Marker Gene Monthly Newsletter   

October, 2005

Volume 5, Number 10

© 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.

Vital Stains for lacZ Transfected Cells.  

LacZ b-galactosidase (b-Gal) is a common marker gene used for monitoring gene transfection efficiency in bacteria, yeast and mammalian cell lines.  Numerous assays for b-Gal activity have been described in the literature for intracellular analyses in fixed or live cells.  Localizing the staining pattern in the transfected cells, for later photomicroscopy or counting, requires that the product remain in the cells after staining, either as a precipitate or through production of a membrane impermeant product.  Staining with the indigogenic substrate 5-bromo-4-chloro-3-indolyl b-D-galactopyranoside (X-Gal) is among the most common.  This substrate produces a blue indigo dye after reaction with galactosidase and subsequent oxidation.  It can be used in fixed (both adherent and suspension cells) as well as live cells in culture and even in live tissues, where staining is dependent upon both lacZ activity as well as permeation into the tissues.  When ß-gal cleaves the glycosidic linkage in X-Gal, a soluble, colorless indoxyl is produced, which then dimerizes nonenzymatically and oxidizes to give a stable, insoluble halogenated indigo dye. The dimerization and oxidation reactions require transfer of an electron, that can be facilitated by electron acceptors of the proper redox potential. The ferric and ferrous ions included in most X-Gal reaction buffers provide this function.  But tetrazolium salts can also serve as the electron acceptors, and if added in conjunction with X-Gal, highly colored formazan compounds are produced.  Nitroblue tetrazolium (NBT) is reduced by the hydride produced when dimerization of two indoxyls result from cleavage of X-Gal forming a formazan,  with a dark purple precipitate.  Other tetrazolium salts include tetrazolium red which  produces a greenish-blue precipitate, but increases background.  Modified indolyl-based substrates that provide alternative colored precipitates include Salmon-Gal (orange/pink precipitate), Magenta-Gal (light pinkish purple precipitate) and Green-Gal (in the presence of NBT gives a purple precipitate).  Phenazine methosulfate (PMS) can further increase the development reaction rate by quantitatively reducing tetrazolium salts.

Fixed cells also can be analyzed by assaying for b-gal activity by fluorescence histochemistry using an azo dye in combination with either X-gal or 5-bromo-6-chloro-3-indolyl b-D-galactopyranoside (5-6-X-Gal, Red-Gal).  A preferred combination is the azo dye Red Violet LB and 5-6-X-Gal, referred to as Fluor-X-Gal.  With this combination, fluorescence micrographs can be obtained using a Rhodamine/Texas Red filter set.  Use of these substrates allows b-Gal-dependent fluorescence to be visualized simultaneously with other fluorescent labels (FITC, AMC, etc.).

A vital fluorogenic substrate, resorufin beta-galactoside bis-aminopropyl polyethylene glycol 1900 (RGPEG) has also recently been described.  This compound can be delivered to cells by microinjection, electroporation or a variety of bulk-loading techniques. Once inside a cell, the substrate is unable to escape through the plasma membrane or by gap junctions and specifically stains lacZ-positive cells.  For more information about these substrates and cell labeling and analysis, please see our web site or the references below.

• Minden, J.S., (1996) “Synthesis of a new substrate for detection of lacZ gene expression in live Drosophila embryos.”BioTechniques 20(1): 122-129.
• Mohler, W. A., Blau, H. M., (1996) “Gene expression and cell fusion analyzed by lacZ complementation in mammalian cells.“ Proc. Natl. Acad. Sci.,USA , 93: 12423-12427

• Rosenberg, W.S., Breakefield, X.O., DeAntonio, C., Isacson, O., (1992) “Authentic and artifactual detection of the E. coli laxZ gene product in the rat brain by histochemical methods.” Brain Research 16:311.
• Krasnow, M.A., S. Cumberledge, G. Manning, L.A. Herzenberg, and G.P. Nolan, (1991) “Whole animal cell sorting of Drosophila embryos.” Science 251: 81.
• Lin, S., S. Yang, and N. Hopkins (1994) “LacZ expression in germline transgenic zebrafish can be detected in living embryos” Dev. Biol. 161: 77.
• Lojda, Z. (1970) “Indigogenic methods for glycosidases.” Histochemie 22: 347.
• Altman, F.P. (1972) “Quantitative dehydrogenase histochemistry with special reference to the pentose shunt dehydrogenases.” Progr. Histochem. Cytochem. 4: 225.

