Article

Dual fluorescent protein reporters for studying cell behaviors in vivo

Department of Genetics, University of Texas M.D. Anderson Cancer Center, Houston, Texas, USA.
genesis (Impact Factor: 2.04). 10/2009; 47(10):708-17. DOI: 10.1002/dvg.20565
Source: PubMed

ABSTRACT Fluorescent proteins (FPs) are useful tools for visualizing live cells and their behaviors. Protein domains that mediate subcellular localization have been fused to FPs to highlight cellular structures. FPs fused with histone H2B incorporate into chromatin allowing visualization of nuclear events. FPs fused to a glycosylphosphatidylinositol anchor signal sequence label the plasma membrane, highlighting cellular shape. Thus, a reporter gene containing both types of FP fusions would allow for effective monitoring of cell shape, movement, mitotic stage, apoptosis, and other cellular activities. Here, we report a binary color-coding system using four differently colored FP reporters that generates 16 distinct color codes to label the nuclei and plasma membranes of live cells in culture and in transgenic mice. As an initial test of this system in vivo, the promoter of the human Ubiquitin C (UBC) gene was used to widely express one of the color-code reporters. Widespread expression of the reporter was attained in embryos; however, both male and female transgenic mice were infertile. In contrast, the promoter of the mouse Oct4/Pou5f1 gene linked to two different color-code reporters specifically labeled blastocysts, primordial germ cells, and postnatal germ cells, and these mice were fertile. Time-lapse movies of fluorescently-labeled primordial germs cells demonstrate the utility of the color-code system to visualize cell behaviors. This set of new FP reporters should be a useful tool for labeling distinct cell populations and studying their behaviors in complex tissues in vivo.

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    • "At the same time, the fertility in homozygous R26-RG mice indicates that the expression level of FFPs is not high enough to be toxic at this locus. Indeed, the intensity of fluorescent signals was lower than that in UBC-HS-GR transgenic mice which were infertile at a heterozygous state (Stewart et al., 2009); the intensity might also be lower than that in Tom-2A-GFP transgenic mice byTrichas et al. (2008) which is fertile. Using R26-RG embryos, we were able to observe the nuclear movement in the epiblast cells, located deeply within the embryos, in live-imaging with a conventional confocal microscope, but the quality was not high enough. "
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