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.

[1]  R Y Tsien,et al.  Biochemistry, mutagenesis, and oligomerization of DsRed, a red fluorescent protein from coral. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[2]  S. Barolo,et al.  A Notch-Independent Activity of Suppressor of Hairless Is Required for Normal Mechanoreceptor Physiology , 2000, Cell.

[3]  B. Glick,et al.  Rapidly maturing variants of the Discosoma red fluorescent protein (DsRed) , 2002, Nature Biotechnology.

[4]  Polyubiquitin-regulated DsRed marker for transgenic insects. , 2001, BioTechniques.

[5]  T. Maniatis,et al.  An amino acid sequence motif sufficient for subnuclear localization of an arginine/serine-rich splicing factor. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[6]  R. Tsien,et al.  A monomeric red fluorescent protein , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[7]  S. Barolo,et al.  GFP and beta-galactosidase transformation vectors for promoter/enhancer analysis in Drosophila. , 2000, BioTechniques.

[8]  Kei Ito,et al.  An Enhanced Mutant of Red Fluorescent Protein DsRed for Double Labeling and Developmental Timer of Neural Fiber Bundle Formation* , 2001, The Journal of Biological Chemistry.

[9]  S. Lukyanov,et al.  Fluorescent proteins from nonbioluminescent Anthozoa species , 1999, Nature Biotechnology.

[10]  E. Wurmbach,et al.  The Enhancer of split complex of Drosophila melanogaster harbors three classes of Notch responsive genes , 1999, Mechanisms of Development.

[11]  G. Rubin,et al.  Genetic transformation of Drosophila with transposable element vectors. , 1982, Science.

[12]  O. V. Stepanenko,et al.  High stability of Discosoma DsRed as compared to Aequorea EGFP. , 2003, Biochemistry.

[13]  E. Lai,et al.  Antagonism of notch signaling activity by members of a novel protein family encoded by the bearded and enhancer of split gene complexes. , 2000, Development.

[14]  A. Brand,et al.  Ectopic gene expression in Drosophila using GAL4 system. , 1998, Methods.

[15]  E. Lai,et al.  The enhancer of split complex of Drosophila includes four Notch-regulated members of the bearded gene family. , 2000, Development.