Measuring Hypoxia in Tumor Cells.

Solid tumors containing hypoxic regions exhibit a more malignant phenotype by increasing the expression of genes encoding angiogenic and metastatic factors. The Hypoxia-Inducible Factor-1 (HIF-1) is a master transcriptional activator of these genes, and thus, imaging and targeting hypoxic tumor cells where HIF-1 is active has become an important method for measuring the levels of hypoxia in solid tumors.  HIF-1 activity has been monitored via optical in vivo imaging systems by using several marker gene constructs (luciferase, GFP or hCG) under the regulation of an HIF-1-dependent promoter.  Human chorionic gonadotropin (hCG) is a glycoprotein secreted during pregnancy that has been used to monitor tumor burden in xenografts engineered to express this marker.  Another reporter gene construct using a herpes simplex virus 1-thymidine kinase (HSV1-tk) fused with the enhanced green fluorescent protein (eGFP) under the regulation of an artificial hypoxia-responsive enhancer/promoter, HIF-1 has also been used in these studies.

Immunohistochemical analysis of the xenografts with a hypoxia marker, pimonidazole, have confirmed that marker gene-expressing cells under control of the HIF-1 promoter are hypoxic. Evaluation of the efficacy of a hypoxia-targeting prodrug, TOP3, using these optical imaging systems have also revealed that hypoxic cells were significantly diminished by TOP3 treatment.   Immunohistochemical analysis of the TOP3-treated xenografts confirmed that hypoxic cells underwent apoptosis and were removed after TOP3 treatment.  These results demonstrate that this model system using an HIF-marker gene construct can provide qualitative information (hypoxic status) of solid tumors and enable convenient evaluation of the efficacy of cancer therapies on hypoxia in malignant solid tumors.  For more information on these new marker gene strategies, please visit our website or see the references below.

•   Harada H., Kizaka-Kondoh S.,  Hiraoka M., (2005)  “Optical imaging of tumor hypoxia and evaluation of efficacy of a hypoxia-targeting drug in living animals.” Mol. Imaging 4(3): 182-93.
• Wen B., Burgman P., Zanzonico P., O'donoghue J., Cai S., Finn R., Serganova I., Blasberg R., Gelovani J., Li GC., Ling C.C. (2004)  “A preclinical model for noninvasive imaging of hypoxia induced gene expression; comparison with an exogenous marker of tumor hypoxia.” Eur. J. Nucl. Med. Mol. Imaging  31(11): 1530-8. 
• Gross J., Fuchs J., Machulik A., Jahnke V., Kietzmann T., Bockmühl U. (2005) “Apoptosis, necrosis and hypoxia inducible factor-1 in human head and neck squamous cell carcinoma cultures.” Int. J. Oncol. 27(3): 807-14.
• Nelson D.W., Cao H., Zhu Y., Sunar-Reeder B., Choi CY., Faix JD., Brown J.M., Koong A.C., Giaccia A.J., Le Q.T , (2005)  “A noninvasive approach for assessing tumor hypoxia in xenografts: developing a urinary marker for hypoxia.” Cancer Res.  65(14): 6151-8. 

Near Infrared Dyes for Tumor Monitoring.

