternatively spliced transcripts in the gene trap direction, homozygous mutants for this locus were viable with no obvious phenotypic differences from their wild-type littermates . Numerous conditionally activated genes for gain of function studies, reporter cassettes for cell lineage tracing, or suicide genes for genetic cell ablations have been inserted into this locus -. In addition, strategies to accelerate the exploitation of the R26 locus have been developed , .
Here we describe the generation of a mouse strain bearing a Cre activable dual fluorescent reporter gene in the R26 locus. We use a dual fluorescent protein reporter, which encodes for a self-cleavable, bipartite, complex fusion protein that is composed of a chromatin-associated H2B-EGFP fusion protein and a plasma membrane-bound mCherry-GPI (glycosyl-phosphatidyl-inositol signal sequence)...