In Vivo Imaging with Red Fluorescent Proteins

Application: Noninvasive Optical Tracking of Breast Cancer Metastasis with tdTomato Red Fluorescent Protein

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 1; 1).

Read more about in vivo imaging with tdTomato

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.

Red and far red fluorescent proteins are ideal for in vivo imaging due to their reduced autofluorescence. We recommend the following fluorescent proteins for plant and animal imaging studies:

Far Red Fluorescent Proteins

• mPlum was developed in Dr. Roger Tsien’s lab by directed mutagenesis of mRFP1, a monomeric mutant of DsRed (2–5).
• E2-Crimson matures quickly, and has high photostability, high solubility, and low cytotoxicity. It was designed was designed specifically for in vivo and stem cell applications (6).
• HcRed is a far-red fluorescent protein derived from a nonfluorescent chromoprotein found in the Anthozoa-class sea anemone Heteractis crispa. HcRed1 has a low tendency to form aggregates in living cells.

Red Fluorescent Proteins

• mCherry has been successfully fused to many proteins and widely used for quantitative imaging techniques including fluorescence resonance energy transfer (FRET). mCherry was developed in Dr. Roger Tsien’s lab by directed mutagenesis of mRFP1, a monomeric mutant of DsRed (2–5).
• tdTomato (also developed in Dr. Roger Tsien’s lab) 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. tdTomato can be seen in SCID mice, as deep as 1 cm below the surface (7). tdTomato has also been used to quantify breast cancer tumor growth in response to target gene activation (1). 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 (8).
• DsRed-Express2 has accelerated maturation and high solubility, and was designed specifically for in vivo and stem cell applications (9).

References

1. Winnard Jr., P. T. et al. (2006) Neoplasia 8(10):796–806.
2. Shaner, N. C. et al. (2004) Nature Biotechnol. 22(12):1567–1572.
3. Wang, L. et al. (2004) Proc. Nat. Acad. Sci. USA 101(48):16745–16749.
4. Shu, X. et al. (2006) Biochemistry 45(32):9639–9647.
5. Campbell, R. E. et al. (2002) Proc. Nat. Acad. Sci. USA 99(12):7877–7882.
6. Strack, R. L. et al. (2009) Biochemistry 48(35):8279–8281.
7. Johnstone, C. N. et al. (2008) Mol. Cell Biol. 28(2):687–704.
8. Muzumdar, M. D. et al. (2007) Genesis 45(9):593–605.
9. Strack, R. L. et al. (2008) Nat Methods. 5(11):955–957.