Near-infrared (NIR) fluorescence imaging has the potential to revolutionize human cancer surgery by providing sensitive, specific, and real-time intraoperative visualization of normal and disease processes.  Because tissues are transparent in the so-called “therapeutic window (650-1000nm), these new labels have found applications for real-time diagnostic or therapeutic procedures.  Recently several new NIR dyes have made it possible to target tumor tissues for in vivo detection.  Indocyanine Green (ICG) is a FDA approved dye that has already been used in numerous angiography or retinal and choroids diagnostic tests.  Now an indocyanine green derivative (ICG-sulfo-OSu) has been developed as an infrared fluorescent labeling substance suitable for detection by an IR fluorescence endoscope.  By combining NIR dye labels with antibody, or specific antigen labels (CEA, HER2), real-time visualization of structures in live tissues can be obtained.  Additional NIR labels include the use of cypate or nanoparticle substances.  A Cypate-Bombesin Peptide Analogue Conjugate (Cybesin) has been used as a prostate tumor receptor-targeted contrast agent. The absorption and fluorescence spectra of Cybesin were measured and shown to exist in the NIR tissue "optical window".  The spectral polarization imaging of Cybesin-stained prostate cancerous and normal tissues has shown that prostate cancerous tissue takes-up more Cybesin than that of prostate normal tissue, making Cybesin a potential marker of prostate cancer.  Nanoshells are another novel class of optically tunable NIR particles that consist of a dielectric core surrounded by a thin gold shell.  Nanoshells may be designed to scatter and/or absorb light over a broad spectral range including the near-infrared (NIR), a wavelength region that provides maximal penetration of light through tissue. When combined with an anti-HER2 antibody, a clinically relevant cancer biomarker has been developed.  For more information about these new NIR labeling techniques, please see our website or the references below.

• Pu Y., Wang W.B., Tang G.C., Zeng F., Achilefu S., Vitenson J.H., Sawczuk I., Peters S., Lombardo J.M., Alfano R.R. (2005)  “Spectral polarization imaging of human prostate cancer tissue using a near-infrared receptor-targeted contrast agent.” Technol. Cancer Res. Treat. 4(4): 429-36.

• Inayama K., Ito S., Muguruma N., Kusaka Y., Bando T., Tadatsu Y., Tadatsu M., Ii K., Shibamura S., Takesako K., (2003)  “Basic study of an agent for reinforcement of near-infrared fluorescence on tumor tissue.” Dig. Liver Dis. 35(2): 88-93.
• Ito S., Muguruma N., Kusaka Y., Tadatsu M., Inayama K., Musashi Y., Yano M., Bando T., Honda H., Shimizu I., Ii K., Takesako K., Takeuchi H., Shibamura S., (2001)  “Detection of human gastric cancer in resected specimens using a novel infrared fluorescent anti-human carcinoembryonic antigen antibody with an infrared fluorescence endoscope in vitro.” Endoscopy 33(10): 849-53. 
•  Loo, C., Lowery, A., Halas, N., West, J.,  Drezek, R., (2005) “ImmunotargetedNanoshells for Integrated Cancer Imaging and Therapy” Nano. Lett. 5(4): 709 – 711.

New Fluorogenic Substrate for Caspase 3/7.

Homeostasis in multicellular organisms is maintained by a careful and coordinated cellular self-destruction process termed apoptosis.  This process is mediated by a number of cell surface receptor signal transduction events based on antagonistic or agonistic ligand receptor interactions or by intracellular acting molecules affecting functions within the mitochondria.  The resulting sequential,energy-dependent,enzymatic cascade involving proteolytic enzymes called caspases leads to destruction of integral intracellular DNA repair elements, structural polypeptides and signaling intracellular kinases.  Two of the primary enzymes,   caspases-3 and -7,are widely used in reliable detection of apoptotic events.  Marker Gene now produces a specific Caspase 3/7 fluorogenic substrate for detection of these important enzymes.  The reagent can be delivered either manually or by a robotic liquid-handling system.  After incubation, cleavage of the substrate releases the fluorescent dye rhodamine 110 that can be read in standard fluorometric fashion using conventional microplate readers equipped with a 485 ±20nm excitation source and 530 ±25nm emission collector.

