Marker Gene Monthly Newsletter   

May, 2007

Volume 7, Number 5

© Copyright MGT, Inc., 2007.  Published by Marker Gene Technologies, Inc., The University of Oregon Riverfront Research Park, 1850 Millrace Drive, Eugene, Oregon 97403-1992 USA.  All rights reserved.  For information on the use or copying of the material contained in this document, please contact us at techservice@markergene.com.  Please see below for subscription information and updates.  This newsletter is labeled as an ADVERTISEMENT in accordance with the CAN-SPAM act of 2003, S.877 Public Law: 108-187.

 GFP Complementation Assays for RNA analysis.

Complementation assays have provided a simple and sensitive means of detecting the interaction between two proteins or peptides within living cells. Their use has resulted in the isolation of many new proteins, and has facilitated the identification of important targets for pharmaceutical intervention of diseases that have aided in the development of many new drugs.  Recent extrapolations of this technique have been developed that utilize two subunits of Green Fluorescent Protein GFP which are complementation fragments that, when placed in near proximity and allowed to interact, provide a functional fluorescent protein that can easily be analyzed within living cells using standard fluorescence techniques.  Use of this technique to detect specific RNA sequences in living cells has recently been developed in several laboratories.  The method involves fusing the reading frames for the two GFP fragments to proteins that strongly and specifically bind to adjacent RNA sequences.   Since the two RNA sequences are in close proximity, upon binding, the two GFP-RNA-binding proteins will interact, producing fluorescence.  In one example, Rackham and Brown introduced an MS2 coat protein binding motif and a “zip-code” b-actin mRNA sequence into an artificial mRNA construct and imaged it using corresponding RNA-binding proteins.  In another example, Valencia-Burton and coworkers split a eukaryotic initiation factor 4A protein into two halves and fused them to two sections of GFP.  Since the two eIF4A  proteins bind to a single RNA aptamer sequence, the expression of eIF4A mRNA in E. coli caused GFP fluorescence.  Finally, in another recent example, Ogawa, and coworkers have developed a method to alter the RNA binding specificity of PUMILIO1 in a predictable manner so that two different PUMILIO1 proteins could be used to bind to adjacent 8-nucleotide sequences on a stretch of mRNA in the mitochondria of HeLa cells and detect its expression.   They fused a targeting signal (MTS) to the N-terminal of each protein derived from subunit VIII of cytochrome c oxidase to target the RNA probes to the mitochondrial matrix.   The two pEGFP fragments were obtained by PCR amplification of  the vector with specific primers giving a C-terminal and N-terminal pair (residues 1-158 and 159-238 respectively).    This latter technique opens the possibility to develop mRNA complementation assays for nearly any natural sequence using GFP complementation methods.  For more information about these new techniques, please visit our website or see the references below.

• "A novel genetic system to detect protein-protein interactions." (1989) S. Fields, O.-K. Song, Nature, 340: 254-6.
• "Genome-wide protein interaction maps using two-hybrid systems." (2000) P. Legrain, L. Selig, FEBS Letters, 480: 32-6
• “Visualization of RNA-protein interactions in living cells: FMRP and IMP1 interact on mRNAs.” (2004) Rackham, O., Brown, C.M.,  EMBO J 23(16): 3346-55.
• “RNA visualization in live bacterial cells using fluorescent protein complementation.” (2007) Valencia-Burton M ; McCullough RM ; Cantor CR ; Broude NE Nature Methods 4(5): 421-7.
• “Imaging dynamics of endogenous mitochondrial RNA in single living cells.” Ogawa, T., Natori, Y., Sato, M., Umezawa, Y., (2007) Nature Methods 4(5): 413-419.
  

DiI(C18:5) as a Long-Term Membrane Probe and Counterstain.

Long-chain dialkylcarbocyanines, like 1,1'-Dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine perchlorate (M1269,DiIC18:5, DiD) have been widely used for  neuron labeling in both living and fixed cells and tissues. They can measure transport either away from the cell body to the microtubules and synapse (anterograde) or in the opposite direction (retrograde labeling).  DiI is non-toxic and labeled cells have been found to remain viable for up to four weeks in culture or up to one year in vivo.   DiIlabels neurons by means of lateral diffusion in the plasma membrane with diffusion rates of between 0.2 to 0.6 mm per day in fixed specimens and up to 6 mm per day in living tissue.  The quicker diffusion in living cells is likely due to active dye transport processes.In fixed tissues, diffusion of DiI has been monitored for up to two years.  Interestingly, transfer from labeled to unlabeled cells does not normally occur, although transfer has been known to occur if the membrane is disrupted in tissue sectioning.  DiD is an analog of DiI with longer wavelength fluorescence excitation and emission, making it useful for two-color labeling.  This property can also prevent background cellular autofluorescence or phototoxic effects of UV excitation sources.  For more information about DiI labeling, please see the references below or visit our website.

