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Red Fluorescent Protein Vectors

Red Fluorescent Protein Vectors

Red fluorescent proteins are ideal for in vivo imaging due to reduced autofluorescence. Like all fluorescent proteins, they can be detected in cells without adding cofactors or substrates, making them valuable, noninvasive tools for investigating biological events in living cells (1).

mCherry

mCherry is one of the Fruit Fluorescent Proteins, which were developed in Dr. Roger Tsien’s lab by directed mutagenesis of mRFP1, a monomeric mutant of DsRed (2–5).

mCherry Fusion Constructs

mCherry has been successfully fused to several proteins, including actin and tubulin (Figure 1). mCherry fusions have been reported in Arabidopsis (6), zebrafish (7), E. coli (8), HIV virions (9), and yeast (10). These fusions have also been used for quantitative imaging techniques including fluorescence resonance energy transfer (FRET; 11), fluorescence recovery after photobleaching (FRAP; 12), and fluorescence lifetime imaging microscopy (FLIM; 13).

Cells expressing mCherry-actin and mCherry-tubulin.

Figure 1. mCherry fusion constructs. HeLa cells were transiently transfected, via a lipid-based method, with mammalian expression vectors encoding mCherry fused to either human cytoplasmic actin (Panel A) or tubulin (Panel B). Cells were fixed using 4% paraformaldehyde and imaged 36 hr posttransfection with a 40X objective on a Zeiss Axioskop microscope using the 575/50, 610, 640/50 filter set.

Stable Cell Lines

We have established three stably-transfected HEK 293 cell lines with different levels of mCherry expression (as measured by flow cytometry). Transfected cells were observed to grow at a rate similar to nontransfected control cells, without increased cell death, as determined by visual inspection.

tdTomato

tdTomato is another Fruit Fluorescent Protein (2–5). It is a genetic fusion of two copies of the dTomato gene (5) which was specifically designed for low aggregation. Its tandem dimer structure plays an important role in the exceptional brightness of tdTomato. Because tdTomato forms an intramolecular dimer, it behaves like a monomer.

Outstanding In Vivo Imaging with tdTomato

tdTomato’s emission wavelength (581 nm) and brightness make it ideal for live animal imaging studies. In one xenograft mouse model of metastatic breast cancer, tdTomato was easily detected as deep as 1 cm below the surface, and extremely small lesions could be detected and tracked over time (Figure 2; 14). A second model used tdTomato to quantify breast cancer tumor growth in response to target gene activation (15). Transgenic mouse models have also been developed, including one where tdTomato was used as a reporter for Cre recombination. This model was also useful as a tool for cell lineage tracing, transplantation studies, and analysis of cell morphology in vivo (16). tdTomato has also been used very effectively in fusion protein applications (17) and as a promoter reporter (18).

tdTomato detected 1 cm deep in the SCID mouse cadaver phantom model.

Figure 2. tdTomato, but not GFP, can be detected in the SCID mouse cadaver phantom model. False-color overlay images (regions of interest encircled) indicate that the imaging system could detect tdTomato fluorescence in the cadaver model, but not GFP fluorescence. Panel A. Implanted tube with 100 x 106 MDA-MB-231-tdTomato-expressing cells, imaged with the DsRed filter set. Exposure time: 1 sec. Panel B. Implanted tube with 100 x 106 MDA-MB-231-GFP-expressing cells, imaged with the GFP filter set. Exposure time: 1 sec.

DsRed-Express & DsRed-Express2

DsRed-Express is a rapidly maturing variant of Discosoma sp. red fluorescent protein (DsRed) with mutations that enhance its solubility, reduce its green emission, and accelerate its maturation. Although DsRed-Express most likely forms the same tetrameric structure as wild-type DsRed, DsRed-Express displays a reduced tendency to aggregate (19). DsRed-Express2 has even higher solubility, and was designed to be better suited for cell and stem cell applications (20).

DsRed-Express2 expression in mouse stem cells and progenitor cells.

