Marker Gene Monthly Newsletter   

December, 2005

Volume 5, Number 12

© 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.

Improved Transfection Reagents.

The promise of gene therapy for the treatment of genetic diseases has been tempered by issues of poor gene delivery across cell membranes, problems with endosomal trafficking as well as harmful side-effects.  Retroviral or lentiviral vectors commonly used for such transfections can elicit immune responses from the transferred DNA as well as other toxic effects.  For this reason, non-viral  methodologies utilizing cationic liposomes and polymers are being investigated for DNA and siRNA delivery to influence gene expression in vivo.  Typically, lipofectamine^TM reagents (dioleoylphospatidylcholine and it’s polypropyleneimine derivative) and polyethylenimines (PEIs) are used in such gene transfers.  Recent work from the laboratory of Dr. Alexander M. Klibanov and co-workers at The Department of Chemistry and the Center for Cancer Research, Massachusetts Institute of Technology, has investigated the purity of commercial PEI preparations with respect to their effect on transfection efficiency.  It was found that common linear PEI’s contain up to 11% residual acylated amino groups (propionyl groups) left over from the commercial preparation process.  Removal of these extra groups by acid hydrolysis resulted in clean PEI’s that were up to 21 times more efficient for in vitro transfections, and up to 10,000 times more efficient in vivo, with a 15,000-fold greater specificity for lung. These improvements seem to stem from an increase in the number of protonatable nitrogens, that causes tighter binding with plasmid DNA and better endosomal escape of the PEI-DNA complexes.  Fully deacylated PEIs of MW 22-, 87- and 217 kDa prepared from poly-2-ethyl-oxazolines (PEOZ’s) were even more efficient for in vitro and in vivo transfections. The 87-kDa PEI, for example, mediated the delivery of an siRNA against the influenza nucleocapsid protein, resulting in a 94% drop in the virus titer of infected mice.  For more information about these new techniques, please see the references below, or visit our website.

• Thomas M, Ge Q, Lu JJ, Chen J, Klibanov AM. (2205) “Cross-linked small polyethylenimines: while still non-toxic, deliver DNA efficiently to mammalian cells in vitro and in vivo.” Pharm Res 22: 373-380.
•    Thomas M, Klibanov AM. (2003) “Conjugation to gold nanoparticles enhances polyethylenimines transfer of plasmid DNA into mammalian cells.” Proc Natl Acad Sci USA 100: 9138-9143.
• Thomas, M.,  Lu, J.J., Ge, Q. Zhang, C., Chen, J., and Klibanov, A.M.,  (2005) “Full deacylation of polyethylenimine dramatically boosts its gene delivery efficiency and specificity to mouse lung” Proc. Natl. Acad. Sci. USA 102 (16): 5679–5684.
•   Thomas, M., Klibanov, A. M. (2003) “Non-viral gene therapy: polycation-mediated DNA delivery.” Appl. Microbiol. Biotechnol. 62: 27–34.
• Kaiser, J. (2004) “Gene therapy. Side effects sideline hemophilia trial.“ Science 304: 1423–1425.
  

Cell / Tissue Permeant D-Luciferin.

The luciferase enzyme cloned from the North American firefly (Photinus pyralis: [1.13.12.7]) oxidizes ATP-activated D-luciferin through a dioxetane intermediate, producing oxyluciferin and a bright yellow-green light (560 nm) with high quantum yield (0.88) at the site of activity.   The firefly luciferase gene luc and several codon optimized versions have been widely used as marker genes, especially in live cell analysis of transcription and regulatory mechanisms in bacterial, yeast, mammalian and plant cells.  Measurement of the light emission can be monitored using common microplate readers, luminometers, scintillation counters or with high-speed film or digital cameras.  Recently, the use of the luciferase marker gene in whole organisms has been developed, by combining luciferase transfection in specific tissues with systemic application of D-luciferin.  The ability to measure light emission from luciferase activity inside living tissues and in vivo by injecting live animals with D-luciferin (M0237) is aided by the use of cooled CCD or photon counting cameras that can easily analyze the low-light levels emitted in whole animal studies.  The potential of measuring tumor growth and tumor burden in vivo as well as the possibility of streamlining development of many types of therapies, including DNA-based gene therapies and gene vaccines using these new techniques is exciting. 