• "Fibre optic sensor for the detection of potassium using fluorescence energy transfer." Roe JN, Szoka FC, Verkman AS., Analyst 115: 353-358 (1990).
• "Carbocyanine dyes with long alkyl side-chains: broad spectrum inhibitors of mitochondrial electron transport chain activity." Anderson WM, Trgovcich-Zacok D., Biochem. Pharmacol. 49: 1303-1311 (1995).
• "Optical sectioning-- slices of life." Paddock S., Science 295: 1319-1321 (2002).
• "Photodamage to intact erythrocyte membranes at high laser intensities: methods of assay and suppression." Bloom JA, Webb WW., J. Histochem. Cytochem. 32: 608-616 (1984).
• "Iontophoretic dye labeling of embryonic cells." Fraser SE., Methods Cell. Biol. 51: 147-160 (1996).
• "Multicolor "DiOlistic" labeling of the nervous system using lipophilic dye combinations." Gan WB, Grutzendler J, Wong WT, Wong RO, Lichtman JW., Neuron 27: 219-225 (2000).
• "Chemical imaging of tissue in vivo with video-rate coherent anti-Stokes Raman scattering microscopy." Evans CL, Potma EO, Puoris'haag M, Cote D, Lin CP, Xie XS., Proc. Natl. Acad. Sci. U. S. A. 102: 16807-12 (2005).
• "Rapid labeling of neuronal populations by ballistic delivery of fluorescent dyes." Grutzendler J, Tsai J, Gan WB. Methods 30: 79-85 (2003).
• "Lateral Diffusion of Lipids and Proteins in Bilayer Membranes." Vaz WLC, Goodsaid-Zaluondo, F., Jacobson K., FEBS Lett. 174: 199 (1984).

Combined Rose Bengal - Luciferase System for Photodynamic Therapy

Photodynamic therapy (PDT) is a treatment method where a photosensitive compound, like Rose Bengal (M1277) or Rose Bengal diacetate (M0780) is targeted to cancer cells, and a high-intensity  light source is used to excite the dye and produce cytotoxic intermediates, like singlet oxygen or other  free radicals.  One of the main drawbacks of PDT remains poor accessibility of light to more deeply situated malignancies, and it has therefore been mainly applied to either surface cancers (melanomas) or intestinal malignancies with the use of fiber optic light sources using lasers or UV light endoscopic light sources.   But even using these approaches, the light distribution over the tumor is not homogeneous and some metastatic disease is often left untreated. 

Recently the laboratory of Dr. Theodossis Theodossiou and collaborators at the Department of Surgery, University College - London have combined the bioluminescent intracellular light emission from specific firefly luciferase expression with Rose Bengal administration, as a targetable alternative to external sources of illumination and cell ablation.  The in vitro photodynamic effect of Rose Bengal was activated by intracellular generation of light, in luciferase-transfected NIH 3T3 murine fibroblasts after addition of D-luciferin (M0237) in a cell-culture test system.  The potential application of this type of combined chemiluminescent – photodynamic therapy system offers a new weapon in the arsenal of techniques that can be utilized for treatment of malignancies.  For more information about these new techniques, please see our website of see the references below. 

• "Firefly Luciferin-activated Rose Bengal - In Vitro Photodynamic Therapy by Intracellular Chemiluminescence in Transgenic NIH 3T3 Cells" (2003) Theodossiou, T., Hothersall, J.S., Woods, E.A., Okkenhaug, K., Jacobson J., MacRobert A.J., Cancer Research 63: 1818-1821.
• Khajehpour M, Troxler T, Vanderkooi JM (2004) Probing the Active Site of Trypsin with Rose Bengal: Insights into the Photodynamic Inactivation of the Enzyme. Photochemistry and Photobiology: Vol. 80, No. 2 pp. 359–365.

Carboxyfluorescein di-Acetate Succinimidyl Ester (CFDA-SE) as a Viability Probe for FACS Analysis.

Carboxyfluorescein diacetate succinimidyl ester (CFDA-SE, M0013) is a lipophilic viability probe that freely passes through the cell membrane and is non-fluorescent until inside cells, where ubiquitous esterase can remove the acetyl groups and produce fluorescence.  In addition, the succinimidyl ester group binds covalently to amino groups on intracellular proteins, anchoring the dye and making it well retained intracellularly. CFDA-SE is the main ingredient in

Since its introduction in the flow cytometric analysis of lymphocyte proliferation by serial halving of fluorescence intensity, CFDA-SE (also called CFSE) has become widely used in immunological laboratories around the world.  Daughter cells inherit half of the label after each cell division, resulting in the sequential halving of mean fluorescence with each generation.  The probe has also been used for the quantitative analysis of in vitro natural killer cell proliferation detected by flow cytometry and to evaluate several class I MHC receptors for their ability to activate or inhibit NK cell division. CFDA-SE has also been used to follow fibroblast or bacterial proliferation or as an indicator of bacterial activity.  CFDA-SE labeling offers a means for the rapid detection of other kinds of cells by flow cytometric analysis, including those undergoing apoptotic or necrotic cell death.  Researchers have reported using CFDA-SE to label hepatocytes for localization following transplantation or to localize transplanted or engrafted human Schwann cells in the spinal cord of nude rats in vivo.  It also provides a rapid, reproducible and simple method for the fluorescent labeling of murine blood cells in situ following intravenous injection of CFDA-SE.  CFDA-SE is also suitable for analyzing the cytolytic activity of cytotoxic T lymphocytes (CTLs). 

