FUCCI - Fluorescent, Ubiquitination-based Cell Cycle Indicator
The ability to monitor phase transitions in the cell cycle can be important in many types of research including analysis of differentiation, morphogenesis, apoptosis or cell-death. The most common method of monitoring G1/S phase transition has routinely been flow cytometric analysis of DNA levels, which increase upon cell division, using bromodeoxyuridine or propidium iodide staining. Recently, work from the laboratory of Dr. Atsushi Miyawaki at the RIKEN Basic Science Institute and coworkers at Tokyo University of Pharmacy and Life Science and Nagoya University Graduate School of Medicine, have developed an elegant method of monitoring the cell cycle in live cells or tissues based upon its tight regulation by the specific nuclear complexes. The APC Cdh1
complex is active in the late M and G1 phases, while
the SCF
Skp2
complex is active in the S and G2 phases. Two direct substrates of the APC and SCF complexes are Cdt1 and Geminin, respectively. Cdt1 levels are highest
during G1, while Geminin levels are highest during the S, G2,
and M phases of the cell cycle. By fusing the Cdt1 and Geminin factors to either red or green fluorescent proteins respectively, which also included ubiquitin sites, they were able to kinetically monitor cell cycle changes by transient expression of these fluorescent fusion proteins. During G1 phase, the cells were found to fluoresce red (mRFP1) and in the S Phase they produce the green fluorescent protein (mEGFP). In addition, in the G1/S Phase transition, a yellow color of fluorescence was observed. They compared their results to standard BrdU staining and found the analysis to be correct. They were also able to use the vectors to produce transgenic mouse lines with the system expressing in vivo in a variety of tissues and monitor proliferation and cell cycle changes during mouse development. They were also able to make similar protein constructs for zebrafish embryo analysis. For more information about these techniques, please see the references below or visit our website
- Sakaue-Sawano A, Kurokawa H, Morimura T, Hanyu A, Hama H, Osawa H,
Kashiwagi S, Fukami K, Miyata T, Miyoshi H, Imamura T, Ogawa M,
Masai H, Miyawaki A, (2008) "Visualizing Spatiotemporal Dynamics
of Multicellular Cell-Cycle Progression." Cell 132: 487–498.
- Nishitani H, Lygerou Z, Nishimoto T (2004). "Proteolysis of DNA replication
licensing factor Cdt1 in S-phase is performed independently of geminin
through its N-terminal region." J. Biol. Chem. 279: 30807–30816.
- Sugiyama M, Sakaue-Sawano A, Iimura T, Fukami K, Kitaguchi T,
Kawakami K, Okamoto H, Higashijima S, Miyawaki A (2009) "Illuminating cell-cycle progression in the developing
zebrafish embryo." PNAS 106(49): 20812–20817.
- Sakaue-Sawano A, Ohtawa K, Hama H, Masako Kawano M, Masaharu Ogawa M, Miyawaka A (2008) "Tracing the silhouette of individual cells in S/G2/M
phases with fluorescence." Chem Biol 15:1243–1248.
- Evanko D, (2008) "Protein suicide highlights the cell cycle." Nature Methods 5: 283.
New near-IR Pdot Fluorescent Nanoparticles
 The design of probes for use in biological tissues continues to be an important area of research for diagnosis of disease or for use in varied bioimaging applications. Because of the significant interference from autofluorescence of native compounds in tissues as well as absoption of incident visible light wavelengths, methods that can be used in the near-IR are of particular interest. But many of the common dyes used in the near-IR region (carbocyanines, phthalocyanines, etc.) suffer from poor solubility, self-aggregation and losses in planarity that limit their quantum yields in vivo. Recent work from the laboratories of Dr. Daniel Chiu and coworkers at the University of Washington has outlined the development of several new semiconductor Pdots that contain a silicon-naphthalocyanine dye encapsulated in a polyfluorene (PFBT) matrix. These new Pdots can be excited with blue light (457 nm) and emit in the near infrared (777 nm). It was found that this matrix not only stabilized
the hydrophobic dyes but also helped act as an efficient light-harvesting
agent to transfer energy to the
internalized dyes and improve their fluorescence brightness. Applications of these new Pdots for single particle
imaging, cellular imaging, flow cytometry and in vivo labeling of medulloblastoma tumors in mice indicated a much higher fluorescence
brightness of Pdots compared to Alexa-fluor dye conjugates or quantum dot probes. To find out more about these methods, please see references below or visit our website.
- Jin Y, Ye F, Zeigler M, Wu C, Chiu DT(2011) "Near-Infrared Fluorescent Dye-Doped
Semiconducting Polymer Dots."ACS Nano 5 (2): 1468–1475 .
- Wu C, Schneider TS, Zeigler M, Yu J,
Schiro PG, Burnham DR, McNeill JD, Chiu DT(2010) "Bioconjugation of Ultrabright Semiconducting Polymer Dots
for Specific Cellular Targeting." J. Amer. Chem. Soc. 132: 15410–15417.