Our (Ac-DEVD)2-Rhodamine 110 substrate, enables a simple "add-mix-read" format for the detection of caspase-3 and -7 in adherent, suspension, and primary culture cells, or in purified caspase preparations. The homogeneous format eliminates tedious washing, concentration, and multiple freeze-thaw steps required by conventional caspase detection assays, resulting in a dramatic reduction in sample preparation time. The rhodamine 110-based substrate allows for exquisite sensitivity previously unobtainable with conventional colorimetric or fluorometric assays.  Furthermore, the assay is inherently flexible for use in a variety of volumes and formats from cuvettes to 384 well plates.  For more information about this new Caspase substrate for use in ultrasensitive measurement of apoptosis initiation please see our website or the references below.

• Learish, R.L., Bruss, M.D., and Haak-Frendscho, M. (2000) Dev. Brain Res. 122, 97–109. 
• Nicholson, D.W. et al. (1995) Nature 376, 37–43. 
• Garcia-Calvo, M. (1999) Cell Death Differ. 6, 362–9.
•   Slee, E. A., C. Adrain, and S. J. Martin. 1999. Serial Killers: ordering caspase activation events in apoptosis. Cell Death and Differ. 6:1067-1074.
• Walker, N. P., R. V. Talanian, K. D. Brady, L. C. Dang, N. J. Bump, C. R.Ferenz, S. Franklin, T. Ghayur, M. C. Hackett and L. D. Hammill. 1994. Crystal Structure of the Cysteine Protease Interleukin-1ß-Converting Enzyme: A (p20/p10)₂ Homodimer. Cell 78:343-352.
• Wilson, K. P., J. F. Black, J. A. Thomson, E. E. Kim, J. P. Griffith, M. A.Navia, M. A. Murcko, S. P. Chambers, R. A. Aldape, S. A. Raybuck, and D. J.Livingston. 1994. Structure and mechanism of interleukin-1 beta converting enzyme. Nature 370: 270-275.
• Rotonda, J., D. W. Nicholson, K. M. Fazil, M. Gallant, Y. Gareau, M. Labelle,E. P. Peterson, D. M. Rasper, R. Ruel, J. P. Vaillancourt, N. A. Thornberry and J.W. Becker. 1996. The three-dimensional structure of apopain/CPP32, a key mediator of apoptosis. Nature Struct. Biol. 3(7): 619-625.
• Kumar, S. (1999) "Mechanisms mediating caspase activation in cell death." Cell Death and Differ. 6: 1060-1066.
  
  

Fluorescently Labeled Avidin and Streptavidin Assays.

Avidin is a tetrameric glycoprotein of MW 68K daltons that represents about 0.05% of the total protein content of the hen egg white.  The great affinity of avidin for d-biotin (Ka = 10^-15/M) results in a great number of applications in biochemistry (immunoassays, receptor and histochemical studies, bacteriophage inhibitions).   It combines stoichiometrically with biotin. The toxic effect of uncooked egg white which causes a syndrome similar to that of vitamin B deficiency led to the discovery of the vitamin biotin. The toxic factor, first isolated by Eakin, who named it avidin, combines with the essential growth factor resulting in a "non-digestible" avidin-biotin complex that is not absorbed from the intestine or from the surrounding medium by microorganisms.  Avidin is a highly cationic glycoprotein that can selectively bind to a component in human and rodent mast cell granules in fixed-cell preparations, and can be used to identify mast cells in normal and diseased human tissue without requiring a biotinylated probe.  It does not contain the RYD sequence found in Streptavidin, that is homologous of some integrins, causing nonspecific binding that is observed in some detection systems with Streptavidin.