Figure 3. Robust expression of DsRed-Express2 in mouse bone marrow hematopoietic stem and progenitor cells. Mononuclear bone marrow cells were transduced with retroviral vectors encoding DsRed-Express (Panel A), DsRed-Express2 (Panel B), or EGFP (Panel C); and fluorescent cells were sorted 87 hr later. Red and green fluorescence signals were detected using the PE and FITC filter sets, respectively. The lines represent gates defined by analyzing untransduced cells.

DsRed-Monomer

DsRed-Monomer is a true monomeric mutant of our red fluorescent protein from Discosoma sp. reef coral, which makes it the optimal choice for use as a red fluorescent fusion tag. DsRed-Monomer is well tolerated by mammalian cells, and has been successfully used to create stably transfected, clonal cell lines. The DsRed-Monomer chromophore matures rapidly and is readily detected 12 hours after transfection and the fluorescent protein is extremely stable, allowing you to monitor fluorescence over extended periods of time.

An Ideal Fusion Tag

Ideally, when you label a protein of interest, the fluorescent tag itself should not interfere with the biological function of the target protein. If the fluorescent protein has a strong tendency to form oligomers, it is more likely to alter or hinder the original function of the tagged protein. Because DsRed-Monomer is a true monomer, it is the optimal choice for use as a red fluorescent fusion tag. When expressed in mammalian cells, the protein is highly soluble and homogeneously distributed within the cytosol, with no detectable aggregation (Figure 4).

DsRed-Monomer is soluble and displays homogeneous distribution in mammalian cells.

Figure 4. DsRed-Monomer is soluble and displays even, consistent, and homogeneous distribution in HeLa cells. DsRed-Monomer has been expressed as a fusion with a large panel of diverse proteins with diverse functions and subcellular locations. The localization of the resulting tagged protein was monitored and all the tested proteins localized properly. For example, the DsRed-Monomer-Actin fusion protein correctly incorporates into the actin filament system—the cytoskeletal network, ruffling edges, and filipodia.

A True Monomer

The monomeric nature of the DsRed-Monomer protein (28 kDa, calculated molecular weight based on amino acid sequence) has been confirmed by two independent methods:

  • FPLC gel filtration chromatography of recombinant DsRed-Monomer yields a single elution peak at a retention time consistent with a 28 kDa molecular weight. The elution profile does not display higher molecular weight species, and provides strong evidence that DsRed-Monomer is a true monomer. In contrast, recombinant DsRed-Express protein elutes earlier than DsRed-Monomer because of its tetrameric structure.
  • Pseudonative gel electrophoresis yields a fractionation profile that is consistent with a monomeric protein, and similar to the monomeric green fluorescent protein AcGFP1.
The monomeric nature of DsRed-Monomer protein (28 kDa) has been confirmed FPLC and gel electrophoresis.

Figure 5. Living Colors DsRed-Monomer is a monomeric protein. Panel A. Recombinant DsRed-Express and DsRed-Monomer (100 µg) were analyzed by FPLC gel filtration chromatography. Overall absorbance (A280) and chromophore excitation (A557) of the eluted material were monitored simultaneously. DsRed-Monomer elutes from the column at a retention time (39 min) corresponding to a molecular weight of 28 kDa. The calculated molecular weight of DsRed-Monomer is 26.8 kDa. DsRed-Express is a tetrameric protein that elutes at an earlier retention time (33 min) corresponding to a molecular weight of 89 kDa. Panel B. Pseudonative gel analysis of proteins. The oligomeric structure of proteins is preserved during SDS PAGE analysis if samples are kept at 4°C and not boiled prior to loading on a gel. Boiled and unboiled recombinant proteins (7.5 µg) were separated by SDS PAGE electrophoresis (12% acrylamide). In both the boiled (denatured) and unboiled (nondenatured) samples, DsRed-Monomer runs as a uniform band of ~30 kDa due to its monomeric structure. The unboiled (nondenatured) DsRed-Express runs at a much higher molecular weight than its boiled (denatured) counterpart due to its tetrameric structure.