- Wu C, Hansen SJ, Hou Q, Yu J, Zeigler M, Jin Y,
. Burnham DR, . McNeill JD, Olson JM, Chiu DT (2011) "Design of Highly Emissive Polymer Dot Bioconjugates for In Vivo
Tumor Targeting." Angew. Chem. Int. Ed. 50: 3430-3434.
- Chan YH, Wu C, Ye F, Jin Y, Smith PB, Chiu DT (2011) "Development of Ultrabright Semiconducting Polymer Dots for Ratiometric pH Sensing" Anal. Chem. 83: 1448-1455.
- Ye F, Wu C, Jin Y, Chan YH, Zhang X, Chiu DT (2011) "Ratiometric temperature sensing with semiconducting polymer dots" J. Amer. Chem. Soc. 133: 8146-8149.
RubyGlowTM Cell Proliferation, Viability, Cytotoxicity Kits
Rapid and accurate measurement of viable cell number and cell growth is often required in cell biological applications. In addition, traditional assessment of cell viability has been made via membrane integrity (e.g., trypan blue exclusion), and cell proliferation via the incorporation of labeled nucleotides (e.g., [3H]-thymidine) into newly synthesized DNA during cell division. An alternative technology used in quantitating all of these parameters including cell number, proliferation, growth, viability or cytotoxicity is based on the generation of ATP in metabolically active cells. Studies have shown that the level of intracellular ATP per cell is highly regulated and remains essentially constant in a cell population. The cell responds to the increased demand for ATP by increasing the production or turnover rate of ATP. Increasing levels of ATP is therefore a reflection of cell proliferation and an increase in the number of viable cells per well. In addition, controlled studies have shown that ATP measurements correlate well with traditional tritiated thymidine incorporation methods. Any form of cell injury results in a rapid decrease in cytoplasmic ATP levels, and thus measuring ATP in drug treated cells can assess the viability of the cells as well as cytotoxicity of the drug.
The most successful technique for ATP measurement has proven to be the luciferin-luciferase bioluminescent assay. The use of the luciferase enzyme has become highly valuable as a genetic marker gene due to the convenience, high sensitivity and linear range of the luminescence assay. The reaction involved is described as:
Luciferase + ATP + D-Luciferin + O2 -> Oxyluciferin + AMP + PPi + CO2 + Light.
Thus measuring the units of light output will accurately reflect the amount of ATP in a sample or reaction. Using genetic engineering, we have generated a new luciferase which exhibits long-wavelength light emission, as well as improved thermostability, compared to the native firefly luciferase often used in these assays. The light output is stable over several hours and thus eliminates the necessity for substrate injection. We have adapted this new luciferase into a series of kits for use in a microtiter plate format and thus made them amenable to high-throughput applications. Our new RubyGlowTM luciferase kits (M1574, M1575 and M1576) produce a red light (EM 619 nm) upon luciferin conversion, while most of the commercially available luciferase kits still use older green light emitting (562 nm) firefly analogs. This unique feature enables the possibility of multiplexing. The new kit components contain no radioisotopes, are not toxic to cells and are environmentally safe. The kits provide enough reagents and a detailed protocol sufficient for 100 reactions in 96-well microplate format. For more information about these new kits, please see the references below, or visit our website.
- Cunningham BA. (2001) “A growing issue: cell proliferation assay” The Scientist 15(13): 26.
- Tatsumi H, Masuda T, Kajiyama N, Nakano E. (1989) "Luciferase cDNA from Japanese firefly, Luciola cruciata: cloning, structure and expression in Escherichia coli." J Biolumin Chemilumin 3(2): 75-78.
- Masuda T, Tatsumi H, Nakano E. (1989) "Cloning and sequence analysis of cDNA for luciferase of a Japanese firefly, Luciola cruciata." Gene 77(2): 265-270.
- de Wet JR, Wood KV, Helinski DR, DeLuca M. (1985) "Cloning of firefly luciferase cDNA and the expression of active luciferase in Escherichia coli." Proc Natl Acad Sci U S A. 82(23): 7870-7873.
- Mamaev SV, Laikhter AL, Arslan T, Hecht SM (1996) "Firefly Luciferase: Alteration of the Color of Emitted Light Resulting from Substitutions at Position 286." J. Amer. Chem. Soc. 118: 7243-7244.
- Kajiyama N, Nakano E. (1991) "Isolation and characterization of mutants of firefly luciferase which produce different colors of light." Protein Eng. 4: 691.
- Kajiyama N, Nakano E. (1993) "Thermostabilization of Firefly Luciferase by a Single Amino Acid Substitution at Position 217." Biochem 32: 13795-13799.
- Crouch SP, Kozlowski R, Slater KJ, Fletcher J. (1993) "The use of ATP bioluminescence as a measure of cell proliferation and cytotoxicity," J. Immunol. Methods 160(1): 81-88.
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