Marker Gene Monthly Newsletter   

March, 2005

Volume 5, Number 3

© 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.

Genetically engineered cell lines for detecting viruses.

Recent work from the laboratory of Dr. Paul Olivo and co-workers at Washington University and Apath, LLC have utilized cell lines that have been engineered to contain a reporter gene (E. coli LacZ, CAT or luciferase) under the control of an inducible viral promoter.   These stably transfected cell lines (e.g. BHK, Vero, 293) are then used to detect viruses, as they will express the marker gene only upon infection.  Simple histochemical, chemiluminescent or fluorescence-based assays for the reporter enzyme can then be used to quantitate viral titer.   Methods have been described that utilize a herpes simplex virus (HSV) promoter-reporter gene chimeric construct cloned into BHK cell lines.  Herpes promoters have included the HSV-1 ICP6 promoter and the HSV-2 ICP10 promoter. Reporter genes used include the lacZ (beta-galactosidase) gene, firefly luciferase gene and many others.  For RNA viruses such as Respiratory Syncytial Virus (RSV) transcription signals flanking the lacZ or CAT gene are used to promote virus-specific induction.  Although chromogenic detection of virus particles has been quite efficient, fluorogenic substrates, including FDG (M0250) or chemiluminescent detection using D-Luciferin (M0237) are also described.  Among the viruses that can be detected by these methods are several medically important viruses including herpes simplex, influenza, and RSV.  These virus-inducible systems have been used in diagnostic virology to detect viruses in clinical samples (ELVIS-HSV, Diagnostic Hybrids, Inc. Athens, OH) and to determine antiviral susceptibility. The techniques have also been translated into high-throughput antiviral screening platforms for use in antiviral drug development for other viral targets including several flaviviruses (West Nile, Dengue, Yellow Fever), filoviruses (Ebola, Marburg) and togaviruses (VEE).  For more information about these virus detection strategies, please see the references below or visit our website.

•   Olivo, P.D. (1996) “Transgenic cell lines for detection of animal viruses” Clinical Microbiology Reviews 9(3): 321-334, Vol 9, No. 3
• Stabell, E.C., O'Rourke, S.R. Storch, G.A. and Olivo P.D., (1993) “Evaluation of a genetically engineered cell line and a histochemical beta-galactosidase assay to detect herpes simplex virus in clinical specimens.” J. Clin. Microbiol. 31:2796-2798.
•   Ashley R.L., Dalessio J., Sekulovich R.E., (1997) “A novel method to assay herpes simplex virus neutralizing antibodies using BHKICP6LacZ-5 (ELVIS) cells.” Viral Immunol. 10(4): 213-20.
• Proffitt M.R., Schindler S.A., (1995) “Rapid detection of HSV with an enzyme-linked virus inducible system (ELVIS) employing a genetically modified cell line.” Clin. Diagn. Virol. 4(2):175-82.
• Olivo, P.D., (1994) “Detection of herpes simplex virus by measurement of luciferase activity in an infected-cell lysate.” J Virol Methods 47(1-2): 117-28.
• Olivo PD, Collins PL, Peeples ME, Schlesinger S.  (1998) Detection and quantitation of human respiratory syncytial virus (RSV) using minigenome cDNA and a Sindbis virus replicon: a prototype assay for negative-strand RNA viruses. Virology. 251(1):198-205.
• Tebas P, Scholl D, Jollick J, McHarg K, Arens M, Olivo PD.  (1998) A rapid assay to screen for drug-resistant herpes simplex virus.
  J Infect Dis. 177(1): 217-20.
• Andrew Lutz, Julie Dyall, Paul D. Olivo and Andrew Pekosz (2005) Virus-inducible reporter genes as a tool for detecting and quantifying influenza A virus replication.Journal of Virological Methods, In Press, , Available online 12 February 2005,
  

DNA Quantitation using Fluorescent Dye Staining Assays.

Fluorescent Dye	Linear Range	Excitation (nm), Emmission (nm)
4',6-Diamidino-2-Phenylindole (DAPI)	To 2 µg/ml DNA & 50 µg/ml RNA	372, 454
Ethidium Bromide (EtBr)	0.5-3 µg/ml	370, 620
Hoechst 33258	0-150 ng/ml	356, 492
OliGreen	0.0001-1 µg/ml	480, 520
PicoGreen	0.025-1 µg/ml	480, 520
RiboGreen	0.001-1 µg/ml	480, 520

Quantitation of DNA or RNA is extremely important to many protocols in Molecular Biology.  Common techniques that utilize samples of DNA or RNA, such as sequencing, labeling, cDNA synthesis, cloning, RNA transcription and transfection protocols, all require a defined DNA or RNA concentration measurement.  The common UV absorbance technique (Absorbance at 260 nm) is often used to determine nucleic acid concentration.  Typically, a solution having an absorbance of one unit at 260 nm with a path length of 1 cm is said to correspond to a concentration of 30-37 ug DNA per ml.   However, in order to accurately and reliably quantitate low nucleic acid concentrations, UV absorbance alone can be difficult due to the technique's limited sensitivity and susceptibility to background interferences (protein interference, etc.).  Fluorometric analysis provides high sensitivity, selectivity and reliability that make it an excellent choice for low level DNA or RNA quantitation.  Accurate concentration measurements can easily be achieved using a fluorometer, fluorescence microplate reader or simple spot analyses, using one of many commercially available fluorescent dyes.  Because many fluorescent dyes intercalate with the DNA, the specificity of readings is extremely accurate.  See the table above for a list of common dyes and visit our website for more information about these techniques and new products from Marker Gene.