Marker Gene has now developed a membrane permeable analog of D-luciferin, D-Luciferin, ethyl ester (M0906) to aide with these studies.  This analog of the primary substrate for the firefly luciferase light producing system has been found to produce up to 30% higher light intensity in vivo.  The esterification of the D-luciferin carboxyl group as an ethyl ester increases the uptake of the substrate by intact mammalian cells in live-cell luciferase assays.  After entering the cell, the ethyl ester is quickly removed by ubiquitous esterase activity inside living cells and tissues.  For more information about this new substrate or our other products for live cell luciferase assay, please visit our website or see the references below.

• "Membrane-permeable Luciferin Esters for Assay of Firefly Luciferase in Live Intact Cells." (1991) F.F. Graig, et al. Biochem. J. 276: 637.
• "Applications of the Direct Imaging of Firefly luciferase Expression in Single Intact Mammalian Cells Using Charge-coupled Device Cameras." M.R.H. White, et al.Bioluminescence and Chemiluminescence: Current Status. P.E. Stanley and L.J. Kricka (Eds), John Wiley, Chichester, pp. 357-360.
• "CCD Imaging of Luciferase Gene Expression in Single Mammalian Cells." (1990) C.E. Hooper, et al. J. Bioluminescence and Chemiluminescence. 5:123 (1990).
• "Real-time Analysis of the Transcriptional Regulation of HIV and hCMV Promoters in Single Mammalian Cells." (1995) M.R.H. White, et al. J. of Cell Science 108: 441.

ROS measurement in live cells.

The generation of reactive oxygen species (ROS) is ubiquitous in aerobic organisms, and in healthy cells it occurs at a controlled rate.  But under conditions of oxidative stress, ROS production can increase, resulting in damage to membrane lipids, proteins, and nucleic acids.   This oxidative damage has been associated with a variety of pathological conditions including neurodegenerative disorders, carcinogenesis, injuries during ischemic reperfusion and atherosclerosis.   Dichlorofluorescin, diacetate (H₂DCFDA, M0807) is a cell-permeant indicator for reactive oxygen species that is nonfluorescent until the acetate groups are removed by intracellular esterases and oxidation occurs within the cell.  The reduced substrate releases the highly fluorescent dye 2',7'-dichlorofluorescein and allows easy detection of peroxidase activity (oxidative metabolism, intracellular regulation of reactive oxygen species or oxidative burst) inside living cells.  When the cell begins to produce reactive oxygen species, the highly fluorescent dye 2',7'-dichlorofluorescein is produced, with EX: 495nm and EM: 529 nm.  Marker Gene now provides this reagent in a easy to use kit form in the MarkerGene^TM Live Cell Fluorescent Reactive Oxygen Species Detection Kit (M1049), that includes an inducer (for positive control applications), the H₂DCFDA substrate, standards and a detailed protocol for quick and easy discrimination of oxidatively stressed and nonstressed cells by fluorescence microscopy, microtiterplate assay or photomicroscopy.  For more information about this new kit, please see the references below or visit our website:

•  Ubezio P., Civoli F., (1994) "Flow cytometric detection of hydrogen peroxide production induced by doxorubicin in cancer cells." Free Radic Biol Med 16: 509-516.
• Bailey S.R., Mitra S., Flavahan S., Flavahan N.A., (2005) "Reactive oxygen species from smooth muscle mitochondria initiate cold-induced constriction of cutaneous arteries." Am J Physiol Heart Circ Physiol 289: H243-50.
• Halliwell B., Whiteman M., (2004) "Measuring reactive species and oxidative damage in vivo and in cell culture: how should you do it and what do the results mean?" Br J Pharmacol 142: 231-55.
• Kutuk O., Adli M., Poli G., Basaga H., (2004) "Resveratrol protects against 4-HNE induced oxidative stress and apoptosis in Swiss 3T3 fibroblasts." Biofactors 20: 1-10.
• Myhre O., Andersen J.M., Aarnes H., Fonnum F., (2003) "Evaluation of the probes 2',7'-dichlorofluorescin diacetate, luminol, and lucigenin as indicators of reactive species formation." Biochem Pharmacol 65: 1575-82.