Streptavidin is a neutral bacterial analog of Avidin obtained from Streptomyces avidinii that is non-glycosylated, and does not show the non-specific cell surface binding found with the native avidin.  Streptavidin is also a tetrameric protein (4 x 13kDa) that has been used in various biochemical applications for detection in tissues or cells.  Streptavidin also has a high affinity for biotin (Ka ~ 10¹³ M^-1).  Each monomer of streptavidin binds one molecule of biotin.  Although streptavidin and avidin are similar, each has distinct properties that should be taken into consideration when developing an assay. Streptavidin has physical properties that allow higher signal-to-noise ratios than is possible with the biotin-avidin complex. But it is much less soluble in water than avidin, and reduced nonspecific binding to cell surface antigens.

Fluorescein, Texas Red ^TM as well as TAMRA conjugates of Avidin and Streptavidin have become increasingly useful for DNA and RNA microarray analyses, as a method of detecting biotin-labeled antibodies, or in ELISA assays.  Marker Gene is now developing these important labeled avidin and streptavidin conjugates for your assay development.  Our Texas Red^TM Avidin is a highly fluorescent conjugate of Avidin and sulforhodamine 101.  This red fluorescent product excites at 595 nm with an emission maximum at 615 nm.  Since the excitation and emission maxima are well separated from those of fluorescein, Texas Red^TM Avidin can be employed with Fluorescein Avidin or other fluorescein conjugates to simultaneously localize two antigens in the same tissue section.  Texas Red^TM Avidin is ideal for flow cytometry applications using instruments equipped with dye lasers.  For more information about these new products and for technical assistance in developing your assays, please visit our website or contact our technical assistance department at techservice@markergene.com or by telephone at 1-888-218-4062.

• Alon, R., Bayer, E. A., and Wilchek, M., (1990) "Streptavidin Contains An RYD Sequence Which Mimics The RGD Receptor Domain of Fibronectin" Biochemical and Biophysical Research Communications 170: 1236-1241
• Chaiet, I. and Wolf, F.J. (1964). "The properties of streptavidin, a biotin -binding protein produced by Streptomycetes.". Arch. Biochem. Biophys. 106: 1-5.
• Gitlin, G., Bayer, E.A. and Wilchek, M. (1987) "Studies of the biotin -binding site of avidin." Biochem. J. 242: 923-926.
• Wood, G.S. and Warnke, R. (1981) “Suppression of endogenous avidin-binding activity in tissues and its relevance to biotin-avidin detection systems.” J. Histochem. Cytochem. 29: 1196-1204.
• Wood, G.S., and Warnke, R. (1981) J. Histochem. Cytochem. 29:1196.
  
  

Compare Our Quality. 

Marker Gene strives to offer our customers products of the highest quality and at the best possible prices.  Our years of experience allow us to provide timely products for less cost to you.  See our latest Price Comparison Chart that compares our prices with those from several alternate sources, to see if you can save money by switching to Marker Gene (http://www.markergene.com/crossref.htm).  Or visit our website at www.markergene.com and click on the link “COMPARE”.  We think you will appreciate our efforts to keep costs low and maintain excellent quality of our products for your research.  For more information about any of our products, simply telephone us toll free at 1-888-218-4062 or contact us by e-mail at techservice@markergene.com.  We will be happy to send you more about our products and their specifications.

CONTRACT  RESEARCH@markergene.com

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

Marker Gene Accepts Major Credit Cards.

Place your orders now, using Master Card or Visa and save time and money!  Our Customer Assistance Staff can now accept either Master Card or Visa Credit Card orders, securely by telephone (toll-free) at 1-888-218-4062 (Domestic orders only).   We will continue to accept Institutional Purchase Orders for our products, online or by FAX at 1-541-342-1960.  International customers should contact us by e-mail, post or telephone for more information about International Distributors and ordering.  For information on pricing for individual products, or for a quote on bulk quantities of our products or kits, please contact our technical assistance staff at techservice@markergene.com.   We will be happy to assist you. 

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