For deep imaging of animal tissues, the optical window favorable for light penetration is in near-infrared wavelengths, which requires proteins with emission spectra in the far-red wavelengths. Here we report a far-red fluorescent protein, named Katushka, which is seven- to tenfold brighter compared to the spectrally close HcRed or mPlum, and is characterized by fast maturation as well as a high pH-stability and photostability. These unique characteristics make Katushka the protein of choice for visualization in living tissues. We demonstrate superiority of Katushka for whole-body imaging by direct comparison with other red and far-red fluorescent proteins. We also describe a monomeric version of Katushka, named mKate, which is characterized by high brightness and photostability, and should be an excellent fluorescent label for protein tagging in the far-red part of the spectrum.
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Chapman, S., Oparka, K.J. & Roberts, A.G. New tools for in vivo fluorescence tagging. Curr. Opin. Plant Biol. 8, 565–573 (2005).
Chudakov, D.M., Lukyanov, S. & Lukyanov, K.A. Fluorescent proteins as a toolkit for in vivo imaging. Trends Biotechnol. 23, 605–613 (2005).
Hoffman, R.M. Advantages of multi-color fluorescent proteins for whole-body and in vivo cellular imaging. J. Biomed. Opt. 10, 41202 (2005).
Passamaneck, Y.J., Di Gregorio, A., Papaioannou, V.E. & Hadjantonakis, A.K. Live imaging of fluorescent proteins in chordate embryos: from ascidians to mice. Microsc. Res. Tech. 69, 160–167 (2006).
Stewart, C.N., Jr. Go with the glow: fluorescent proteins to light transgenic organisms. Trends Biotechnol. 24, 155–162 (2006).
Seitz, G. et al. Visualization of xenotransplanted human rhabdomyosarcoma after transfection with red fluorescent protein. J. Pediatr. Surg. 41, 1369–1376 (2006).
Wacker, S.A., Oswald, F., Wiedenmann, J. & Knochel, W. A green to red photoconvertible protein as an analyzing tool for early vertebrate development. Dev. Dyn. 236, 473–480 (2007).
Lukyanov, K.A., Chudakov, D.M., Lukyanov, S. & Verkhusha, V.V. Innovation: photoactivatable fluorescent proteins. Nat. Rev. Mol. Cell. Biol. 6, 885–891 (2005).
Chudakov, D.M. et al. Kindling fluorescent proteins for precise in vivo photolabeling. Nat. Biotechnol. 21, 191–194 (2003).
Stark, D.A. & Kulesa, P.M. An in vivo comparison of photoactivatable fluorescent proteins in an avian embryo model. Dev. Dyn. 236, 1583–1594 (2007).
Bertera, S. et al. Body window-enabled in vivo multicolor imaging of transplanted mouse islets expressing an insulin-Timer fusion protein. Biotechniques 35, 718–722 (2003).
Mirabella, R., Franken, C., van der Krogt, G.N., Bisseling, T. & Geurts, R. Use of the fluorescent timer DsRED-E5 as reporter to monitor dynamics of gene activity in plants. Plant Physiol. 135, 1879–1887 (2004).
Konig, K. Multiphoton microscopy in life sciences. J. Microsc. 200, 83–104 (2000).
Gurskaya, N.G. et al. GFP-like chromoproteins as a source of far-red fluorescent proteins. FEBS Lett. 507, 16–20 (2001).
Wang, L., Jackson, W.C., Steinbach, P.A. & Tsien, R.Y. Evolution of new nonantibody proteins via iterative somatic hypermutation. Proc. Natl. Acad. Sci. USA 101, 16745–16749 (2004).
Shkrob, M.A. et al. Far-red fluorescent proteins evolved from a blue chromoprotein from Actinia equina. Biochem. J. 392, 649–654 (2005).
Merzlyak, E.M. et al. Bright monomeric red fluorescent protein with an extended fluorescence lifetime. Nat. Methods 4, 555–557 (2007).
Petersen, J. et al. The 2.0-A crystal structure of eqFP611, a far red fluorescent protein from the sea anemone Entacmaea quadricolor. J. Biol. Chem. 278, 44626–44631 (2003).
Wiedenmann, J. et al. Red fluorescent protein eqFP611 and its genetically engineered dimeric variants. J. Biomed. Opt. 10, 14003 (2005).
Mohun, T.J., Garrett, N. & Gurdon, J.B. Upstream sequences required for tissue-specific activation of the cardiac actin gene in Xenopus laevis embryos. EMBO J. 5, 3185–3193 (1986).
Nieuwkoop, P.D. & Faber, J. Normal Table of Xenopus laevis (Daudin). (Elsevier, Amsterdam, 1967).
Martynova, N. et al. Patterning the forebrain: FoxA4a/Pintallavis and Xvent2 determine the posterior limit of Xanf1 expression in the neural plate. Development 131, 2329–2338 (2004).
Ermakova, G.V., Solovieva, E.A., Martynova, N.Y. & Zaraisky, A.G. The homeodomain factor Xanf represses expression of genes in the presumptive rostral forebrain that specify more caudal brain regions. Dev. Biol. 307, 483–497 (2007).
Shaner, N.C. et al. Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat. Biotechnol. 22, 1567–1572 (2004).
Rizzo, M.A., Springer, G.H., Granada, B. & Piston, D.W. An improved cyan fluorescent protein variant useful for FRET. Nat. Biotechnol. 22, 445–449 (2004).
Yanushevich, Y.G. et al. A strategy for the generation of nonaggregating mutants of Anthozoa fluorescent proteins. FEBS Lett. 511, 11–14 (2002).
Terskikh, A.V., Fradkov, A.F., Zaraisky, A.G., Kajava, A.V. & Angres, B. Analysis of DsRed mutants Space around the fluorophore accelerates fluorescence development. J. Biol. Chem. 277, 7633–7636 (2002).
Bevis, B.J. & Glick, B.S. Rapidly maturing variants of the Discosoma red fluorescent protein (DsRed). Nat. Biotechnol. 20, 83–87 (2002).
Wiedenmann, J. et al. A far-red fluorescent protein with fast maturation and reduced oligomerization tendency from Entacmaea quadricolor (Anthozoa, Actinaria). Proc. Natl. Acad. Sci. USA 99, 11646–11651 (2002).
We thank N.E. Yelina (University of East Anglia, UK) for critical reading of the manuscript. Supported by grants from Howard Hughes Medical Institute 55005618, Molecular and Cell Biology, Russian Academy of Sciences, European Commission FP-6 Integrated Project LSHG-CT-2003-503259, Russian Foundation of Basic Research 07-04-12189 and the US National Institutes of Health GM070358. D.M.C. and K.A.L. are supported by Grant of the President of Russian Federation MK-8236.2006.4, and Russian Science Support Foundation. Partially supported by grants from Howard Hughes Medical Institute 55005630, MCB Russian Academy of Sciences, Russian Foundation of Basic Research 07-04-00466 and Civilian Research and Development Foundation RUB1-2826-MO-06 to A.G.Z.
Katushka and mKate are property of Evrogen JSC, Moscow, Russia. K.A.L., S.L. and D.M.C. have interest in Evrogen JSC. E.M.M. and T.V.C. are employed by Evrogen JSC.
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Shcherbo, D., Merzlyak, E., Chepurnykh, T. et al. Bright far-red fluorescent protein for whole-body imaging. Nat Methods 4, 741–746 (2007). https://doi.org/10.1038/nmeth1083
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