Biotin Labeling for Immunochemistry Applications.

Biotin is a commonly used hapten that can be detected through it's strong affinity for the proteins avidin (from egg white) or streptavidin (from Streptomyces avidinii).   Attaching a biotin to a protein or antibody allows easy detection since many avidin or streptavidin conjugates are available that have been fluorescently labeled or are conjugated to a second protein or other detection reagent.  Biotin labeling has been used in many detection schemes for peptides, proteins, organelle components, monoclonal antibody analyses, oligonucleotide and microbead labelings.  Marker Gene now provides a quick and easy kit for labeling of analytes with D-biotin.  The MarkerGene^TM Biotin-X Protein Labeling Kit (M1138) provides all of the reagents and a detailed protocol for efficient biotin labeling of antibodies or other proteins of interest.  This new kit is perfect for labeling your important proteins or antibodies with biotin for the application in immunohistochemistry, in immunoblotting, or in ELISA applications.  The kit can also be used for labeling of aminoallyl labeled DNA or RNA samples.  The principle of labeling involves reaction with free amino groups on the protein with the highly selective reagent D-biotinoyl-e-aminocaproic acid, N-hydroxy-succinimidyl ester (M0783: biotin-X-NHS) by forming a stable amide bond.  Unreacted biotin-X-NHS is easily separated from conjugated protein on a Sephadex  G-25 spin-column provided with the kit.  The long spacer between the biotin and the reactive group in biotin-X succinimidyl ester enhances the ability of the conjugated biotin to interact with biotin-binding sites on avidin or streptavidin.  Also included in the kit are a gel filtration spin-column and Sephadex packing to purify protein from excess biotin,  Each kit contains enough biotinylation reagents for several reactions or for labeling of up to 5–20 mg of protein.  For more information about biotin labeling or the new MarkerGene^TM Biotin-X Protein Labeling Kit (M1138), please see the references below, or visit our website. 

• "Peptide biotinylation with amine-reactive esters: differential side chain reactivity." Miller BT, Collins TJ, Rogers ME, Kurosky A. Peptides 18, 1585-1595 (1997)
• "Phorbol 12-myristate 13-acetate down-regulates Na,K-ATPase independent of its protein kinase C site: decrease in basolateral cell surface area." Beron J, Forster I, Beguin P, Geering K, Verrey F. Mol Biol Cell 8, 387-398 (1997)
• "Identification and characterization of O-biotinylated hydroxy amino acid residues in peptides." Miller BT, Rogers ME, Smith JS, Kurosky A. Anal Biochem 219, 240-248 (1994)
• "The biotin/avidin-mediated microtiter plate lectin assay with the use of chemically modified glycoprotein ligand." Duk M, Lisowska E, Wu JH, Wu AM. Anal Biochem 221, 266-272 (1994)
• "In Vitro Synthesis of Biotinylated RNA Probes from A-T Rich Templates: Problems and Solutions." Luehrsen KR, Baum MP. Biotechniques 5, 660 (1987)
• "Novel biotinylated nucleotide--analogs for labeling and colorimetric detection of DNA." Gebeyehu G, Rao PY, SooChan P, Simms DA, Klevan L. Nucleic Acids Res 15, 4513-4534 (1987)
• "Homogeneous functional insulin receptor from 3T3-L1 adipocytes. Purification using N alpha B1-(biotinyl-epsilon-aminocaproyl)insulin and avidin-sepharose." Kohanski RA, Lane MD. J Biol Chem 260, 5014-5025 (1985).
• "Immunogenicity of biotinylated hapten-avidin complexes." Scott D, Nitecki DE, Kindler H, Goodman JW. Mol Immunol 21, 1055-1060 (1984)
• "Avidin-biotin affinity chromatography: application to the isolation of human placental insulin receptor." Finn FM, Titus G, Horstman D, Hofmann K. Proc Natl Acad Sci U S A 81, 7328-7332 (1984)
• "Rapid and sensitive colorimetric method for visualizing biotin-labeled DNA probes hybridized to DNA or RNA immobilized on nitrocellulose: Bio-blots." Leary JJ, Brigati DJ, Ward DC. Proc Natl Acad Sci U S A 80, 4045-4049 (1983)
  
  

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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