Spectral Properties

DsRed-Monomer preserves key spectral features of other DsRed variants, in particular DsRed-Express. DsRed-Monomer has an excitation maximum of 556 nm and an emission maximum of 586 nm. Its spectral profile is virtually identical to our other DsRed fluorescent protein variants, allowing DsRed-Monomer to be detected using standard existing filter sets or with custom-tailored, optimized sets such as those available from Chroma Technology Corporation (see http://www.chroma.com/ for details). Although DsRed-Monomer is somewhat less bright than DsRed-Express, it is nevertheless an excellent choice for fluorescence microscopy imaging and flow cytometry. As with our other red fluorescent proteins, DsRed-Monomer performs well when multiplexed with compatible fluorescent proteins such as AcGFP1 (Figure 6).

Fluorescence excitation and emission spectra of DsRed-Monomer and AcGFP1.

Figure 6. Fluorescence excitation and emission spectra of DsRed-Monomer and AcGFP1.

DsRed2

DsRed2 is a variant of our original red fluorescent protein (DsRed), modified with six point mutations to improve solubility and decrease the time from transfection to detection. DsRed2 retains the benefits typical of red fluorescent proteins, such as a high signal-to-noise ratio and distinct spectral properties for use in multicolor labeling experiments.

mStrawberry

mStrawberry is another Fruit Fluorescent Protein (2–5). Its excitation and emission maxima are 574 nm and 596 nm, respectively.

AsRed2

AsRed2 is a novel fluorescent protein that has been adapted from the corresponding full length cDNA for higher solubility, brighter emission, and rapid chromophore maturation (8–12 hours). It has also been human codon optimized to enhance translation efficiency in mammalian cells. It is a very good reporter, and is useful in two-color analyses with AmCyan1 or ZsGreen1.

References

  1. Matz, M. V. et al. (1999) Nature Biotechnol. 17(10):969–973. Erratum in: Nature Biotechnol. (1999) 17(12):1227.
  2. Campbell, R. E. et al. (2002) Proc. Nat. Acad. Sci. USA 99(12):7877–7882.
  3. Shaner, N. C. et al. (2004) Nature Biotechnol. 22(12):1567–1572.
  4. Wang, L. et al. (2004) Proc. Nat. Acad. Sci. USA 101(48):16745–16749.
  5. Shu, X. et al. (2006) Biochemistry 45(32):9639–9647.
  6. Song, L. et al. (2007) Proc. Nat. Acad. Sci. USA 104(13):5437–5442.
  7. Pisharath, H. et al. (2007) Mech. Dev. 124(3):218–229.
  8. Pradel, L. et al. (2007) Biochem. Biophys. Res. 353(2):493–500.
  9. Campbell, E. M. et al. (2007) Virology 360(2):286–293.
  10. Snaith, H. A. et al. (2005) EMBO J. 24(21):3690–3699.
  11. Anderson, K. I. et al. (2006) Cytometry A. 69(8):920–929.
  12. Picard, D. et al. (2006) Exp. Cell Res. 312(19)3949–3958.
  13. Tramier, M. et al. (2006) Microsc. Res. Tech. 69(11)933–939.
  14. Winnard Jr., P. T. et al. (2006) Neoplasia 8(10):796–806.
  15. Johnstone, C. N. et al. (2008) Mol. Cell Biol. 28(2):687–704.
  16. Muzumdar, M. D. et al. (2007) Genesis 45(9):593–605.
  17. Bjørkøy, G. et al. (2005) J. Cell Biol. 171(4):603–614.
  18. Alandete-Saez, M. et al. (2008) Mol. Plant 1(4):586–598.
  19. Bevis, B. J. & Glick, B. S. (2002) Nat. Biotechnol. 20(1):83–87.
  20. Strack, R. L. et al. (2008) Nature Methods. 5(11):955–957.

 

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