• Sambrook, J., Fritsch, E.F., Maniatis, T., (1989) “Molecular Cloning: A Laboratory Manual, 2ndEc. Plainview, NY: Cold Spring Harbor Laboratory Press.

• Huberman, J.A. (1995). “Importance of measuring nucleic acid absorbance at 240 nm as well as at 260 and 280 nm.” BioTechniques 18(4): 636-636.
• Katouzian-Safadi, M., et al, (1989). “Limitation of DNA-4',6-diamidine-2-phenylindole assay in the presence of an excess of tRNA.” Anal. Biochem.176: 416-419.
•  Brunk, C.F., et al. (1979). “Assay for nanogram quantitites of DNA in cellular homogenates.” Anal. Biochem. 92: 497-500.
• Daxhelet, G. A., et al. (1989). “Spectrofluorometry of dyes with DNAs of different composition and conformation.” Anal. Biochem.179: 401-403. 
• Fisher, Robert, (1997). “Rapid, accurate RNA quantitation using ethidium bromide and a fluorometer.” Biomedical Products, February, p16-17.
• Cesaroni, C. F., et al. (1979). “Improved microfluorometric DNA determination in biological material using 33258 Hoechst.” Anal. Biochem. 100 188-197.

Recombinant Arabidopsis detects explosives residue.

A genetically engineered strain of Arabidopsis thaliana that has the ability to detect explosives in soil, including buried landmines, by changing color from green to red has been developed by Dr. Carsten Meier of the Department of Plant Molecular Biology, University of Copenhagen, Denmark, and Dr. Simon Østergaard of Aresa Biodetection ApS, a Danish company.  The plant is sensitive to nitrogen dioxide gas, which is released by underground explosives in contact with soil through degradation processes.  The team doesn't yet know how sensitive the plants are  to nitrogen dioxide levels, and therefore are not sure how much of the gas is needed to make it turn red.  Because the Arabidopsis plant is quite shallow-rooted, it will likely only detect explosives or mines near the surface.  But this is often  where most landmines are found.  The researchers achieved the color change by manipulating the naturally occurring machinery that produce the red pigment, anthocyanin.  This process is normally switched off during most of the year, except during the Autumn. But the team inserted a gene that turns on this red pigment-making apparatus in the presence of nitrogen dioxide gas.  For more information about these new genetically engineered plants, see the references below or visit our website.

• http://www.nature.com/news/2004/040126/pf/040126-10_pf.html
• http://europa.eu.int/comm/research/headlines/news/article_04_02_11_en.html

Routine Coliform Detection using Fluorogenic Substrates.

The detection of pathogenic bacteria in large volume samples (i.e. drinking water) continues to provide a challenge to researchers.  Proposed indicator bacteria and total coliform (TC) levels often do not accurately indicate a health risk as their presence may result from re-contamination in the distribution system or from natural environmental sources. The group of thermotolerant or "fecal" coliforms (FC) also represents a poor indicator of fecal contamination because it includes species of the genera Klebsiella and Enterobacte that lack significance for human health. Only E.coli, the main representative of the thermotolerant coliforms, always originates from animal sources and is generally considered to be the preferred indicator of fecal contamination of drinking water.  Total coliforms (TC) continue to be maintained as a parameter of general water quality in the legislation of most countries.  Recently, enzymatic tests for the detection of total coliforms and E.coli, based on the demonstration of both ß-galactosidase and ß-glucuronidase, respectively, have become increasingly popular as compared to the fermentation procedures.  Laser scanning methods (ChemScan) have resulted in tests for E. coli and TC that are quick (3.5 h) and sensitive.  These procedures involve enzyme induction (3 h) in the target cells, followed by labelling (30 min) using fluorescein-di-beta-D-glycosides (M0250 FDG; M0969: FDGlcU(OMe) and laser scanning (3 min) of the membrane filter.  Application of the ChemScan E. coli test to naturally contaminated well and surface, as well as uncontaminated water samples has indicated a >90 % agreement, and equivalence with reference methods, with low false-negative rates versus Chromocult Coliform and agar methods.

• Nelis, H., Van Poucke, S,. (2000) “Enzymatic detection of coliforms and Escherichia coli within 4 hours.” Water, Air, and Soil Pollution 123(1-4):  43-52. 
• Allen,M.A., Edberg,S.C (1997),in Coliforms and E.coli .Problem or Solution?The Royal Society of Chemistry, Cambridge,176-181.
• Hofstra,H., Huis in ’ t Veld,J.H.J.:1988 ,J. Appl. Bacteriol. Symp. Suppl ., 197S-212S.
• Edberg,S.C.,  Edberg,M.M.:1988,Yale J. Biol. Med. .61: 389-39 .
• Brenner, K.P.,Rankin, C.C.,Roybal, Y.R.,Stelma, G.N.,Scarpino, P.V., Dufour, A.P.(1993) Appl.Environ.Microbiol .59: 3534-3544.
• Jermini,M., Domeniconi,F., Jäggli,M., (1994) Lett. Appl. .Microbiol .19: 332-335.
  
  

New 2005-2006 Catalog Now Available.

The new 2005-2006 edition of the Marker Gene catalog is now available.  Visit our website or use the link www.markergene.com/catalog2005-2006.pdf for an electronic version.  Many new products and kits, additional literature references, data and protocols have been included, as well as new information about our old products.  If you haven’t already received yours, be sure to add your name to our mailing list.  Visit our Customer Information Form, or e-mail us at techservice@markergene.com and we will have a copy sent out to you as soon as possible.

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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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