The optimal concentration of CFDA-SE for cell labeling varies according to cell type. CFDA-SE has been found to be essentially non-toxic to cells (cell death rate below 5%). The optimal cell labeling time has been determined to be only 5-15 min of incubation with CFDA-SE.  Labeling is stopped by addition of heat-inactivated fetal calf serum (FCS) for one minute and washing with PBS before returning to media for FACS analysis.  Propidium iodide (10 μg/mL) can also be added as a counter stain to mark dead cells for counting.  CFDA-SE labeling can be measured for up to 24 hours after labeling, allowing long-term detection of labeled cells. However, during the first 4 h after labeling, there is a decline in fluorescence intensity, most likely due to degradation of certain intracellular CFDA-SE-protein conjugates.  Some protein conjugates remain stable for prolonged periods while others are degraded in the first few hours after labeling.  After this initial change, labeling intensity remains essentially stable for more than 24 hours.  Despite this change, CFDA-SE is a brighter viability label than other fluorescent probes, including FITC.  For more information about this important new viability probe, please see our website, or see the references below.

• “Analyzing cell division in vivo and in vitro using flow cytometric measurement of CFSE dye dilution.” Lyons AB. (2000) J. Immunol. Methods 243: 147–154.
• “The fate of thymocytes labeled in vivo with CFSE.” Graziano M, St-Pierre Y, Beauchemin C, Desrosiers M, Potworowski EF. (1998) Exp, Cell Res. 240: 75–85.
• “A novel technique for the fluorometric assessment of T lymphocyte antigen specific lysis.” Sheehy ME, McDermott AB, Furlan SN, Klenerman P, Nixon DF. (2001) J Immunol Methods, 249: 99–110.
• “Fluorescence for lymphocyte migration and proliferation studies.” Parish CR. (1999) Immunol Cell Biol, 77: 499–508.
• “Insulin has a limited effect on the cell cycle progression in 3T3 L1 fibroblasts.”  Khil LY, Kim JY, Yoon JB, Kim JM, Keum WK, Kim ST, Yoon Y, (1997) Mol. Cells  7: 742–748.
• “Flow cytometric analysis of Lactobacillus plantarum to monitor lag times, cell division and injury.” Ueckert JE, Nebe von-Caron G, Bos AP, ter Steeg PF. (1997) Lett. Appl. Microbiol, 25: 295–299.
• “A comparative study of carboxyfluorescein diacetate and carboxyfluorescein diacetate succinimidyl ester as indicators of bacterial activity.” Hoefel D, Grooby WL, Monis PT, Andrews S, Saint CP. (2003) J Microbiol Methods, 52: 379–388.
• “Labeling Schwann cells with CFSE-an in vitro and in vivo study.” Li X, Dancausse H, Grijalva I, Oliveira M, Levi AD. (2003) J. Neurosci. Methods, 125: 83–91.
• “Tracking of leukocyte recruitment into tissues of mice by in situ labeling of blood cells with the fluorescent dye CFDA SE.”  Becker HM, Chen M, Hay JB, Cybulsky MI. (2004) J. Immunol. Methods, 286: 69–78.
• “5, 6-carboxyfluorescein diacetate succinimidyl ester-labeled apoptotic and necrotic as well as detergent-treated cells can be traced in composite cell sample. Dumitriu IE, Mohr W, Kolowos W, Kern P, Kalden JR, Herrmann M. (2001) Anal. Biochem.  299: 247–252.
• “A flow-cytometric NK-cytotoxicity assay adapted for use in rat repeated dose toxicity studies.” Toxicology Marcusson-Stahl M, Cederbrant K. (2003) 193: 269–279.
• “A novel flow cytometric assay for quantitation and multiparametric characterization of cell-mediated cytotoxicity.” J Lecoeur H, Fevrier M, Garcia S, Riviere Y, Gougeon ML. (2001). Immunol. Methods, 253: 177–187.

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Marker Gene Technologies, Inc. has the expertise to perform contract research with you on your project. We have worked with many biotechnology and pharmaceutical companies on successful, proprietary and patented projects.

Contract Research and Development Capabilities in the following areas:

• Established in 1993 at the University of Oregon Riverfront Research Park.
• Screening Assay Development for HTS and uHTS
• Chemical and Cellular Assays – High-Content Screening.
• DNA/RNA (genomics) and protein (proteomics) labeling and assay development.
• Pharmaceutical Intermediates - design, synthesis, and in vitro testing in mammalian cell culture.
• Specializing in Carbohydrate, Lipid, Peptide, and Nucleic Acid Chemistries.
• Fully equipped laboratories (Biochemistry, Chemical Synthesis, Tissue Culture, Analytical).
• Confidentiality, help in patent preparation and filings.

Contact us by telephone at (888) 218-4062 or (541) 342-3760 or FAX us at (541) 342-1960 or you can write to us at  Contract Research, Marker Gene Technologies, Inc., 1850 Millrace Drive, Eugene, Oregon 97403-1992 or contact us by e-mail at: techservice@markergene.com

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