New Drosophila transgenic reporters: insulated P-element vectors expressing fast-maturing RFP.
In vivo green fluorescent protein (GFP)/red fluorescent protein (RFP) double-labeling studies have been hampered by several inconvenient properties of DsRed, the first described RFP. These disadvantages include a very slow (> 24 h) maturation time, emission of contaminating green light, and low solubility. A recently developed variant of DsRed, called DsRed.T4, has a much shorter maturation time, no significant green emission, and improved solubility. We have constructed Drosophila P-element transformation vectors encoding DsRed.T4 for promoter/enhancer analysis, labeling of living cells, or RFP tagging of proteins. These new vectors have all of the features of the widely used Pelican/Stinger GFP vectors, including insulator sequences to reduce position effects, an extensive polylinker, and both cytoplasmic and nuclear-localized forms of the reporter. We have also constructed an upstream activating sequence (UAS)-DsRed.T4 vector, for GAL4 activation of the reporter. We find that DsRed.T4 is very easily detected in transgenic flies without contamination of the GFP signal and that it matures to its fluorescent form nearly simultaneously with GFP. This advance in Drosophila reporter technology makes timed double-labeling experiments in developing transgenic animals possible for the first time.
Barolo
Scott
S
University of California San Diego, La Jolla, CA, USA.
Castro
Brian
B
Posakony
James W
JW
D000818:Animals; D030801:Animals, Genetically Modified / Q000378:metabolism*; D004330:Drosophila / Q000166:cytology / Q000235:genetics* / Q000378:metabolism*; D029721:Drosophila Proteins / Q000235:genetics / Q000378:metabolism; D020869:Gene Expression Profiling / Q000379:methods*; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D038101:Insulator Elements / Q000235:genetics; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D018613:Microscopy, Confocal / Q000379:methods; D008856:Microscopy, Fluorescence / Q000379:methods; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D029721:Drosophila Proteins; D008164:Luminescent Proteins; D009687:Nuclear Proteins; C092281:PSI protein, Drosophila; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; C413663:fluorescent protein 583; D049452:Green Fluorescent Proteins