Flow cytometric procedures are described to detect a "humanized" version of a new red fluorescent protein (DsRed) from the coral Discosoma sp. in conjunction with various combinations of three Aequorea victoria green fluorescent protein (GFP) variants--EYFP, EGFP, and ECFP. In spite of overlapping emission spectra, the combination of DsRed with EYFP, EGFP, and ECFP generated fluorescence signal...

Genetic transformation of most insect systems requires dominant-acting markers that do not depend on reverting a mutant phenotype in a host strain, andfor this purpose GFP has proven to be useful in several insect orders. However, detection of multiple transgenes and reporters for gene expression requires the development of new visible markers that can be unambiguously detected when co-expresse...

Here we report stable gene transfer in cord blood-derived CD34(+) hematopoietic stem cells using a hyperactive nonviral Sleeping Beauty (SB) transposase (SB100X). In colony-forming assays, SB100X mediated the highest efficiency (24%) of stable Discosoma sp red fluorescent protein (DsRed) reporter gene transfer in committed hematopoietic progenitors compared with both the early-generation hypera...

Rat embryonic stem cells (ESCs) offer the potential for sophisticated genome engineering in this valuable biomedical model species. However, germline transmission has been rare following conventional homologous recombination and clonal selection. Here, we used the CRISPR/Cas9 system to target genomic mutations and insertions. We first evaluated utility for directed mutagenesis and recovered clo...

GFP from Aequorea victoria is a standard genetic marker widely used to visualize cellular events in a noninvasive manner. For simultaneous imaging of different processes, in vivo mutants of GFP with shifted wavelength spectra (e.g., blue fluorescent protein) are conventionally used. The recently reported red fluorescent protein from Discosoma sp., DsRed, represents a new marker that can be used...

Gene expression of intrinsically fluorescent proteins in biological systems offers new noninvasive windows into cellular function, but optimization of these probes relies on understanding their molecular spectroscopy, dynamics, and structure. Here, the photophysics of red fluorescent protein (dsRed) from discosoma (coral), providing desired longer emission/absorption wavelengths, and an improve...

We describe here a dual-labeling technique involving the green fluorescent protein (GFP) and the red fluorescent protein (DsRed) for in situ monitoring of horizontal gene transfer via conjugation. A GFPmut3b-tagged derivative of narrow-host-range TOL plasmid (pWWO) was delivered to Pseudomonas putida KT2442, which was chromosomally labeled with dsRed by transposon insertion via biparental matin...

Bones consist of a number of cell types including osteoblasts and their precursor cells at various stages of differentiation. To analyze cellular organization within the bone, we generated Col1a1CreER-DsRed transgenic mice that express, in osteoblasts, CreER and DsRed under the control of a mouse 3.2kb Col1a1 promoter. We further crossed Col1a1CreER-DsRed mice with Prx1CreER-GFP mice that expre...

DsRed, a brilliantly red fluorescent protein, was recently cloned from Discosoma coral by homology to the green fluorescent protein (GFP) from the jellyfish Aequorea. A core question in the biochemistry of DsRed is the mechanism by which the GFP-like 475-nm excitation and 500-nm emission maxima of immature DsRed are red-shifted to the 558-nm excitation and 583-nm emission maxima of mature DsRed...